SUZANNE ABRAFI ADJOA
DETERMINANTS .GENETIQUES DE LA PERFORMANCE AEROBIE
ET DE LA REPONSE A L'ENTRAINEMENT PHYSIQUE
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presentee
a la Faculte des etudes superieures
de l'universite Laval
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pour I'obtention
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du grade de Philosophiae Doctor (Ph.D.)
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Departernent d'Education Physique
FACULTE DES SCIENCES DE L'EDUCATION
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UNIVERSITE LA VAL
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JANVillR 1996
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B UNlVERSITE
$lAVAL
ATTESTATION
Faculte des etudes superieures
Ce
26
jour du mois de fevri er
19 ~ , les personnes soussignees , en
leur qualite de membres du jury de la these de _Su_z_a_n_n_e_A_b_r_a_f_i_A_d....,:J'-·a_a
_
ont assiste ala soutenance de cette these.
NOMS
UNIVERSITE
SIGNATURE
France T. Di anne
Uni versite Lava 1
Marcel Boulay
Universite Laval
Claude Bouchard
Universite Laval
Jean-Aime Simaneau
Universite Laval
,lames S
Skj noer
National Institllte for
Fitness & Sport
(/~c6~j/~
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A Olivia Lebry
A Christian Berger
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11
BREF RESU1\\-tE
Cette these pone sur les determinants genetiques qui pourraient influencer la sensibilite a
1'entrainement physique en endurance. Les resultats de nos travaux indiquent que des
variations dans la sequence du gene de la sous-unite Va de la cytochrome c oxidase
(COXVa) et de l' ADN mitochondrial (ADNmt) ainsi que les variations de la proreine
phosphoglucomutase-l (PGMl), peuvent influencer la reponse a l'entrainement. Ainsi, la
distribution
des
genotypes
de
la
COXVa
chez
les
athletes
d'endurance
etaic
significativement differente de celle d'un echantillon de sujets non entraines
(P<0.05).
Quant a l' ADNmt, les patrons d' ADN qui avaient ete associes a une \\'0 max
de
2
preentrainement elevee et a une bonne reponse de la \\'0 max a l'entrainement, etaient
2
egalement associes au statut d'athletes d'endurance (P<0.05). Par ailleurs, au niveau de la
PGMl, certains genotypes identifiables par focalisation isoelectrophoretique etaient associes
aune faible reponse ala performance, definie par un test de 90 minutes (P=O.057), d'autres,
aune bonne trainabilite de la \\'0 max (P<O.OOl). Finalement, il decoule des resultats de
2
l'analyse du polymorphisme des genes que les Noirs Sud-Africains etaient significativement
differents des Caucasiens Canadiens et Sud-Africains pour la distribution des genotypes des
genes de l'adenylate kinase 1 musculaire (AKIM) (P<0.005) et de la COXVa (P<0.005).
/fALYV'6d ]2>-0';'''':'-
,
I
'-Suzanne A. Adj~a, M.
France T. Dfonne, Ph. D.
\\

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ill
RESUME
11 est bien etabli qu'il existe une grande variabilire clans la reponse des individus a
l'entrainement. n est en outre bien connu que ces differences de reponses sont
genetiquement detenninees. Pour definir les bases genetiques qui detenninent ces
differences individuelles de trainabilite, nous avons en premier lieu identifie des variations
clans la sequence de certains genes candidats des voies de production et de regeneration de
l' adenosine triphosphate (ATP). En second lieu, nous avons recherche des associations entre
le polymorphisme de sequence de ces genes et la reponse de la V0 max a l'entrainement
2
d'une pan et la perfonnance d'autre part. L' ADN total a donc ere isole des globules blancs
et les variations de sequence ont ete analysees par la technique du polymorphisme de
longueur de fragments de restriction (RFLP). De plus, la technique de focalisation
isoelectrophoretique (IEF) a ete utilisee pour caracteriser l' enzyme phosphoglucomutase-l
(PGMl). Nos resultats ont permis d'identifier des variations dans la sequence de l' ADN des
genes de la glycogene synthase (GS) et de la sous-unite Va de la cytochrome c oxidase
(COXVa). Ces 2 RFLPs sont donc des marqueurs genetiques du polymorphisme de leur
gene respectif. Des etudes d'association, il ressort que la variabilite du gene de la COXVa
etait significativement associee au statut d'athlete d'endurance puisque la distribution des
genotypes de la COXYa-SstI des athletes etait significativement differente de celle des
sedentaires (P<O.05). Enfin, les variations clans la sequence des genes de 1'adenylate kinase
1 musculaire (AKIM) et de la phosphofructokinase musculaire (PFKM) n'etaient pas
associees au niveau de performance des sujets. Quant al' ADN mitochondrial (ADNmt), les
variations au niveau de l' ARN de transfert de la threonine detectees avec l'enzyme de
restriction MspI et de la sous-unite 5 de la NADH deshydrogenase detecrees avec NciI, se
retrouvaient a une plus haute frequence (P<O.05) chez les athletes d'endurance. Ces
\\
variations avaient ere prealablemenc associees a une \\'0 max de preentrainemenr elevee.
2
La variation dans la region D-loop de l' ADNmt detectee avec l' enzyme KpnI, presentait
egalement une frequence de 5 fois superieure (P<O.05) acelle observee chez les sedentaires.
Cette variation avait ete prealablement assoclee a'une bonne reponse de}a V0 max a
2
.
"
",_., .,;.~.:.:

---

iv
l'entrainement. Par ailleurs, au niveau de la proteine PGMl, les genotypes 1-1- et 1+2-
etaient significativement associes aune faible reponse de la perfonnance (P=O.057) evaluee
par un test de 90 minutes. De plus, les genorypes 1+2- et 1+2+ etaient significativement
associes a une bonne trainabilite de la \\10 max (P<O.OI). Finalement, 3 groupes
2
d'echantillons de population ont ete compares sur la base de la distribution des frequences
alleliques et genotypiques des genes de l' AKIM, de la PFKM et de la COXVa. Les
resultats revelaient que les 2 groupes de Caucasiens (Canadiens Fran<;ais et Sud-Africains)
etaient similaires malgre leur difference d'origine et que les Noirs Sud-Africains eraient
differents d'eux pour les genes de l'AKIM (P<O.OOS) et de la COXVa (P<O.OOS).
.
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---

v
AVANT·PROPOS
L'achevement de cette these qui s'est deroule a un moment difficile de ma vie, constirue
pour moi un nouveau depart. C'est une etape qui aura ete tres de"tenninante pour moL C'est
pourquoi je tiens adire merci acelles et ceux qui m'ont peJ;111is de la franchir.
Au Docteur France T. Dionne, ma direcuice de these, j'adresse mes remerciements pour
m 'avoir accueillie au sein de son laboratoire et pour avoir mis ama disposition le materiel
necessaire ala realisation de ces travaux. Ses conseils et sa patience ont su me guider dans
les rigueurs de la recherche scientifique. le lui exprime egalement une profonde et sincere
gratitude, elle a su par ailleurs m'assister dans mes demarches aupres des Services de
I'immigration. Elle a toute mon estime et mon respect.
Je remercie aussi le Docteur CIaude Bouchard, l'interet qu'il a accorde a mon travail a
grandement contribue a la qua!ite de cette these.
Quant au Docteur Angelo Tremblay, je ne le remercierai jamais assez d'avoir accepte de
m' accueillir dans le departement et me faire visiter Le Laboratoire des Sciences de
l' Activite Physique. C'est lui qui m 'a pennis de rencontrer le Docteur France T. Dionne.
\\ \\

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VI
Je tiens a temoigner ma reconnaissance aux coauteurs des differents articles, a Lucie
Turcotte qui m 'a initiee aux techniques de laboratoire, a Monique Chagnon et a Yves
Gelinas pour leur aide et conseils, et a Claude Leblanc pour son aidedans l'analyse
statistique des donnees.
Un merci bien special a ma famille, en particulier ma soe~ Marie-Blanche Lebry et a
Mobiot Gabin, pour leur soutien moral.
Je remercie tout ceux qui m'ont aidee a rendre agreable et utile mon sejour a Quebec, en
paniculier Rode et Gaetan Boivin, Augustin Nouveau, Alex Nague, et Kraidi et Denise
Miessan.
Je remercie egalement le Ministere de la Fonction Publique de C6te-D'!voire pour m'avoir
accorde le temps necessaire pour l'achevement de cene these.
Le Docteur Claude Bouchard et la Fondation de I'Universite Laval ne peuvent etre oublies
pour l'aide financiere apponee sous forme de bourse et de Fonds de soutien.
Je remercie tous les membres du jury qui, malgre leurs nombreuses occupations, m'ont
permis de soutenir ma these a temps. Je remercie particulierement le Dr M. Boulay, qui
\\
a bien voulu faire la prelecture de ma these dans un delai assez bref.
.
,
:..

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vu
Sous la direction du Or France T. Dionne et du Or Claude Bouchard, j'ai rectige 6 articles,
dont 2 ont ete publies. Les quatre autres sont en voie d'etre soumis pour publication. La
realisation des travaux qui composent cette these a ete possible en partie grace 11 une
banque de donnees 11 laquelle j'ai eu acces. J'ai cependant activement participe 11 plusieurs
autres etapes de ces travaux de recherches. J'ai d'abord passe plusieurs mois 11 m'initier
aux techniques de laboratoire consistant apreparer les sondes des genes qui ont fait l'objet
de nos etudes et arechercher des variations au niveau du gene de la glycogene synthase et
des sous-unites IV, Vb et Va de la cytochrome c oxidase. Les resultats de ces travaux ont
fait l'objet des etudes des chapitres 3 et 4. Les protocoles d'entrainement des sujets utilises
dans ces etudes se sont acheves avant mon arrivee a Quebec.
Les donnees sur les
sedentaires de l' Arizona et les sujets Sud-Africains nous ont ete foumies par le Or lames
Skinner et le Dr Thirnothy Noakes respectivement. J'ai detennine les genotypes du gene
de la PFKi\\1 des cyclistes canadiens et des marathoniens Sud-Africains.
J'ai egalement
caracterise les types de genes de la CoxVa et de I'AKIM de l'ensemble des sujets
sedentaires et des athletes. Dans I'etude du chapitre 6, j'ai recherche les variants au niveau
de l' ADNmt des athletes avec l'aide de Marie-Claude Vohl qui emit en stage au laboratorre.
L'estimation de la V0 max du reste des marathoniens a ete faite avec la collaboration du
2
Or Denis Prud'homme et de M. Richard Chouinard. L'etude du chapitre 7 a ete realis6e
a partir des donnees obtenues de Prud'homme et al. (1984), Lortie et al. (1984) et
Simoneau et al. (1986, 1987).
Leurs donnees m' ont pennis de faire des analyses
statistiques additionnelles pour rediger le manuscrit. Enfin, dans la demiere etude (chapitre

---

·
'.
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Vlll
8), une banque d'ADN de sujets participant a l'etude sur les familles Quebecoises (QFS)
et un nombre additionnel de 45 marathoniens Sud-Africain m 'ont pennis de caracteriser
l' ADN des sujets.
La realisation de ce programme de recherche a ere possible grace ai' etroite collaboration
du Laboratoire des Sciences de l'Activire Physique de l'Universite Laval et du MRC/MCf
Bioenergetics of Exercise Research Unit, Depanment of Physiology, University of Cape
Town, Cape, South-Africa.
." ., ..-~ ".- '.--:".

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IX
TABLE DES MATIERES
PAGE
LES RESUNlES
11
AVANT-PROPOS
v
TABLES DES MATIERES
IX
LISTE DES AB REVIATIONS
xii
LISTE DES FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Xlll
CHAPITRE I
INTRODUCTION GENERALE
1
CHAPITRE II PROBLEMATIQUE DES TRAVAUX
5
2.1
_Systemes de regeneration de l' ATP . . . . . . . . . . . . . . . . . . . . . . ..
5
2.1.1
Metabolisme anaerobie
6
2.1.2
Metabolisme aerobie . . . . . . . . . . . . . . . . . . . . . . . . . . ..
9
2.2
Puissance aerobie maximale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2.1
Definition et facteurs d'influence
11
2.2.2
Variabilite de la \\10 max
12
2
2.3
L'entralnement
12
2.3.1
Adaptations metaboliques a 1'entrainement
et performance
. . . . . . . . . . . . . . . . . . . . . . . 12
2.3.2
Adaptation cardiovasculaire a l'entrainement
et performance
. . . . . . . . . . . . . . . . . . . 16
2.3.3
Differences individuelles
17
2.4
Heritabilite de la capacite d'adaptation a un entrainement en
endurance
18
2.4.1
Heritabilite, des determinants de la performance
19
2.4.1.1
Metabolisme energetique et systeme
cardiovasculaire . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.4.1.2
Heritabilite de la \\10 max
20
2
2.5
Variations genetiques
20
2.5.1
Types de variations
21

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x
PAGE
2.5.2
Recherche de genes candidats
22
2.6
Genes candidats erudies ou en cours d'erude
27
2.6.1
Etudes anterieures sur les genes candidats
28
2.6.1.1
Polymorphisme au niveau des proteines
28
2.6.1.2
Polymorphisme au niveau de l'ADN nucleaire
28
2.6.1.3
Polymorphisme au niveau de l' ADN
mitochondrial
'
29
2.6.2
Genes candidats de l' ADN nucleaire selectionnes pour
nos etudes
30
2.7
Approche experimentale
33
2.7.1
Technique d'analyses des fragments de restriction
34
2.7.2
Amplification genetique ou "Polymerase Chain Reaction" .. 36
2.7.3
Focalisation isoelectfophoretique
36
CHAPITRE III
EcoRI restriction fragment length polymorphism in human glycogen
synthase gene
38
CHAPITRE IV
Nuclear encoded subunits of human cytochrome c oxidase: SstI restriction
fragment length polymorphism
45
CHAPITRE V
Muscle adenylate kinase 1, muscle phosphofructokinase and subunit Va of
cytochrome c oxidase DNA sequence polymorphism, and variation in V0 max
2
and its response to training
57
CHAPITRE VI
Mitochondrial DNA sequence polymorphism and V0 max in endurance
2
athletes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
82
CHAPITRE VII
Phosphoglucomutase-1 polymorphism and relationship with aerobic
performance and response to exercise training
106
...
.
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xi
PAGE
CHAPITRE VIII
Comparison of DNA fragment length polymorphisms in French-Canadians
and in South-African Caucasians and Blacks
129
CHAPITRE IX DISCUSSION ET CONCLUSIONS
152
LES REFERENCES
163
"
.-.:.

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xii
LISTE DES ABREVIATIONS
ADN
Acide deoxyribonucleique
ADNmt
ADN mitochondrial
ADP
Adenosine diphosphate
AKl
Adenylate kinase 1
AMP
Adenosine monophosphate
ATP
Adenosine triphosphate
COX
Cytochrome c oxidase
CTP
Cytidine triphosphate
EP
Perfonnance en endurance evaluee par le test de 90 minutes
FABP
Fatty acid-binding proteins
FAD
Flavine adenine dinucleotide
GALE
Galactose epimerase
GDH
Glucose deshydrogenase
GLUTl
Transponeur de glucose 1
GS
Glycogene synthase
H
Hydrogene
IEF
Focalisation isoelectrophoretique
MCK
Creatine kinase musculaire
NAD
Nicotinamide adenine dinucleotide
OGDH
Oxoglutarate deshydrogenase
6PGD
6 phosphogluconate cteshydrogenase
PGM
Phosphoglucomutase
Pi
Phosphate inoganique
PFK
Phosphofructokinase
RFLP
Polymorphisme de longueur de fragments de restriction
UTP
Uridine triphosphate
V0 max
Consommation maximale d'oxygene
2

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Xl11
LISTE DES FIGURES
PAGE
Figure 1:
Systemes de re generation de I' ATP . . . . . . . . . . . . . . . . . . . . . . .. 7
Figure 2:
Differentes composantes de la \\10 max et leurs genes candidats ... 22
2
Figure 3:
Illustration des methodes "phenotype down" et "genotype up" ..... 23
Figure 4:
Technique d'analyse de l' ADN (Southern Blotting)
34
Figure 5:
Technique de focalisation isoelectrophoretique
37

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CHAPITRE I
INTRODUCTION GENERALE
11 est bien etabli que la reponse a l'entrainement en endurance et la performance en
endurance sont genetiquement detenninees (Prud'homme et al., 1984; Bouchard et al.,
1986b). La contribution des composantes genetiques a la variation de la performance
aerobie (Bouchard, 1992, Bouchard and Lanie, 1984) et a la reponse a l'entrainement
(Bouchard et aI., 1988a; Bouchard et al., 1992) a fait l'objet de plusieurs investigations dans
notre laboratoire depuis une decennie. Puisque les analyses de l'heterogeneite genetique au
niveau des proteines n' ont pas pennis l'identification de loci significativement associes a
la variabilite de la puissance aerobie maximale CV0 max) et au statut d'athlete d'elite
2
(Marcotte et aI., 1987; Bouchard et aI., 1988b; Chagnon et aI., 1984; Couture et al., 1986),
des etudes ont ete entreprises pour identifier directement au niveau de I' ADN, les variations
de sequence associees a ces phenotypes. La strategie du gene candidat (Lusis, 1988;
Warden and Fisler, 1994) a ete retenue pour ces etudes. Nous cherchons par consequent a
etablir une association entre les phenotypes d'interet et les variations de sequence de l' ADN
des genes candidats selectionnes pour leur role potentiel au ni veau des phenotypes etudies.
Les objectifs de cette these sont issus d'un programme qui a pour objectif global
d'identifier les marqueurs genetiques qui rnodulent la n5ponse a l'entrainement DU qui sont
associes a la performance.
. .- "~ .' .
.,
,
;.::.

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2
Les objectifs specifiques de la these visem a :
1)
identifier des variants de sequence par la technique d'analyse de polymorphisme de
longueur de fragments de restriction (RFLPs) de l' ADN nucl6aire des genes des
enzymes impliquees dans la production et la regeneration de l'ATP. Les enzymes
musculaires selectionnees
sont: l'adenylate kinase 1 musculaire (AK1M), la
glycogene synthase (GS) et la cytochrome c oxidase (COX);
2)
verifier s'il y a une association d'une part entre les divers RFLPs des genes de
l' AKIM, de la phosphofructokinase musculaire (PFKM) et de la COXVa et la \\'02
max initiale des sujets sedentaires et d'autre part entre ces RFLPs et la reponse de
la \\'0 max de ces sujets a un entrainemem en endurance;
2
3)
elargir ces etudes a deux groupes d'athletes Caucasiens composes de cyclistes
Canadiens et de marathoniens Sud-Africains;
4)
comparer les patrons d'ADNmt qui avaient ere prealablement associes a une \\'02
max de preentrainement elevee et a sa bonne reponse, entre des sujets sedentaires
et des athletes entraines en endurance;
5)
verifier au moyen de la methode de focalisation isoelectrophorerique (IEF), la
contribution du polymorphisme de la phosphoglucomutase-l musculaire (PGMl)
dans la performance et la reponse de la puissance aerobie maximale de sujets
sedentaires soumis a divers programmes d'entrainement.
Ces travaux sont essentiels pour definir les mecanismes fondamentaux qui modulent la
perfonnance et la reponse a l'entrainement.
Par ailleurs, l'analyse genetique au niveau meme de l' ADN a revolutionne l'etude de la
phylogenese des populations au cours de la derniere decennie. L_'ADNmt a ete largement

---

3
utilise a cette fin. Quand aI' ADN nucleaire, le nombre croissant des RFLPs a pennis de
reexaminer
les questions touchant les relations entre les populations qui dans le passe
avaient ere evaluees a l'aide de marq~eurs tels que les groupes sanguins, les enzymes du
globule rouge, les proreines etc...Les marqueurs de l' ADN sont generalement plus utiles
parce que leur nombre est plus eleve que les marqueurs de proteines. En ce qui conceme
cette these, nous avons utilise les donnees sur la variation de sequence de l' ADN des genes
etudies chez differents groupes ethniques ou raciaux, afin de comparer la distribution des
frequences allelique et genotypique entre ces divers groupes. A notre connaissance, il
n'existe aucune etude comparant les RFLPs des enzymes de voies metaboliques telles que,
l'AK1M, la PFKM et la COXVa, entre differents echantillons de population. C'est
pourquoi, notre demier objectif etait de:
6)
comparer les variations genetiques de l'AK1M, la PFKM et la COXVa, entre trois
groupes ethniques: les Caucasiens Canadiens Franc;ais, les Caucasiens Sud-Africains
et les Noirs Sud-Africains.
Le corps de la these est compose d'une serie de 6 etudes qui sont publiees au qui seront
soumises pour publication. Elles sont presentees du chapitre III au chapitre VIII. La liste
apparait ci-dessous.
Chapitre lll: Suzanne A. Adjoa, Claude Bouchard and France T. Dionne. EcoRI restriction
fragment length polymorphism in human glycogen synthase gene. Hum. Genet. 92: 632,
1993.
Chapitre IV: Suzanne A. Adjoa, Claude Bouchard, P. Coetzer, D. Timothy Noakes and
France T. Dionne. Nuclear encoded subunits of human cytochrome C oxidase: SstI
restriction fragment length polymorphism. Hum.Genet. 93: 347-348, 1994.
.. - .. '.~, ......;--:.

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4
Chapitre V:
Suzanne A. Adjoa, M.-C. Thibault, J.-A. Simoneau, Pieter Coetzer, Timothy
D. Noakes, Marce1 R. Boulay, lames S. Skinner, Claud.e Bouchard and F. T. Dionne.
Muscle adenylate kinase 1, muscle p~osphofructokinase and subunit Va of cytochrome c
oxidase DNA sequence polymorphism, and variation in V0 max and its response to
2
endurance training.
Chapitre VI: Suzanne A. Adjoa, Marie-Claud.e Vohl, Pieter Coetzer, Timothy D. Noakes,
Marcel R. Boulay, Claude Bouchard and F. T. Dionne. Mitochondrial DNA sequence
polymorphism and V0 max in endurance athletes.
2
Chapitre VII:
Suzanne A. Adjoa, Jean-Aime Simoneau, Monique Chagnon, France T.
Dionne and Claude Bouchard. Phosphoglucomutase-1 polymorphism and relationship with
aerobic performance and response to exercise training.
Chapitre VIII: Suzanne A. Adjoa, M.-C. Thibault, Pieter Coetzer, Timothy D. Noakes,
Marcel R. Boulay, Claude Bouchard and F. T. Dionne. Comparison of DNA fragment
length polymorphisms for three genes in French-Canadians and South-African Caucasians
and Blacks.
•
<. ~.~: .;-.;", ,

---

CHAPITRE IT
PROBLEMATIQUE DES TRAVAUX
2.1
Systemes de regeneration de l'adenosine triphosphate (ATP)
L' ATP est la source d'energie sollicitee pour la contraction musculaire. L'energie est
liberee lorsque l' ATP est hydrolyse en adenosine diphosphate (ADP) et en phosphate
inorganique (Pi) au niveau des ponts croises entre l' actine et la myosine du muscle actif
(Saltin and Gollnick, 1983; Geeves, 1991). La concentration d'ATP dans le muscle est
relativement limitee, soit 5-8 mmoles par kg de masse de tissu (Saltin and Gollnick, 1983~
Noakes, 1991). C'est la concentration requise pour une contraction musculaire explosive,
equivalant a environ une seconde de sprint (Noakes, 1991). L'ATP doit donc etre regenere
continuellement durant les longues periodes d'exercice. 11 existe une etroite correlation
entre l'intensite de l'exercice et la consommation cellulaire d' ATP (Dudley et al., 1982;
Saltin and Gollnick, 1983). L 'activite des enzymes impliquees dans la regeneration de
l' ATP augmente donc en fonction de l'intensire de I'exercice (Henriksson and Reionan,
1977; Dudley et al., 1982; Saltin and Gollnick, 1983). La resynthese de l'ATP se fait donc
par un accroissement de la liberation d'energie via
differentes voies metaboliques: le
metabolisme anaerobie et le metabolisme aerobie.
c.
' . . . "~ . '. -':'C',

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6
2.1.1
Le metabolisme anaerobie
Les systemes energetiques dont les reactions sont catalysees par les enzymes creatine kinase
(CK) et adenylate kinase (AK) et par les enzymes de la glycolyse, sont les 3 principales
voies de regeneration de I' ATP via le metabolisme anaerobie.
Une des sources immediates de phosphate pour la regeneration de l' ATP est la creatine
phosphate, un compose chimique £res riche en energie et, qui est environ 3 fois plus
abondant dans la cellule musculaire que l'ATP. Dans le muscle squelettique, le Pi est separe
de la CP par l'enzyme CK musculaire (MCK) (Figure I-A). L'energie liberee au cours de
cette reaction sen a combiner le Pi avec l' ADP pour fonner l' ATP (Brooks and Fahey
1984; Noakes, 1991; SjOdin, 1992; Wilmore and Cos till, 1994). n en resulte 1 mole d' ATP
par mole de creatine phosphate. La CK est presente dans le cytosol et les mitochondries du
coeur, du muscle squelettique et du cerveau (Bessman and Carpenter, 1985). La CK du
cytosol est composee de 2 sous-unites: celle du muscle (M) et celle du cerveau (B), dont
les combinaisons
donnent
3 isozymes qui sont: la CK-MM, la CK-MB et la CK-BB
(Bessman and Carpenter, 1985). La CK-MM (MCK) est liee 11 la bande M des myofibrilles.
Une quatrieme isozyme, la CK mitochondriale, est localisee dans l'espace intennembranaire
de la mitochondrie (SjOdin, 1992). 11 existe un processus de navette creatine-creatine
phosphate encre la mitochondrie et les myofibrilles qui contr6le la distribution d'energie et
la reponse cellulaire a la demande d'energie (Bessman and Carpenter, 1985). Dans ce
processus la creatine phosphate qui est synthetisee dans la mitochondrie diffuse 11 travers
les myofibrilles Oll est liee la MCK. Au fur et amesure que la contraction genere de I'ADP,
la MCK catalyse la resynthese de l'ATP pour soutenir la contraction musculaire. La
creatine liberee durant cette reaction retoume dans la mitochondrie pour etre rephosphorylee
(Bessman and Carpenter, 1985; SjOdin, 1992). Soderland et al. (1992) apres avoir soumis
8 sujets aun sprint de 30 s sur ergocycle ont estime que le taux de regeneration de I'ATP,
calcule apartir des changements des concentrations de I'ATP, de la phosphocreatine et du
lactate, etait significativement reduit apres les 30 s de sprint et la capacite totale de travail,
reduite de 17%. Les auteurs concluent que la baisse de la force pourrait etre reliee a la

---

I
Figure 1: Systemes de regeneration et
de production de I'ATP
o Systeme creatine kinase
o Systeme adenylate kinase
e Glycolyse
(;) B-Oxydation
Cl Cycle de Krebs
G Chaine de transport des electrons
(B-Ox=Beta oxydation; figure modifiee de Sj6din, 1992)
.- .. ~.~,.; ..;-;,
" , ••• ,_ ~ "';.':0:".

---

8
capacite a regenerer I' ATP. Ces resultats sont en accord avec ceux de Bogdanis et al.
(1995) qui ont soumis 40 sujets miles a 2 sprints de 30 s sur ergocycle, sepms par un
intervalle de 1,5,3 et 6 min de repos. ,Leurs resultats montrent une fone correlation de 0.86
entre le pourcentage de resynthese de la creatine phosphate et le pourcentage de
recuperation de la puissance maximale et de la vitesse maximale de pedalage entre 2
sprints. Cette source d'energie est anaerobie alactacide, c'est-a-dire que l'oxygene n'est pas
directement utilise et il ne se fonne pas d'acide lactique. Ce systeme est surtout sollicite
au tout debut d'un exercice physique intense. Cependant, la capacite de la creatine
phosphate a regenerer une grande quantite d' ATP est limiree. Au dela de ce temps, la
glycolyse et le systeme aerobie doivent etre sollicites.
Dans la reaction catalysee par l'enzyme AK (Figure I-B), 2 moles d'ADP sont converties
en une mole d' ATP et une mole d'adenosine monophosphate (AMP) au niveau du
cytoplasme. Cette reaction est reversible. L' AK existe en 3 isofonnes, on retrouve
l'isofonne AKl dans les globules rouges et dans la mitochondrie, les isofonnes AK2 et
AK3 (Bessman and Carpenter, 1985; Savabi et al., 1986). L'isofonne AK1M, appele
myokinase, est presente dans le muscle squelettique. Cet isofonne est egalement localise
pres du complexe actomyosine dans les myofibrilles (Savabi et al., 1986). La fonnation
d' ATP est favorisee lorsque la concentration d' ADP est elevee, comme lors d'un exercice
intense (SjOdin, 1992). L'activite de l'AK est par consequent tres elevee dans cette
condition, a cause de l'accumulation d'ADP. Ce systeme energetique catalyse par l'AK est
egalement sollicite lorsque le glycogene musculaire est epuise (Nonnan et al; 1987).
La glycolyse qui rerere a la degradation du glucose ou du glycogene en pyruvate et en
lactate par la voie d 'Embden-Meyerhof, est catalysee par 1'action successive de onze
enzymes. Ces enzymes sont localisees dans la porrion soluble du cytoplasme (Essen, 1979).
La glycolyse est caracterisee par deux grandes etapes: le glucose est d'abord phosphoryle
et scinde en deux pour donner naissance au glyceraldehyde (1ere etape), qui est ensuite
transfonne en pyruvate et lactate (2eme etape). La glycolyse, regenere l' ATP sous raction
de la degradation des glucides, principalement du glycogene, en a~ide lactique (Figure I-C).
......
"
, ..•.. ":-.

---

9
L'A1P produit clans ces conditions est de source anaerobie lactacide, avec formation de 2
moles de lactate et de 4 moles d' ATP par unite de glucose.
Ces differents systemes energetiques, CK, AK, et glycolyse, sont les sources majeures
d'energie durant la premiere minute d'un exercice a haute intensite. Ainsi, Boulay et al.
(1995) ont rapporte que les concentrations musculaires d'ATP, de phosphocreatine et de
glycogene etaient significativement reduites chez 5 sujets sedentaires soumis a 3 tests de
haute intensite de 10, 30 et 90 s sur ergocycle.
2.1.2
Metabolisme 3erobie
Les voies anaerobies de regeneration d'ATP ne peuvent subvenir seules aux besoins
energetiques des cellules. D'autres voies metaboliques, en l'occurrence celles du systeme
oxydatif, doivent par consequent prendre la releve.
La production oxydative d' ATP se fait clans les mitochondries. Au cours du metabolisme
aerobie, la degradation des carburants tels le glycogene, le glucose hepatique, les acides
gras libres circulants, et les proteines en ATP, se fait en presence d'oxygene. Toutefois,
pour que cette voie metabolique opere de maniere efficace, les mitochondries des cellules
musculaires doivent etre convenablement approvisionnees en oxygene.
L'oxydation des glucides, initiee par la glycolyse, se poursuit dans le cycle de Krebs et la
chaine respiratoire. Avant d'entrer dans le cycle de Krebs, le pyruvate doit etre transporte
dans la mitochondrie gr:lce aun transporteur specifique du pyruvate situe clans la membrane
mitochondriale exteme. Dans la mitochondrie, le pyruvate est transfonne en acetyl-CoA
grace a l'enzyme pyruvate deshydrogenase, cette reaction s'accompagne de la reduction
d'un transporteur NAD+ en NADH. L'acetyl-CoA fonne entre dans le cycle de Krebs pour
subir une serie de reactions successives, qui permettent la formation de 3 moles de NADH,

---

11
pour fonner de l'eau (Figure I-F). Les electrons separes de l'hydrogene subissent une serie
de reactions, d'oll le nom de chaine de transpon des electrons, pour fournir de l'energie
pour la phosphorylation de l' ADP, d?nc pour la formation de l' ATP (Martin et al., 1985).
Ce processus qui se deroule gr:1ce a l'oxygene, est appele phosphorylation oxydative. De
grandes quantites d'energies sous forme d' ATP resultent ainsi de cette derniere etape.
En somme, la mitochondrie est ajuste titre surnommee "la generatrice" de la cellule (Bray
et al., 1988), car c'est clans la mitochondrie que la plus grande partie de l'energie provenant
de l'oxydation des differents substrats est produite sous fonne d' ATP.
Les differents systemes energetiques que nous venons de decrire operent en synergie, de
sone apourvoir convenablement aux besoins du muscle en ATP en fonction des exigences
energetiques musculaires.
2.2
Puissance aerobie maximale
2.2.1
Definition et facteurs d'influence
La capacite de performance depend notamment de la puissance aerobie maximale (\\'02
max) et de la fraction de cette \\10 max pouvant etre maintenue pendant un exercice.
2
Robinson et al. (1938) ont ere les premiers a identifier la \\10 max cornme etant un
2
determinant important de la performance. La \\10 max est generalement cIefinie comme
2
etant l'habilete maximale des muscles actifs d'un individu a capter, transponer et utiliser
l'oxygene (Astrand and Rodhall, 1986). La puissance aerobie maximale peut etre ainsi
quantifiee en tennes de volume par minute (\\1) d' oxygene (VO,) consomme par l' organisme
au caurs d'un exercice progressif jusqu'a epuisement, c'est-a-dire en effort maximal.
Cependant, deux facteurs ont une influence majeure sur la V0 max.. Ces facteurs sont la
2
capacite des tissus autiliser l'oxygene dans la combustion des carburants cellulaires (facteur
penpherique) et la capacite des mecanismes pulmonaire, cardiaque, sanguin et vasculaire
a transporter l'oxygene aux sites actifs (muscle) (facteur central). Ces deux facteurs,

---

Lli;@~;':'
:Fj;:~:'>
12
consideres comme limitant la V02 max font emerger deux opinions, l'une favorisant la
limitation centrale et l'autre, la limitation peripherique. Toutefois, on mentionne de plus en
plus que la 1imite centrale serait predominante (Di Pamprero, 1985). Cet auteur rapporte que
,
lorsqu'un exercice est effectue avec les deux jambes, environ 75% de la V02 max est regie
par le transport de 1'0xygene. Lorqu'une jambe est utilisee, la composante centrale et
peripherique contribuent a egale proportion.
2.2.2
Variabilite de la "02 max
La "02 max vane largement dans une meme population
ou entre differents groupes
ethniques (Andersen et aI., 1960; Hermansen and Andersen, 1965; Rode and Shephard,
1971; Glick and Schwartz, 1974) ayant des niveaux d'activite differents. Elle est egalement
influencee par le mode de vie, plus specifiquement, par le niveau habituel d'activire
physique. Chez un jeune sedentaire, les valeurs de la \\10
max sont de I' ordre de 40
2
ml/kg/min tandis que les valeurs peuvent atteindre 85 ml/kg/min chez les athletes d'elite
(Rowell, 1974). Les valeurs sont plus basses chez les femmes que chez les hommes d'un
meme age et decroissent avec I'age et les maladies cardio-vascu1aires chez les 2 sexes
(Saltin and Strange, 1992). La variabilire genetique de la \\10
max a ere erudiee par
2
Klissouras, (1971); Weber et aI., (1976); Komi and Karlsson, (1977); et Lesage et al.
(1985).
2.3
L 'entrainement
2.3.1
Adaptations metaboliques a I'entrainement et perfonnance
nest bien connu que les individus sedentaires augmentent 1eur endurance par le biais
d'exercices physiques reguliers. Apres quelques semaines d'entrainement en endurance, il
est possible pour ces individus de faire de 1'exercice confortablement, pendant des periodes
l1rotongees, a des intensites qu'ils n'auraient pu tenir plus de quelques minutes avant
l'entrainement. Du point de vue physiologique, les reponses adaptatives favorisent la
.
, . '.~ . -. '.;":
.. ...... .... ..
;-
"';

---

13
resistance ala fatigue et ameliorent la performance. L' exercice prolonge en endurance, telle
que la course de longue distance, induit des modifications importantes du metabolisme
energetique dans le muscle squelettique (Holloszy and Booth, 1976). Les effets les plus
marquants sont l'augmentation du nombre de mitochondries et de l'activite des enzymes
de la mitochondrie, accompagnee d'une augmentation concomitante de la capacite aoxyder
les glucides et les graisses (Gollnick et al., 1973; Coyle, 1986; Saltin and Gollnick, 1983;
Costill, 1979; Davies et al., 1981; Holloszy and Coyle, 1984; Wibom et aI., 1992;
Hagerman, 1992; Starnes, 1994). Ces effets se traduisent donc par une augmentation de
l' activite des enzymes de des voies meraboliques impliquees dans la production et la
regeneration de l' ATP au niveau de la glycolyse et de la phosphorylation oxydative
(Hagerman, 1992; Honig et al., 1992).
L'entralnement augmente le potentiel oxydatif des fibres musculaires. Les fibres lentes sont
preferentiellement recrutees lors des premiers stades d'une course en endurance, mais
lorsque les muscles commencent a etre fatigues, un plus grand nombre de fibres
musculaires rapides est recrute, en commen~ant par les fibres rapides oxydatives (Ffa) puis
les fibres rapides glycolytiques (FIb) (Saltin, 1981; Noakes, 1991) pour compenser
potentiellement la perte de tension musculaire (Costill, 1979; Gollnick et al., 1973). En
effet, les athletes ayant un pourcentage presqu'identique de fibres rapides et de fibres lentes,
c'est-a-dire 40 a 50% de fibres rapides, reussissent probablement mieux au marathon et a
l'ultramarathon, comparativement a ceux qui ont un pourcentage eleve de fibres lentes
(Noakes, 1991).
Chez le lapin, une stimulation electrique a long terme mimant l' entrainement en endurance,
induit une reduction de l'activire des enzymes impliquees dans le transfert de phosphates
a haute energie: l'AK et la CK (Pette et al., 1973; Henriksson et al., 1986). Etant donne
l'importance de la glycolyse pour la production d'ATP lors de l'exercice, plusieurs
chercheurs se sont interesses a la determination des effets de 1'entrainement en endurance
sur l'activite maximale des enzymes de la glycolyse. Les etudes portant sur la PFK
fJportent une baisse de son activire (Holloszy and Coyle, 1984; Green et al., 1979;
,....... - .

---

.!.r',:- '1'·,
:~~.\\, .-
.;1
14
Tremblay et aL, 1994) ou une valeur inchangee (Morgan et al., 1971) apres l'entrainement.
Toutefois, les exercices de haute intensite augmentent significativement l'activite de la PFK
(Hamel et al,
1986, Simoneau,
1995). L'enzyme hexokinase, impliquee clans la
phosphorylation du glucose, augmente significativement apres entrainement aussi bien chez
l'animal (Pette et al., 1973) que chez l'humain (Ramel et al., 1986). L'activire de la
glycogene synthase, enzyme de la glyconeogenese, et de la lactate deshydrogenase, enzyme
du metabolisme du lactate, augmente egalement en reponse a l'entrainement en endurance
(Hamel et al., 1986).
Il existe une etroite correlation entre la capacite du muscle a supponer un exercice aerobie
prolonge et l'activite des enzymes oxydatifs. L'entrainement en endurance peut doubler
l'activite de plusieurs enzymes du cycle de Krebs et de la chaine respiraroire (Brooks and
Fahey, 1984). L'entrainement en endurance augmente egalement l'activite des enzymes de
la ~-oxydation, pennettant par consequent une utilisation accrue des lipides pour la
production d'ATP (Brooks and Fahey, 1984). Une augmentation de 60% de l'activite d'une
des enzymes de de la ~-oxydation, la 3 hydroxyacyl CoA deshydrogenase, a ete d'ailleurs
rapponee par Bylung et al. (1976) et Hamel et al. (1986). Ces resultats son[ en accord avec
ceux de Theriault et aI., (1994), qui ont observe une augmentation de l'activite de cette
enzyme suite a une stimulation electrique chronique de basse frequence chez l'humain.
L'activire des enzymes cle du cycle de Krebs, la citrate synthase, la succinate
deshydrogenase, et la malate deshydrogenase, augmente en reponse a un entrainement en
endurance (Holloszy et al., 1970; Klausen et al., 1981; Davies et al., 1981; Saltin and
Gollnick, 1983; Henriksson et aI., 1986; Gollnick et al.,1973; Henriksson and Reitman,
1977; Cos till, 1979).
L 'activire de la COX, enzyme de la chaine respiratoire, augmente egalement en reponse a
l'entrainement. Une augmentation de 78% de l'activite de la COX a ete observee par
Wioom et al., (1992) chez des sujets soumis a 6 semaines d'entrainement en endurance.
Trois semaines apres ce meme entrainement (Wibom et al., 1992), l'activite de la COX
,
•••• ~, -.0-".;"';".

---

15
demeurait significativement superieure (50%) aux valeurs de preentrainement. Theriault et
al., (1994) ont egalement observe une augmentation de 25% de 1'activite de la COX suite
a une stimulation electrique chronique de basse frequence chez l'humain.
Selon Henriksson and Reitrnan (1977); Saltin and Gollnick (1983); Wibom et al. (1992);
Wibom and Hultman (1990), 1'augmentation de 1'activite des enzymes s'accompagne d'une
augmentation concomitante de la \\'0 max. En effet, chez les sujets entraines, il existe une
2
relation entre 1'augmentation de 1'activite des enzymes de la mitochondrie, telle que la
citrate synthase, et la \\'0 max (Holloszy and Coyle, 1984)
2
La production mitochondriale d' ATP semit environ 80% plus elevee chez les sujets hien
entraines en endurance que chez les sujets sedentaires (Wibom et al., 1992; Wibom and
Hultrnan, 1990). Dans le but d'evaluer jusqu'a quel point l'augmentation de 1'activite des
enzymes glycolytiques et mitochondriales du muscle squelettique rtfletait 1'augmentation
de la production d' ATP de la mitochondrie, Wibom et al.(l992) ont mesure la production
d' ATP dans les mitochondries isolees, par la methode bioluminometrique. Apres 6 semaines
d'entrainement, la production mitochondriale d' ATP avait augmentee de 70% pour une
combinaison de substrats composee du pyruvate, du palmityl-L-carnitine, de 1'a-
cetoglutarate et du malate,
de 50% pour la composition comprenant le pyruvate et le
malate et enfin, de 92% pour la combinaison du palmityl-L-carnitine et du malate. Ces
resultats montrent 1'ampleur de la production d' ATP de la mitochondrie en fonction de
l'activite des enzymes impliquees dans les differentes voies metaboliques.
Les proteines sont utilisees dans des conditions extremes telles que le jeGne ou l'exercice
prolonge, sptcialement lorsque le glycogene musculaire est epuise (Lemon and Mullin,
1980). Cependant, comme mentionne precedemment, leur contribution a1'exercice prolonge
n' excede pas 10% des substrats oxydes. Dans ces conditions, le role majeur des proteines
,
est de fournir au foie des substrats a partir desquels il peut produire du glucose lorsque 1es
reserves de glycogene sont basses (Lemon and Mullin, 1980).
.... " "~"..'~':': ,
........ ,
.
.~~.~~

---

16
Pour realiser de bonnes perfonnances, une bonne reserve en glycogene musculaire est
necessaire (Hagenn an , 1992). L'entrainement augmente la capacite des muscles actifs a
entreposer le glycogene. Dans les muscles entraines, les reserves peuvent exceder 2 a 3
fois les valeurs nonnales. Le taux d'utilisation du glycogene depend de l'intensite de
l'exercice et est eleve durant les
15 a 20 premieres minutes de l'exercice. Les
concentrations baissent ensuite progressivement durant l'exercice (Saltin and Karlsson,
1971). L'exercice prolonge d'une intensite de 85% de la V0 max (marathon standard) ou
2
de 70 a 75% de la V0 max (ultramarathon), entraine un epuisement plus imponant du
2
glycogene musculaire (Noakes, 1991; Shennan, 1992). Les reserves de glycogene seraient
completement epuisees lars d'une course d'une duree comprise entre 140 et 215 minutes,
a70-85% de la V0 max. Les reserves en glycogene musculaire constituent d'ailleurs un
2
facteur detenninant de la perfonnance pour des courses dont la duree est comprise entre 90
min et 3h 30 environ, (Peronnet, 1991).
2.3.2
Adaptations cardiovasculaires a I'entrainement et perfonnance
L'entrainement en endurance, induit aussi des adaptations cardiovasculaires qui pennettent
d'avoir un meilleur appon musculaire en oxygene. La capacite respiratoire du muscle est
2 fois plus elevee chez les sujets entraines comparativement aux non entraines. Le debit
cardiaque et le debit d'ejection systolique sont accrus sous l'effet de l'entrainement La
frequence cardiaque de repos et sous maximale ainsi que la pression anerielle quam ae1les,
decroissent avec l'entrainement (Costill, 1979). La frequence cardiaque maximale demeure
constante, mais, on observe par contre une augmentation marquee du debit d'ejection
systolique (Astrand and Rodahl, 1986). Pour cette raison, Saltin (1967) soutient que le debit
d'ejection systolique est le facteur qui differencie l'athlete d'elite en endurance des
individus bien entraines. L'augmentation du debit d'ejection systolique serait due a un
volume cardiaque de 30 a 40% plus grand chez les sujets entrames comparativement aux
sedentaires du meme age, taille et poids (Costill, 1979) et aun volume ventriculaire gauche
plus large chez les entraines. Cette caracteristique se Itflete par un volume teled.iastolique
accru, observe au repos et a l'exercice chez les entraines. L'augmentation du debit
0 ' ,
• • • • • , ••••: . : .

---

17
d'ejection systolique est donc responsable de l'augmentation du debit cardiaque. De plus,
l'enrrainement augmente le volume sanguin sous l'influence de l'augmentation du volume
plasmatique et de I'hemoglobine totale (Costill,1979). Cette adaptation facilite l'appon
d'oxygene
gcice a l'augmentation de la dynamique de la circulation et de la
thermoregulation. L'enrrainement accroit egalement la quantite d'oxygene extraite de la
circulation sanguine par les muscles actifs. Cela est reflete par l'augmentation de la
difference arterio-veineuse en oxygene qui est plus elevee chez les enrraines (McArdle et
al., 1986). Le debit sanguin total du muscle augmente durant l'exercice, cette augmentation
etant due a I'accroissement du debit cardiaque max. et a une meilleure redisrribution
sanguine (McArdle et al., 1986). Le bas quotient respiratoire resultant de I' enrrainement
indique une plus grande participation de l'oxydation des acides gras a la regeneration de
l'energie (McArdle et al., 1986; Wilmore and Costill, 1994).
Les sujets bien enrraines peuvent d'ailleurs travailler ades intensites pres de leur \\10 ma.;'(
2
beaucoup plus longtemps que des sujets sedentaires. Les sujets bien enrraines, les
marathoniens par exemple, courent enrre 70 et 85% de leur \\10 max pendant des p6iodes
2
plus longues que les sujets non enrraines (Saltin and Gollnick, 1983). La \\10 max des
2
sedentaires etant d'environ 60% plus basse que celle des athletes (Noakes, 1991) et la
fraction de la \\10 max qui peut etre soutenue durant une course etant en etroite correlation
2
avec la performance (Leger et al., 1984; Davies and Thompson, 1979; Maughan and Leiper,
1983; Noakes, 1991). Par consequent, quelque soit l'enrrainement suivi, il est peu probable
qu'un sujet sedentaire atteigne une perfonnance proche de celle des athletes.
2.3.3
Differences individuelIes
La capacite d'adaptation a un enrrainement est tres heterogene enrre les individus. Le
niveau de pre-enrrainement et le genotype seraient les causes majeures de la variabilite
observee en reponse aI'entrainement (Bouchard et al., 1990).

---

18
Plusieurs etudes ont ete entreprises pendant la demiere decennie dans le but d'identifier les
facteurs ou les mecanismes pouvant expliquer les differences de sensibilite al'entrainement.
La "02 max et les perfonnances aerobies sont des phenotypes qui s'adaptent a l'exercice
(Bouchard, 1986; Bouchard et al., 1986). Cependant, tous les individus ne repondent pas
de la meme maniere a un meme type d'entrainement (Prud'homme et al., 1984; Lartie et
al., 1984). En effet, il existe une tres grande variabilite parmi les individus dans la reponse
al'entrainement. Cette variabilite se manifeste par une reponse a un entrainement presque
nulle pour certains et tres elevee pour d'autres (Prud'homme et al., 1984; Bouchard and
Malina, 1983; Boulay et al, 1986). A titre d'exemple, Lonie et al. (1984) om soumis des
sujets sedentaires a 20 semaines d'entrainement standardise sur ergocycle. L' augmentation
moyenne dela "02 max etait de 33% mais variait de 7 a 87% entre les individus. Dans les
etudes effectuees chez des jumeaux monozygotes, une aussi grande variabilite a egalement
ete observee entre les paires de jumeaux (Prud'homme et al., 1984; Hamel et al., 1986).
2.4
HeritabiJite de la capacite d'adaptation a I'entrainement en endurance
Les perfonnances dans les sports d'endurance se sont considerablement ameliorees. Pate
and Branch (1992) rapponent que depuis 1960, le record mondial masculin de marathon
s'est ameliore de 6%. Ces ameliorations seraient d'une pan attribuables au fait que
l'augmentation du nombre de participants ait amene dans les competitions de marathon,
beaucoup d'athlt~tes qui ont un bagage genetique plus approprie ace type de performance,
et d'autre pan, au fait que les entrainements soient devenus plus rigoureux et raffmes (Pate
and Branch, 1992; Bouchard and Malina, 1984). Ces derniers mentionnent que les athletes
olympiques actuels et ceux du futur seraient des individus phenotypiquement superieurs
avant l'entrainement et qu'ils seraient en outre extremement sensibles a l'entrainement
.. "~ : .;.. -:'.

---

"
19
2.4.1
Hentabilite des detenninants de la performance
2.4.1.1
Metabolisme energetique et systeme cardiovasculaire
Plusieurs detenninants de la perfonnance sont sous l'influence d'un effet genetique
(Robens, 1985; Bouchard, 1992). Une ressemblance familiale a d'ailleurs ete observee pour
plusieurs de ces detenninants (Adams et al., 1986; Bouchard, 1986; Komi and Karlsson,
1977; Theriault et al., 1986; Bouchard et al.,1988a). Ainsi', l'heritabilite des dimensions
cardiaques est estimee a moins de 25% (Theriault et al., 1986; Fagard et al., 1987) tandis
que celui du pouls maximal d'oxygene est superieure a 50% (Bouchard et al., 1986a;
Fagard et al., 1991). Au niveau de l'heritabilite du pourcentage du type de fibres
musculaires squelettiques, les resultats des differents auteurs divergent. Ainsi, Komi et al.
(1977) ont rappone une heritabilire de 98 a 99% des types de fibres musculaires chez les
jumeaux. Cependant, ces resultats n'ont pu etre reproduits dans une des etudes de notre
laboratoire (Bouchard et al., 1986). Les coefficients d'heritabilite etaient beaucoup plus bas.
Enfin, Simoneau et Bouchard (1995) ont recemment publie que le coefficient d'heritabilite
de la proponion de fibres de type I est de l'ordre de 45% de la variance. Quand au
potentiel oxydatif, et au taux d'utilisation des lipides du muscle squelettique, l'heritabilite
est respectivement de moins de 50% et de plus de 50% (Bouchard et al., 1986a; Bouchard,
1988; Fagard et al., 1991). Bouchard et al.(1986a) om etudie l'heritabilite de differents
marqueurs du metabolisme energetique, entre jumeaux monozygotes, c'est-a-dire des
jumeaux issus de la fragmentation d'un meme oeuf et qui dispose m donc d'un panimoine
genetique identique et entre jumeaux dizygotes, qui ne sont pas genetiquement identiques.
Les resultats montrent que des facteurs genetiques comptent pour 25 a 50% de la variation
phenotypique totale de l'activite maximale de la phosphofructokinase (PFK) et de
l'oxoglutarate deshydrogenase (OGDH), et du ratio PFKlOGDH. Ces donnees montrent
l'imponance de l'heritabilite de la variation des enzymes cle du metabolisme aerobie et
anaerobie du muscle squelettique.
.
.
' ... :.", ". ~.:':
. -
,
;'.

---

~Elj~:" '.
• "<,,".,
"
i
,
"
20
2.4.1.2
Heritabilite de la V0 max
1
Dne ressemblance familiale de la V0 max exprime par kg de masse corporelle ou de masse
2
maigre a ete rapponee (Montoye and Cayle, 1978; Lesage et al., 1985). Cette ressemblance
est plus marquee entre freres et soeurs et entre jumeaux. Les resultats d'une etude conduite
chez 27 paires de freres, 33 paires de jumeaux dizygotes et 53 paires de jumeaux
monozygotes (Bouchard et al., 1986b) montrent un effet genetique sur la V0 max/kg de
2
poids corpore!. Les auteurs estiment que l'heritabilite de la \\'0 max serait d'environ 25%.
2
Dne ressemblance familiale tres significative a ete egalement observee chez des individus
soumis au test de performance aerobie de 90 minutes (Boulay et al., 1984). n decoule des
resultats que l'heritabilite de cette performance aerobie de 90 minutes serait d'environ 60%
(Bouchard et al., 1986b), done plus elevee que celle de la V0 max.
2
En somme, les resultats de ces etudes supponent le concept selon lequel le genotype joue
un role dans la sensibilite de la puissance aerobie aI' entrainement.
2.5
Les variations genetiques
La somme des informations contenues dans les genes, c'est-a-dire dans le genotype, est
responsable des caracteres apparents representatifs d'un individu, soit son phenotype.
L'heterogeneite genetique entre les individus decoule du polymorphisme de la sequence de
I' ADN. On identifie comme variant generique ou moleculaire une variation de sequence
retrouvee dans plus de 1% de la population. Le polymorphisme permet ainsi de discriminer
et d' etudier la transmissibilite de traits ou genes.
Le polymorphisme de I' ADN fait qu'il peut exister plusieurs versions differentes d'un
meme gene, appelees alleles. Le genotype d'un individu comprend alors non seulement
l'information ponee par les milliers de genes, mais egalement celle ponee par l'un des
multiples alleles possibles du gene.

---

21
2.5.1
Types de variations
Trois types de variations genetiques ~uvent affecter les perfonnances. Le premier type
touche les variations des sequences d' ADN aI'interieur des parties codantes appelees exons.
Ces variations pourraient influencer la perfonnance physique car elles peuvent produire une
modification de la composition en acides amines de la proreine. Ces changements pourraient
affecter par consequent l'activire biologique des proteines concernees ainsi que leur
interaction avec les autres molecules. Le second type de variation est celui que l'on
retrouve dans les parties non codantes de l' ADN ou introns et les regions regulatrices ou
flanquantes, ces variations pourraient influencer l'expression du gene. Les variations dans
les regions non codantes sont beaucoup plus frequentes dans la population et engendrent
des marqueurs polymorphiques plus nombreux et pouvant avoir plus d'un allele. On note
egalement des variations silencieuses de la sequence d'ADN, c'est-a.-dire des variations qui
ne modifient en rien le phenotype etudie car elles n' affectent ni la proteine ni son
expression.
Les 3 types de variations dont nous venons de parler resultent generalement de substitutions
de bases faisant apparaitre ou disparai'tre des sites de reconnaissance pour des enzymes de
restriction. Elles sont done detectees par la digestion de I' ADN avec ces enzymes, ce qui
genere des polymorphismes de longueur de fragments de restriction (RFLPs). Depuis
quelques annees, on parle egalement beaucoup dans la litterature, de variation genomique
entre les individus au niveau des microsatellites. n s'agit de repetitions de 2, 3 ou 4
nucleotides retrouves tout au long des chromosomes et meme a. l'interieur de genes. Dans
le cadre de cette these, pour identifier les facteurs genetiques a l'origine des variations
individuelles de la \\'0 max, nous utiliserons des marqueurs polymorphiques qui peuvent
2
etre soit des variations dans la sequence d' ADN d'un gene (marqueurs RFLPs), soit
l'analyse du polymorphisme des proteines elles meme. L'analyse de microsatellites ne fera
pas partie de cette these. Le choix des marqueurs aetudier se fera selon la strategie du gene
candidat.

---

22
2.5.2
Recherche de genes candidats
Le gene candidat est un gene dont la fonction suggere qu'il pourrait jouer un role dans le
phenotype etudie. Dans notre cas, le phenotype d'interet est la V0 max. Nous recherchons
2
des genes de susceptibilite, qui pourraient influencer la sensibilite de ce phenotype a
I'entrainement.
La V0 max est un phenotype multifactoriel, done ayant plusieurs detenninants. Pour
2
faciliter l'identification des genes impliques dans la reponse de la V0
max a
2
l'entrainement, il serait idealement plus simple de subdiviser la V0 max en differents
2
phenotypes intennediaires tel qu'illustre a la Figure 2. 11 est pennis de croire que ces sous
phenotypes seront regules par un nombre plus resrreint de genes. De plus, la subdivision
des phenotypes pourrait reduire le probleme d'heterogeneite genetique, c'est-a-dire, le fait
qu'entre les individus, un meme phenotype multifactoriel comme la V0 max. puisse etre
2
regule par differents ensembles de genes (Lusis, 1988). La figure 2 suggere aussi quelques
genes candidats utiles pour etudier chacune des cornposantes de la V02 max.
La figure 3 illustre 2 strategies proposees pour etudier les caracteristiques de la performance
hurnaine: l'approche "top-down" et "bottom-up" (Bouchard et al., 1992) aussi appelee le
"phenotype down" et le "genotype up" (Warden and Fisler, 1994). La premiere consiste a
detenniner si le phenotype (par exernple la V0
rnax) est heritable. La methode
2
d'epidemiologie genetique utilisant des familles nucleaires, des families avec enfants
adoptes, des jumeaux monozygotes et dizygotes font l'objet de ce type de strategie. En
observant les differences entre les groupes de sujets, par rapport a celles des sujets non
apparentes, il est possible d'estirner l'heritabilite. Si un effet genetique est identifie pour
le phenotype, on peut alors detenniner le genotype des genes candidats.
La deuxieme methode, le "genotype up", constitue le processus inverse, c'est-a-dire que les
analyses debutent avec les variations de sequence d'un gene. Dans cette strategie, l'on
recherche une association entre la variabilite au locus des genes candidats, detectee par les
.• '. <. ~, .. ", .• ~.:'. ,

---

PHENOTYPE
~
1si SUBPHENOTYPES
~
2nd SUBPHENOTYPES
~
CARDIAC
O TRANSPORT
MUSCLE
AFFECTORS
MUSCLE MT
MUSCLE
2
OUTPUT
CAPACITY
CAPILLARY
OF DIFFUSION
VOLUME
ENZYMES
DENSITY
AND CARRIERS
N
W
I
I
I
3rd SUBPHENOTYPES
~
HEART SIZE
I
MEMBRANE PERMEABILITY
I
PROTEIN CONTENT
I
BLOOD VOLUME
OXYGEN SOLUBILITY
ENZYME ACTIVITIES
[HB} AND TOTAL HB
MUSCLE FIBER SIZE
I
I
I
[MYOGLOBIN] AND TOTAL MB I
I
I
PHOSPHORYLASE;
SELECTED
~
a AND POF HB;
MYOGLOBIN; etc.
CANDIDATE
ERYTHROPOIETIN;
CS; PFK; POH; CPT;
GENES
VASOPRESSIN; elc.
CYTOX; etc.
FIGURE 2:
Vue d'ensemble des relations entre les genes candidats selectionnes et les sous-phenotypes de la \\'02 max.
HB = hemoglobine; MB = myoglobine; CS = citrate syntase; PFK::: phosphofructokinase;
PDH == pyruvate deshydrogenase; CfYf = carnitine palmitoyltransferase; Cytox == cytochrome oxidase.
(D'apres Bouchard et al., 1992).

---

2£+
"PHENOTYPE-DOWN"
PHENOTYPE
(VO: max)
FONCTION PHYSIOLOGIQUE
(mesure la capacite tota1e d'uti1isation
de l'oxygene des mitochondries du
muscle squelettique actif lors d'un
exercice max-imal)
VOlES METABOLIQUES ET VARIATIONS
PHYSIOLOGIQUES ET METABOLIQUES
(glycolyse, beta-oxydation, cycle de Krebs,
chaine res piratoire)
GENES CANDIDATS
(exemple : Cytochrome c oxidase)
VARIATION DE SEQUENCE DE L'ADN
"GENOTYPE-UP"
DES GENES CANDIDATS
(gene de la Cytochrome c oxidase)
FIGURE 3:
Illustration des strategies de "phenotype-down" et de "genotype-up"
(figure adaptee de Warden and Fisler, 1994).
. '. ,
:,

---

25
techniques d'analyse de fragments de restriction ou la technique d'amplification genetique
(Polymerase Chain Reaction, PCR), et le phenotype d'interet. Ces techniques d'analyse sont
elaborees a la section 7.1. Par exemple un gene qui cause une maladie peut etre isole sur
la base de sa localisation chromosomique, au lieu de sa fonction biologique, qui pourrait
etre inconnue (Yager et al., 1994). Cette approche est celle du "genotype up". En premier
lieu, les familles dans lesquelles on identifie une transmission mendelienne de la maladie
sont utilisees. Ensuite, par le biais des analyses de liaison genetiques (linkage), des
marqueurs genetiques anonymes ou microsatellites sont utilises pour localiser sur le
chromosome le gene ayant montre une liaison avec le trait etudie. Tous les genes potentiels
presents dans ces regions sont identifies successivement par "marche sur le chromosome".
Enfin, le gene responsable de la maladie est identifie par analyse de son expression chez
des sujets poneurs de la maladie (Warden and Fisler 1994; Yager et al., 1994). Cette meme
approche est utilisee pour localiser sur le genome humain, tous les loci associes a un trait
multifactoriel tel l'obesite et la reponse a l'entrainement. Pour cela, l'on utilise des
marqueurs anonymes ou microsatellites situes a des intervalles de recombinaison de 10 a
20 centiMorgan tout au long des chromosomes.
Dans cette these, la methode "genorype down" a ete utilisee. Nous avons done etudie
l'association entre le polymorphisme de genes candidats et la performance ou la trainabilire
de la \\10 max. On doit noter ici que Warden et Fisler (1994) considerent l'approche du
2
gene candidat, c'est adire les etudes d'association entre un polymorphisme de sequence et
un phenotype donne, comme un exemple de "phenotype down". 11s reservent le terme
"genotype up" a l'analyse de microsatellites. Nous avons dfi identifier pour cenains genes
des polymorphismes de sequences d' ADN. Pour confumer que la variation identifiee est
bien un RFLP genetiquement transmis, la transmission mendelienne doit etre etablie. Des
familles nucleaires sont par consequent utilisees pour demontrer que les alle:les identifies
par la technique des RH..Ps sont bien transmis des parents aux enfants.
Pour rechercher l'implication d'un gene dans la variation phenotypique, la contribution de
chacun de ses alleles est evaluee par le biais des etudes d'associations. Dans le cas de
. - ,
<. ~ ... , :;- .:'~ •
..
.
' .
-." .~ ~.;,,:

---

26
maladies hereditaires, ces demieres sont basees sur la comparaison d'individus sains non
apparentes et d'individus malades egalement non apparenres. Ces etudes evaluent si un
alIe::le particulier se retrouve 11 une plus haute trequence panni les individus sains (Lander
and Schork, 1994). En ce qui conceme cette these, comme mention ne ci-dessus, certains
genes sont selectionnes comme candidats car ils sont susceptibles d' etre des facteurs
contribuant 11 la variabilite de la V0 max. L'on recherchera alors si panni les differentes
2
am:les de ces genes, l'un d'entre eux est susceptible d'influencer la trainabilite de la V02
max ou la perfonnance. La distribution des alleles du marqueur entre les sedentaires et les
athletes ou les sujets qui repondent peu "low responders" et les sujets qui repondent bien
"high responders" aI'entrainement, sera alors analysee. Par exemple, si l'allele de 16.8 kb
du gene de la COX (gene candidat d'interet) se retrouve a une frequence significativement
plus elevee chez les "low responders" comparativement aux "high responders", cela
suggerera qu'il est associe aune faible reponse de la \\10 max 11 l'entrainement. Cependanc,
2
un gene voisin pourrait etre egalement responsable de l'association observee.
Les etudes d'associations presentent en effet des limites qu'il faut prendre en consideration
clans I'interpretation des resultats. Dne des premieres limites est le desequilibre de liaison.
Plus deux genes sont proches I'un de l'autre, plus la probabilite de recombinaison, c'est-a-
dire les echanges entre chromosomes paternel et maternel lors de la meiose, est faible voire
nulle. La probabilite de recombinaison entre deux loci augmente avec la distance physique
qui les separe. Par consequent, deux alleles situes trap pres I'un de l'autre ne seram pas
distribues de fa~on aleatoire dans la population, ils semnt toujours transmis ensemble: ils
som en desequilibre de liaison. La frequence de recombinaison donne une mesure de la
distance entre deux genes. Un centiMorgan equivaut 11 environ 1000 kb et correspond a 1%
de probabilite de recombinaison genetique entre deux marqueurs durant le meiose. Par
ailleurs, la frequence de recombinaison varie entre les especes, entre l'un et l'autre sexe et
avec la presence de "hot spots" ou " cold spots", regions ou les recombinaisons surviennent
11 des frequences irregulierement elevees ou faibles (Yager et al., 1994; Warden and Fisler
1994). Le desequilibre de liaison semble egalement survenir le plus souvent clans une
population jeune et isolee (Lander and Schork, 1994). Dans ce cas, les alleles des genes de
, ...-: "'~""';"'

---

.~ ;'
27
deux loci etroitement lies ne seront pas distribues de fa~on aleatoire a cause du nombre
insuffisant de generations. 11 n'y aura pas assez de recombinaisons pour atteindre un
equilibre.
Certaines associations positives pourraient aussi decouler d'un artefact dG a un melange de
populations (Lander and Schork, 1994). Ces auteurs expliquent que, dans une population
melangee, n'importe quel trait present a une plus haute frequence dans un groupe ethnique,
sera en association positive avec n'importe queI allele qui est egalement populaire ou
frequent dans le dit groupe.
Une des methOOes pour mettre en evidence l'implication genetique de determinants
genetiques d'un trait multifactoriel, est l'analyse de liaisons genetiques (linkage). Cette
analyse a pour but de localiser le gene etudie sur le chromosome (Dessein et al., 1992;
Yager et al., 1994). Contrairement aux etudes d'associations, les analyses de liaisons
genetiques utilisent des familIes pour etudier la transmission conjointe du phenotype avec
les marqueurs genetiques (Dessein et al., 1992). Les donnees seront plus informatives
lorsque un parent est heterozygote et que le conjoint est homozygote. L'etude de plusieurs
familIes nucleaires augmente la chance de trouver une telle combinaison. La distance entre
2 genes est determinee par la frequence de recombinaison. La methOOe des "loo-scores"
pennet d'accepter ou de rejeter un liaison. Si le phenotype et le gene sont lies, alors la
probabilite de recombinaison doit etre inferieure a 50%. L'analyse de liaisons genetiques
ne fera pas I' objet de cette these car les banques de donnees necessaires pour realiser ces
etudes n 'etaient pas disponibles.
2.6
Genes candidats etudies ou en cours d'etude
Le genome mitochondrial et differents genes candidats du genome nucleaire ont fait l'objet
de plusieurs etudes a notre laboratoire. Void une breve presentation des resulrats obtenus
ace jour.
' • • • • • • ~ 1 '.~.:"7.

---

28
2.6.1
Etudes anterieures sur les genes candidats
2.6.1.1
Polymorphisme au niveau des proteines
L'utilisation de la technique de focalisation isoelectrophoretique pour l'analyse de 9
enzymes du cycle de Krebs (Marcotte et al., 1987) et de 11 enzymes de la glycolyse
(Bouchard et al., 1988b) n 'a pu reveler de variants de charge dans environ 300 echantillons
de tissus musculaires. D'autres recherches effectuees chez les athletes d'elite n'ont pu
etablir de lien entre le polymorphisme des enzymes du globule rouge et le statut d'athlete
olympique (Chagnon et al., 1984; Couture et al., 1986). La relation entre les variants de
charge de la MCK et de l' AKIM et la performance ainsi que la reponse 11 l'entrainement
a ete evaluee apartir d'echantillons musculaires d'environ 95 sujets (Bouchard et al., 1989).
Les resultats demontrent que les variants des 2 enzymes influencent peu la performance et
la reponse a l'entrainement. Ces etudes anterieures ponant sur les proteines musculaires
suggerent qu'il est peu probable que les regions codantes de ces genes soient impliquees
dans les differences de la sensibilite metabolique 11 l'entrainement (Marcone et al., 1987;
Bouchard et al., 1988b).
2.6.1.2
Polymorphisme au niveau de l' ADN nucleaire
La relation entre le polymorphisme du gene de la MCK et la \\10 max a ete analysee chez
2
des sedentaires soumis 11 un entrainement en endurance. Les resultats revelent que cette
relation est infune (Dionne and Bouchard, 1990). Cependant les auteurs observent chez 3
sujets homozygotes pour l'allele de 3,4 kb, un gain moyen d'environ 0,3 1/02 de la \\102
max de plus que les autres sujets controles porteurs de I'autre allele caracterise par un
fragment d' ADN de 2,5 kb. Malgre le faible nombre de sujets, on peut soup~onner
l'implication de ce locus dans la reponse a l'entrainement.
..
.. .
" ,
"~ , .~
;,:

---

29
2.6.1.3
Polymorphisme au niveau de I'ADNmt
Nous savons que: d'une pan la V0 max est associee au potentiel qu'ont les mitochondries
2
du muscle a utiliser les substrats pour generer l' ATP par la phosphorylation oxydative et
d'autre part, les etudes sur I'heritabilire ont demoncre un effet maternel sur la V0 max
2
(Lesage et al., 1985). Puisque l' ADNmt a toujours ete considere comme etant exclusivement
de transmission maternelle (Brown et al., 1979), c'est-a-dire que les descendants rec;oivent
leur ADNmt de leur mere (Giles et al., 1980), l' ADNmt serait alors un marqueur genetique
interessant. Le genome mitochondrial humain est une molecule circulaire de 16569 paires
de bases (Anderson et al., 1981) qui se replique independamment du genome nucleaire.
Chaque molecule d'ADNmt code pour 2 ARN ribosomique, 22 ARN de transfert et 13
sous-unites de la chaine respiratoire soit 7 sous-unites de la NADH deshydrogenase
(complexe I), une sous-unite de la cytochrome c reductase (complexe Ill), 3 sous-unites de
la cytochrome c oxydase (complexe IV) et 2 sous-unites de l' ATP synrherase (complexe
V) (Anderson et al., 1981). Les autres sous-unites de ces proteines sont codees par l' ADN
nucleaire. Cet ADN possede egalement 2 parties non-codantes, 1'une appelee D-Loop sur
laquelle se trouve les sites de l'origine de replication et de transcription. L'autre partie, dont
la fonction n'est pas encore connue est situee aux environs de la base 8275. Enfin, le
concept de la non transmissibilite paternelle de l' ADNmt n'est plus aussi radical a present.
En effet, une etude conduite sur des souris chez lesquelles l' ADNmt paternel a ete amplifie
par la methode de PCR, revele qu 'une infime panie de l'ADNmt paternel est transmis aux
descendants (Gyllensten et al., 1991).
Plusieurs morphes ou patrons de fragments de restrictions de l' ADNmt ont ete detecres avec
15 enzymes de restriction, chez 46 jeunes sedentaires soumis a un programme
d'entrainement en endurance (Dionne et al., 1991a). Trois de ces sujets qui disposaient
chacun d'une substitution de base du gene de la sous-unite 5 de la NADH deshydrogenase
ainsi qu'une substitution dans l' ARN de transfert de la threonine, avaient tous une V02 max
par kg de masse corporelle, significativement plus elevee que les autres avant
l'entrainement. Ceux qui presentaient une substitution de base au niveau du gene de la

---

30
sous-unite 2 de la NADH deshydrogenase avaient une \\'0
max plus basse avant
2
l'entrainement (Dionne et al., 1991a). Trois autres sujets qui disposaient egalement d'une
substitution de base identifiee avec l'enzyme HindI dans la sous-unite 5 de la NADH
deshydrogenase, avaient une reponse de la V0 max ajustee pour le sire de l'entrainement
2
et la V0 max initiale, plus basse que les autres. En outre, un de ces sujets et deux autres
2
qui presentaient un morphe dans la region "D-Loop" de l' ADNmt avaient une reponse de
la V0 max non ajustee significativement elevee. Cette reponse devient par contre non
2
significative lorsqu'elle est ajustee pour le site de l'entrainement et la VOz max initiale des
sujets (Dionne et al., 1991a). Il ressort de cette etude que les variations au niveau de
l' ADNmt
contribueraient
aux
differences
individuel1es
observees
en
reponse
a
1'entrainement.
2.6.2
Genes candidats de I' ADN nucleaire selectionnes pour nos etudes
Dans le but de poursuivre les recherches sur la contribution des variations genetiques dans
la reponse a l'entrainement et la performance, d'autres enzymes impliquees dans le
processus de regeneration et de production de l' ATP et presentant des variations de
sequences d' ADN ont ete selectionnees. Nous presenterons le role de ces proteines ainsi que
les connaissances deja acquises sUr les genes qui codent pour ces proteines.
La glycogene synthase musculaire (GS; E.C.2.4.1.11) est une enzyme cle de la regulation
de la synthese du glycogene. Dans le muscle, la GS est presenre sous 2 formes
interchangeables: la synthase D, dont l'activite depend totalement de la presence du
glucose-6-phosphate et la synthase I dont le Km pour 1'uridine diphosphate glucose (UDPG)
diminue en presence du glucose-6-phosphate. La synthase I est la forme active de I'enzyme.
La GS effectue l'etape fmale de synthese du glycogene a panir du glucose. La
phosphorylation de la GS par l' AMP l'inactive, ce qui a pour resultat d'arreter la synthese
de glycogene. Le gene de la GS a ete clone par Browner et al. (1989). n est localise sur
le chromosome 19 dans la region q13.3 (Lehto et al., 1993). Un polymorphisme de la GS
a ete detecte avec l' enzyme XbaI par Groop et al. (1993). Ces demiers ont observe une

---

31
association entre les alleles 5.1 et 1.7 kb (allele A2) et les patients souffrant de diabete non-
insulino dependant. Un autre polymorphisme a ete egalement identifie
avec l'enzyme
EcoRI par notre equipe (Adjoa et al., 1993; chapitre Ill).
La cytochrome c oxydase (COX; E.C.1.9.3.1) est une proteine d'une importance capitale
pour la respiration cellulaire. Elle est le dernier maillon de la chaine respiratoire localisee
dans le complexe IV de la mitochondrie. Des 13 sous-unites qui composent ce complexe,
les 3 plus grandes au point de vue poids moleculaire sont codees par le genome
mitochondrial et les 10 plus petites, par le genome nucleaire (Kadenbach et al., 1983). 11
existe plusieurs nomenclatures de la COX, mais la plus utilisee est celle de Kadenbach et
al. (1983). L'interaction enrre les 13 proteines est necessaire pour l'obtention d'une enzyme
fonctionnelle (B use et al., 1988; Forsburg and Guarente, 1989; Kharkats and Volkov, 1989).
Les fonctions catalytiques sont assignees aux sous-unites mitochondriales (Montecucco et
al., 1986; Kadenbach et al., 1983). Quant aux sous-unites nucleaires, elles sont impliquees
dans la regulation de la fonction catalytique des sous-unites mitochondriales, en optimisant
l'activite enzymatique de l'oxydase selon les differents besoins metaboliques des tissus
(Kadenbach and Merle, 1981). Les sous-unites IV, Vb, VIb et VIII sont respectivement
situees sur le chromosome 16q22-qter (Darras et al., 1987), le chromosome 2cen-q 13
(Lomax et al., 1991), le chromosome 19q13.1 (Taanman et al., 1991) et le chromosome
llq12-q 13 (Rizzuto et al., 1989). De toutes ces sous-unites, seule la localisation de la
COXVIII est confmnee (Mc Kusick, 1992). La localisation chromosomique des autres sous-
unites de la COX n'est pas encore identifiee. Des variants de sequences ont ete detectes au
niveau des sous unites mitochondriales de la COX (Johnson et al., 1983; Cann et al., 1982).
Quand aux sous unites nuc!eaires, il n'y avait pas de polymorphisme de decrit avant nocre
etude sur les sous unites IV, Va et Vb (Adjoa et al., 1994; chapitre IV).
L' AK (E.C.2.7.4.3) a ete largement presenre a la section 1.1. Le gene de l'AK1 est localise
sur le chromosome 9 dans la region q34 (Roward, 1987). L' AK1 cytosolique est
polymorphique chez l'homme (AK1 *1 et AKl *2). L'AK1 *1 est le plus frequent des deux
phenotypes (Bonne et al., 1971). L' AK1 *2 est rare (Fildes and Harris, 1966; Bonne et al.,

---

32
1971; Luz et al., 1990) et se retrouve uniquement chez les Caucasiens a une frequence de
5% a9% (Bowman et al, 1967; Bonnt~ et al., 1971). La frequence de ce phenotype est tres
faible chez les Americains Noirs et absente chez les Africains de l'ouest du Ghana et du
Nigeria (Bowman et aI., 1967) et egalement absente chez les gens de la Nouvelle Guinee
et les arborigenes de l'Australie (Kirk et al., 1971). L'AK1 *1 represente 95% de l'isozyme
AK1 (Luz et al., 1990). L' AK 1 est une enzyme dont le gene presente des variations de
sequences d' ADN
identifiables avec l'enzyme de restriction TaqI (Bech-Hansen et al.,
1989).
La PFK (E. C.2.7.1.11) intervient au niveau de la premiere etape de la glycolyse. Elle
catalyse la phosphorylation du fructose-6-phosphate en fructose-I, 6 diphosphate. La PFK
est consideree comme 1'enzyme allosterique, ou regulatrice, de la glycolyse, et son activire
in vitro est contr6lee par la concentration de plusieurs substances metaboliques. Elle est
inhibee par l' ATP, 1'uridine triphosphate (UTP), le cytidine triphosphate (CTP), le
phosphoenolpyruvate, la phosphocreatine et le citrate, et elle est stimulee par l' ADP, l' AMP
et le phosphate inorganique (Mansour, 1966). Il existe 3 sous-unites de la PFK: la sous-
unite codee par le gene du muscle (PFKM), du foie (PFKL) et des plaquettes (PFKP) (Mc
Kusick, 1992). La PFKL est situee sur le chromosome 21 dans la region 22.3 et la PFKP
sur le chromosome 10 (Mc Kusick, 1992). Quant a la PFKM, son gene est situe sur le
chromosome 1 dans la region q32 (Vora et aI., 1982). Un polymorphisme de la sequence
du gene de la PFKM identifiable avec KpnI a ete rappone par Dionne et al.(l991b).
La phosphoglucomutase (PGM; E.C.2.7.5.1) est egalement une enzyme du metabolisme des
glucides. Elle catalyse la reaction reversible de transformation du glucose 6-phosphate en
glucose-I-phosphate. Cette enzyme presente 5 isoformes ( PGM1, PGM2, PGM3, PGM4
et PGMS) et est genetiquement contr6lee par 5 loci structurellement differents (Cantu and
Ibarra, 1982). Les loci des 4 premiers isoformes sont localises sur differents chromosomes
connus (Herbich et aI., 1985). Ces genes sont exprimes differemment dans 1es tissus et les
secretions. La PGM1 dont le gene est situe sur le chromosome 1p31 ( BilJardon et al.,
1973), est exprimee dans les globules rouges et dans les tissus. La PGM1 et le PGM3 sont
.
.
,. "~; .,;~.:'";

---

33
des enzymes habituellement polymorphiques. La PGM2 est polymorphique dans certaines
populations seulement (Gloria-Bottini et al., 1994). Quam a la PGM4, elle n'est
polymorphique que chez les Mexicains "Mestizos", population au sein de laquelle ce
polymorphisme a ere identifie (Gloria-Bottini et al., 1994). L'isoenzyme PGM 1 presente des
variations clans la population
(Spencer et al., 1964; Chagnon et al., 1981; Boret et al.,
1991). Elle presente 4 alU:les (Mc Kusick, 1992) et dix phenotypes (Bark et al., 1976;
Sutton and Burgess, 1978). Il existe 3 phenotypes de la PGMl que l'on identifie
couramment: la PGMl 1-1, la PGMl 1-2 et la PGMl 2-2, qui resultent de la combinaison
des 2 alleles les plus frequents qui sont la PGMl 1 et la PGMl 2 (Spencer et al., 1964).
La PGMl 1 a une frequence de 70 a 90% clans plusieurs populations testees (Bonne et al.,
1971). Elle tend a etre plus elevee chez les Nom que chez les Blancs. Au niveau de
l' ADN, la PGMl presente 2 polymorphismes identifiables avec l'enzyme Taq! (Hollyoake
et al., 1992).
2.7
APPROCHE EXPERIMENTALE
Les sciences fondamentales et la contribution de la biologie moleculaire joueront un
role sans cesse croissant pour les specialistes des disciplines cliniques et non cliniques, tels
que ceux qui sont impliques clans tout ce qui touche aussi bien aux rendements clans les
exercices physiques occasionnels qu'aux performances d'athletes d'elite. Les recentes
connaissances acquises de la biologie moh~culaire ont pennis de mettre au point des
techniques rapides d'analyse de structure d' ADN. L'application des techniques moleculaires
a1'etude de la variation des genes physiologiquement importants se fait par le biais de
techniques telles que l'analyse du polymorphisme de fragments de restriction RFLPs par
Southern blot ou par PCR. L'etude des variations phenotypiques des proteines se fait
egalement par la technique de focalisation isoelectrophoretique (IEF) qui est une methode
biochimique.

---

34
2.7.1
Technique d'analyse des fragments de restriction
Pour ce genre d'analyse, des enzymes de restriction sont utilisees pour detecter les
differences dans la sequence de l' ADN genomique. Les enzymes de restriction ont un site
specifique de reconnaissance de sequence sur l'ADN et coupent I'ADN a ce site. Une
grande variete de fragments d' ADN est ainsi produite dont l'un ou un tout petit nombre
correspond a une partie ou a la totalite du gene etudie. Le changement de sequence d' ADN
peut entrainer l'apparition ou la disparition des sites particuliers de clivage, et modifier par
consequent, la longueur de fragments generes d'une region donnee. Les differences de
sequence sont detectees en terme de longueurs de fragments d' ADN. 11 y a un autre
polymorphisme qui se manifeste par une insertion ou une deletion d'un bloc d' ADN entre
2 sites de reconnaissance d'une enzyme, entrainant egalement une modification de la
longueur du fragment de restriction generee par la digestion avec l' enzyme de restriction.
Les differentes longueurs de fragments sont separees par electrophorese sur un gel
d'agarose. Puis, selon les procedures de Southern (1975) presentees ala figure 4, les brins
d' ADN sont separes par denaturation alcaline directement dans le gel. L' ADN total est
ensuite transfere sur un suppon solide qui est un filtre de nitrocellulose ou de nylon. Le
filtre est hybride avec une sonde d' ADN specifique au gene etudie. La sonde marquee au
phosphore radioactif (p32) reconnait l' ADN homologue lie au filtre et forme des hybrides
stables. Ce filtre est lave pour eliminer le surplus de sonde radioactive non hybridee. Le
filtre est ensuite expose a un film rayon X, qui pennet de detecter et de visualiser les
bandes d' ADN. La longueur des bandes est detenninee a l'aide de marqueurs de poids
moleculaires qui migrent en meme temps que les echantillons d' ADN. Les RFLPs sont
communement utilises comme marqueurs genetiques. Cette methode a ete utilisee dans 5
des 6 etudes de cette these.
"_ ••••• , ...... '7.

---

1- ADN total (nuch~aire et
2- Fragments d'ADN obtenus par
mitocho'ndrial) extrait des
clivage avec une enzyme de
cellules.
.
restriction.
+ .------------, haut poids
moleculaire
.---+H- Filtre
Gel d'agarose
petit poids
molEkulaire
4- Transfert sur un filtre de nitrocellulose
3- Electrophorese sur un gel
apres avoir separe les fragments
d'agarose.
d'ADN par denaturation alcaline:
"Southern blotting".
Filtre
'Sonde
+
Sac
,
fi~hyb'ridation
,
,_~:, -01.
,
+
m
+
***
+
+
ccatg
[N]
+
" " " "11
+
ggtac
+ +
+
+ +
ccatg
11 11 " 1111
+
+
ggatc
**
'------~.<,
.5- Hybridation du filtre avec des J~" ~iY\\' .
'fragments d'ADN ou .illJch~aire
}~
:'C , ' ,
"~fr~~~~~l~~i~~~~~~~~t:~~~~~i~~
Figure 4: Technique d'analyse de l'ADN
(Southern blotting)
.. - .,
.
"~' '

---

36
2.7.2
Amplification genHique ou "Polymerase chain reaction, peR"
La PCR est une technique d'amplification d'ADN recemment developpee (Saiki et al.,
1985; Sharf et al., 1986) qui permet d'obtenir rapidement et en grande quantite des
fragments d' ADN pour le gene d'interet. Brievement, les 2 brins d' ADN sont obtenus sous
l'action d'une polymerase thermostable qui synthetise 1,"ADN entre 2 amorces de
nucleotides synthetisees chimiquement qui sont situees de chaque cote de la sequence a
amplifier. Un nombre croissant de copies estOobtenu sous l'action successive et repetee de
cycles de denaturation des brins a amplifier, d'hybridation des amorces et d'extension de
la synthese.
2.7.3
Focalisation isoelectrophoretique
11 existe egalement d'autres methodes d'etude de polymorphismes, en l'occurrence la
methode de la focalisation isoelectrophoretique (IEF) (Figure 5). L'IEF est une technique
permettant de separer les proteines selon leur point isoelectrique par electrophorese dans
un gradient de pH stable. Ce gradient de pH augmente progressivement de l'anode a la
cathode. Le champ electrophoretique est obtenu par l'electrolyse des ampholytes ajoutes au
gel. Plus le pH de gradient est etroit, mieux s'etablit la separation. L'IEF est sensible non
seulement aux changements des residus d'acides amines charges, mais est capable de
detecter les changements subtils dans les points isoelectriques induits par la substitution des
charges neutres qui affectent la conformation des molecules des proteines, rendant par
consequent, chaque locus plus informatif. L'IEF est couramment utilisee en biologie, dans
cenains secteurs de l'industrie et surtout en biochimie clinique. Cette technique a ete
massivement utilisee pour l'etude du polymorphisme de l'enzyme PGM clans la genetique
des populations. Dans le chapitre VIII, nous avons utilise cette technique.
- .. "~' .;-.;"; ,

---

37
ANODE
CATHODE
Figure 5 : Technique de focalisation isoelectrique
.
...... ". ...
"
:~;'.

---

CHAPITRE III
POLYMORPHISME DE LONGUEUR DE FRAGMENTS DE RESTRICTION DETECfES
AVEC L'ENZYME DE RESTRICTION EcoRI DANS LE GENE DE LA GLYCOGENE
SYNTHASE CHEZ L'HUMAIN.
Cette etude rapporte l'identification de 2 alle1es de 10.1 et 8.1 kb du gene de la glycogene
synthase chez l'humain avec l'enzyme de restriction EcoRI.

---

39
EcoRI RESTRICTION FRAGMENT LENGTH POLYMORPHISM IN HUMAN
GLYCOGEN SYNTHASE GENE
Hum Genet 92:632, 1993
Suzanne A. Adjoa, Claude Bouchard, France T. Dionne
Physical Activity Sciences Laboratory, PEPS, Laval University, Ste-Foy, Quebec, Canada,
G1K 7P4
ADDRESS FOR CORRESPONDENCE:
France T. Dionne, Ph.D.
Physical Activity Sciences
Laboratory
PEPS, Laval University
Ste-Foy, Quebec,
Canada GIK
Tel: (418) 656-3696
Fax: (418) 656-3020
Running Title: Glycogen Synthase RR..P

---

40
ABSTRACT
Two alleles of 10.1 and 8.1 of the human glycogen synthase gene have been revealed with
the restriction enzyme EcoRl.
• 0 ,
. . . . . . ; . ; . . .: " ' : .

---

41
Glycogen synthase is a key regulatory enzyme of glycogen synthesis. The human glycogen
synthase gene has been cloned by Browner et al. (1989).
A 3.4 kb cDNA fragment,
containing the human muscle glycogen synthase cDNA from amino acid 90 (nucleotide
414) through the 3' untranslated region (nucleotide 3535) and cloned in the EcoRI site of
the Stratagene bluescript vector, was used to screen DNA sequence polymorophism at this
locus. EcoRI identifies a two-allele polymorphism with fragments of 10.1 kb (allele 1) and
8.1 kb (allele 2) with constant bands of 5.3, 2.9, and 2.2 kb (Fig. 1). The allele frequencies
estimated in 48 chromosomes of unrelated Nonh American Caucasians were: allele 1 (0.75)
and allele 2 (0.25). Codominant autosomal segregation was observed in two families for
a total of nine individuals (Fig. 1). This gene was not polymorphic for: Bgill, BamHI,
Banl, Dral, HindIIl, Mspl, PvuII, Pstl, Seal, Sstl, and Taql using DNA from 18 unrelated
individuals. The human muscle glycogen synthase locus has been mapped to chromosome
19, band q13.3 (Lehto et al., 1993).

---

42
ACKNOWLEDGEMENTS
This probe was a generous donation from Dr. EM. Browner, School of Medicine,
Department of Biochemistry and Biophysics, San Francisco, Calif.
This research was
supported by a grant from Medical Research Council of Canada.
.. ,.:......; ..:.;..
.
..
'.
"
.... ~ -':":".

---

43
REFERENCES
Browner FM, Nakano K, Band A~G, Fletterick JR (1989).
Human muscle glycogen
synthase cDNA sequence: a negatively charged protein with an asymmetric charge
distribution. Proc Ntl Acad Sci USA 86: 1443-1447.
Lehto, M, Stoffel M, Groop L, Espinosa III R, Le Beau MM, Bell Gl (1993). Assignment
of the gene encoding synthase (GYS) to human chromosome 19, band q13,3.
Genomics 15: 460-161.
..... '.~ '. -~'. ":".

---

44
EcoRI
kb
10.1
8.1
5.3
2.9
2.2
Figure 1:
Southern analysis of the EcoRI glycogene synthase restriction fragment
length polymorphism showing codominant autosomal segregation ill two
families.

---

CHAPITRE IV
SOUS-UNITES DE LA CYTOCHROME C OXIDASE CODEES PAR LE GENOME
NUCLEAIRE:
POLYMORPHISME
DE
LONGUEUR
DE
FRAGMENTS
DE
RESTRICTION DETECTES AVEC L'ENZYME DE RESTRICTION Sst!'
Cette etude rappone l'existence de polymorphisme clans la sequence de l' ADN de la sous-
unite Va de la cytochrome c oxidase humain (COX), une enzyme d'imponance capitale
pour la phosphorylation oxydative dans la mitochondrie. Aucun polymorphisme de sequence
n'a ete detecte clans les genes des sous-unites IV et Vb de la meme enzyme.
. .. '.~' ...
.. -.. ..
'"
, .~ :":.

---

46
NUCLEAR-ENCODED SUBUNITS OF HUMAN CYTOCHROME C OXIDASE: SstI
RESTRICTION FRAGMENT LENGTH POLYMORPHISM
Hum Genet 93:347-348, 1994
Suzanne A. Adjoa, Claude Bouchard, Pieter Coetzer, Tim D. Noakes, France T. Dionne.
Physical Activity Sciences Laboratory, PEPS, Laval University, Ste-Foy, Quebec, G1K 7P4,
Canada. Department of Physiology, University of Cape Town, Cape Town, South Africa
ADDRESS FOR CORRESPONDENCE:
France T. Dionne, Ph.D.
Physical Activity Sciences Laboratory
PEPS, Lava! University
Ste-Foy, Quebec, Canada, G1K 7P4
Tel: (418) 656-5174
Fax: (418) 656-3020
Running Title: Subunit Va of cytochrome c oxidase RFLP

---

47
ABSTRACT
A DNA polymorphism of the nuctear-encoded subunit Va of the human cytochrome c
oxidase (COX), a mitochondrial respiratory enzyme, is reported. No polymorphism was
detected in genes for the subunits IV and Vb of the same enzyme.
..
.
'.' '
~ ' .. '. ":'

---

48
INTRODUCTION
Eukaryotic cytochrome c oxidase (COX; EC 1.9.3.1) is a protein of central importance in
the oxidative respiratory system (Kharkats and Volkov, 1989). The mammalian enzyme
is composed of 13 subunits encoded by both the nuclear and the mitochondrial genomes
(Kadenbach et al., 1983). The 3 larger subunits 0, n, and Ill), encoded and expressed in
the mitochondria, must interact with the 10 smaller subunits (IV, Va,b,c, Vla,b,c, VIIa,b,c,
and VIII according to the nomenclature of Kadenbach et al., 1983), encoded by the nuclear
genome, to fonn a functional mitochondrial enzyme. COX catalyses the following reaction:
4 ferrocytochrome c + H+ + O ~ 4 ferricytochrome c + 2~0
2
The catalytic and proton-pumping functions of COX and the site of interaction with
cytochrome c have been assigned to the mitochondrial subunits (Kadenbach and Merle
1981; Azzi and Muller 1990).
The nuclear-encoded subunits may be involved in the
regulation of the catalytic function of the enzyme (Kadenbach et al., 1983; Montecucco et
al., 1986).
While DNA variants have been reported for the mitochondrial sub units I, IT, and III of
COX (Johnson et al., 1983; Cann et al., 1984), no DNA sequence variation has been
reported yet for the nuclear-encoded subunits.
In the present study, we investigated
restriction fragment length polymorphisms (RFLPs) of the nuclear-encoded subunits IV, and
Va and Vb, the two isoforms of the subunit V of COX.
It has been proposed that the

---

49
function of subunit IV is to bind ATP by altering the strUcture of the oxidase and. then.
allowing subunit IT to bind cytochrome c (Montecucco et al.. 1986).
Kadenbach et al.
(1983) suggested that subunit V is inv01ved in the coupling of electron transport and in the
proton pumping action. The specific function of each isofonn is not yet known.
. ,. '.~ ...
,.
-
"~
.~

---

50
MATERIALS AND METHODS
DNA probes
Three specific cDNA probes were used for the RFLP analyses. For COX IV (pCOX 4.11),
the probe was a 680-bp EeoRI fragment, containing the coding region for the mature
protein and the 3' untranslated region of the gene (Zeviani et al., 1987). For the COX Va
isofonn, pCOX 5.11 containing the full-length cDNA (about 650 bp) was used. This clone
was isolated from a human endothelial A gtll library (Rizzuto et al., 1988). As for subunit
Vb, pCOX 5.15, containing the full-length cDNA (500 bp) insened into the EeoRI site of
Bluescribe M13 (Stratagene), was used as a probe (Zevianni et al., 1988).
DNA extraction and RFLP analysis
Total DNA was isolated from white blood cells as previously described by Dionne et al.
(1991).
Samples of 5 ~g of total DNA were cleaved with the enzymes Avail, BamHl,
BellI, Bgill, EeoRI, HindlII, Hinfl, NeoI, PvuII, SstI, TaqI, and XmnI for subunit IV: with
AvaIl, BamHI, BanII, BellI, BglII, DraI, EeoRI, EeoRV, HindlIl, Hinfl, HpaI, MspI, Nez1,
Neoll, PstI, PvuII, SstI, and XmnI for the subunit Vb; and with Avail, BamHl, BellI, Hincll,
MspI, Neil, Pstl, PvulII, Seal, and TaqI for subunit Va of cytochrome oxidase.
The
digested fragments were separated by electrophoresis on horizontal agarose gels (0.8%) in
TEA buffer, pH 8.5 (40 mM Tris, 20 mM Na acetate, 20 mM NaCI, and 2 mM EDTA).
. ...... : ... :.:.
.
..
..
"~' .,;
:-::".

---

51
The DNA fragments were then transferred to nitrocellulose filters (Schleicher & Schuell).
These fragments were visualized by molecular hybridization with the specific probes
labelled with P
using random priming (Feinberg and Vogelstein, 1984) to a specific
activity> 1.2 x 109 Cpm per ~g.
.... ,
:-:..

---

52
RESULTS AND DISCUSSION
No polymorphism was detected in subunit IV of COX using 13 endonuc1eases and in
subunit Vb of COX using 20 endonuc1eases. These enzymes were selected because at least
one restriction site for each of these enzymes was present in the genes. For subunit Va,
Sstl digestion revealed polymorphic fragments of 20.7 kb (allele 1) and 16.8 kb (allele 2)
with constant bands of 8.8 and 4.6 kb (Figure 1).
A light DNA band of 6.6 kb was
sometimes observed with the 20.7: 16.8 and 16.8:16.8 genotypes. The frequency was 0.5
for each allele as determined from a group of 35 unrelated North American Caucasians and
a group of 34 unrelated South African Caucasians. Codominant segregation was observed
in seven informative families of at least four members each. Two of these families are
shown in Fig. 1.
In 18 unrelated individuals, no polymorphism was detected with the other enzymes. To our
knowledge, the human cytochrome c oxidase subunit Va locus has not yet been mapped.
The results suggest that the DNA sequence of these three nuclear subunits of COX is highly
conserved among Caucasians.

---

53
ACKNOWLEDGEMENTS
These probes were generous donations' from Dr. Eric A. Schon, College of Physicians and
Surgeons, Columbia University, New York. This research was supported by a grant from
Medical Research Council of Canada.
-
,
;"':'.

---

54
REFERENCES
Azzi A., Muller M. (1990) Cytochrome c oxidase: polypeptide composition, role of
subunits, and location of active metal centers.
Arch Biochem Biophys 280: 242-
252.
Cann LR, Brown, NW, Wilson AC (1984).
Polymorphic sites and the mechanism of
evolution in human mitochondrial DNA. Genetics 106: 479-499.
Dionne Fr, Turcotte L, Thibault M-C, Boulay MR, Skinner JS, Bouchard C (1991).
Mitochondrial DNA sequence polymorphism, V0 max, and response to endurance
2
training. Med Sci Sports Exerc 23 177-185.
Feinberg AP, Vogelstein B (1984).
A technique for radiolabeling DNA restriction
endonuclease fragments to high specific activity. Anal Biochem 137: 266-267.
Johnson ill, Wallace CD, Ferris DS, Rattazzi CM, Cavalli-Sforza LL (1984). Radiation
of human mitochondrial DNA types analysed by restriction endonuclease cleavage
patterns. J Mol Evol 19: 255-271.
Kadenbach B, Merle P (1981).
On the function of mutiple subunits of cytochrome c
oxidase from higher eukaryotes. FEBS Lett 135: 1-11.

---

55
Kadenbach B, Ungibauer M, Iaraush I, Buge U, Kuhn-Nentwig L (1983). The complexity
of respiratory complexes. Trends Biochem Sci 8:398-400.
Kharkats IY, Volkov AG (1989).
Cytochrome oxidase: molecular mechanism of
functioning. Bioelectrochem Bioener 22:91-103.
Montecucco C, Schiavo G, Bisson R (1986). ATP binding to bovine heart cytochrome c
oxidase: a photoaffinity labelling study. Biochem I 234:241-143.
Rizzuto R, Nakase H, Zeviani M, Di Mauro S, Schon AE (1988). Subunit Va of human
and bovine cytochrome c oxidase is highly conserved. Gene 69:245-256.
Zevianni M, Nakagawa M, Herben J, Lomax IM, Grosman IL, Sherbany AA, Miranda AF,
Di Mauro S, Schon AE (1987). Isolation of cDNA enccxiing subunit IV of human
cytochrome c oxidase. Gene 55:205-217.
Zeviani M, Sakoda S, Sherbany AA, Nakase H, Rizzuto, R, Samit CE, Di Mauro S, Schon
AE (1988). Sequence of cDNA encoding subunit Vb of human bovine cytochrome
c oxidase. Gene 65: 1-11.

---

56
Sstl
kb
20.7
16.8
8.8
6.6
4.6
Figure 1:
SstI resniction fragment length polymorphism at the human cytochrome c
oxidase subunit Va locus.
.
,
-.-; ....;~~.

---

::~i,:--; .
.'.' ~'.,
·r....~ :.
CHAPITRE V
RELATION ENTRE LE POLYMORPHISME DE SEQUENCE DE L'ADENYLATE
KINASE 1 MUSCULAlRE, LA PHOSPHOFRUcrOKINASE MUSCULAlRE, DE LA
SOUS-UNITE Va DE LA CYTOCHROME C OXIDASE ET LA VARIATION DE LA
V0 MAX ET DE SA REPONSE A L'ENTRAINEMENT EN ENDURANCE.
2
Dans
le
but
d'identifier les
facteurs
qui
pourraient
contribuer
aux
differences
interindividuelles dans la perfonnance et la reponse a I'entrainement, la variation de
sequence de I' ADN de 3 genes codant pour des enzymes regulateurs des voies de
regeneration et de production de l' adenosine triphosphate (ATP), a ete investiguee chez 38
sujets sedentaires soumis a un entrainement en endurance et chez 25 athletes d'endurance.
L'ADN a ete isolee des globules blancs, et la variation de sequence de l'adenylate kinase
1 musculaire (AKIM), la phosphofructokinase musculaire (PFK.M) et la sous-unite Va de
la cytochrome c oxidase (COXVa) a ete analysee par la technique de polymorphisme de
longueurs de fragments de restriction (RFLP). La distribution des genotypes de la COXVa-
SstI des athletes etait significativement differente de celle des sedentaires (P<O.05).
Soixante dix huit pourcent des athletes etaient heterozygotes pour ce variant contre
seulement 39% des sedentaires, bien que les frtquences alleliques etaient comparables entre
les 2 groupes. La comparaison des frequences genotypiques de la COXVa revele egalement
la presence d'un plus grand pourcentage (46%) d'heterozygotes panni les "high responders"
al'entrainernent. Ces donnees suggerent un avantage a etre heterozygote pour les alleles
de la COXVa-SstI, sur le plan de l'adaptation a l'entrainement en endurance. Des
recherches plus elaborees sur ce gene et cette mutation particuliere en relation avec la
perfonnance en endurance et la reponse a l'entrainernent sont souhaitables.
._. ,. _.0' .;~.;-; •
... ...... ..
~ .;
:";".

---

:: t
58
MUSCLE ADENYLATE KINASE· I, PHOSPHOFRUCTOKINASE AND SUBUNIT
Va OF CYTOCHROME C OXIDASE DNA SEQUENCE POLYMORPHISM, AND
VARIATION IN \\'0 MAX AND ITS RESPONSE TO ENDURANCE TRAINING
1
Suzanne A. Adjoa, IMarie-Christine Thibault, Jean-Aime Simoneau, 2Pieter Coetzer,
~imothy D. Noakes, Marcel R. Boulay, 3James S.Skinner, Claude Bouchard, France T.
Dionne
Physical Activity Sciences Laboratory, PEPS Laval University, Ste-Foy, Quebec, Canada
G1K 7P4, Ideceased 01/06/1995 2MRC/MCT Bioenergetics of Exercise Research Unit,
Department of Physiology, University of Cape Town, Cape, South-Africa and 3Department
of Kinesiology, Indiana University, Bloomington, IN.
ADDRESS FOR CORRESPONDENCE:
France T. Dionne, Ph.D.,
Physical Activity Sciences
Laboratory, PEPS
Laval University, Ste-Foy
Quebec, CANADA
G1K 7P4
Tel: (418)-656-3696
Fax: (418)-656-3020
Running title: Candidate gene RFLPs and performance
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59
ABSTRACT
In order to identify genetic factors which could account for interindividual
~
differences in performance and in response to training, DNA sequence variation at 3 genes
encoding key regulatory enzymes of the adenosine triphosphate (ATP) regeneration and
production pathways was investigated in 38 sedentary subjects submitted to endurance
training and 25 highly trained endurance athletes. DNA was extracted from white blood
cells and muscle adenylate kinase (AKIM), muscle phosphofructokinase (PFKM), and
subunit Va of cytochrome c oxidase (COXVa) DNA sequence variations were analyzed
using restriction fragment length polymorphism (RFLP) techniques. Differences were
observed for COXVa-SstI genotype distributions (P<O.05) between athletes and sedentary
subjects. Seventy eight percent of the athletes were heterozygotes for this variant but only
39% of the sedentary subjects despite comparable allele frequencies. These data suggest a
heterozygote advantage for the COXVa-SstI allele in terms of adaptation to endurance
training. Futher research is clearly warranted on this gene and this particular SstI mutation
in relation to endurance perfonnance and the response to training.
KEY WORDS: RFLP, AEROBIC PERFORMANCE, TRAINABILITY, MAXIMAL
OXYGEN UPTAKE, CANDIDATE GENES

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60
INTRODUCTION
V0 max is considered an imponant determinant of performance and its response
2
to endurance training was shown to be largely inherited (3,27). Highly trained endurance
athletes exhibit significantly higher muscle mitochondrial enzymes, such as citrate synthase,
which is related to their V0 max (18,31). It has also been reponed that, differences
2
between trained and untrained subjects as well as differences in response to training in
several skeletal muscle enzyme activities (4,15,30), are genetically determined. Previous
studies were not successful, however, in demonsrrating association between red blood cell
enzyme polymorphism and the status of elite athletes (9). In the same way, no charge
variants were detected in 11 enzymes of the glycolytic pathway and 9 enzymes of the
tricarboxylic acid cycle of skeletal muscle (5,23). It then appeared unlikely that sequence
variation of the coding regions of these genes is involved in the individual differences
observed in the response to training.
Several genes are undoubtely involved in determining the response of aerobic
performance to endurance training (4,6). Hydrolysis of ATP is the major factor determining
the energy requirements during exercise (18,38). Since ATP reserve in muscle is limited,
ATP has to be continuously regenerated. The enzymes implicated in ATP regeneration
process are of critical importance since the improvement of their activity is associated with
the capacity to perform (18,31). Training significantly improves the metabolic capacity of
working muscles, resulting in increased V0 max (33,39). These adaptations allow trained
2
subjects to perform endurance exercise at intensities near their \\10 max for longer period
2
of time. However, subjects given the same endurance training do not respond in the same
manner (21,27). Some subjects exhibit little or no change in response to training, while
others exhibit a high response to training. These large inrerindividual differences in
sensitivity to training were shown to be genotype dependent (27). To search for factors
responsible for this variation, we selected candidate genes involved in ATP regeneration
process, in this case, muscle adenylate kinase 1 (AKIM), muscle phosphofructokinase
(PFKM), and subunit Va of cytochrome c oxidase (COXVa) genes.
.. ..
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61
AK1 (E.C.2.7.4.3) catalyses the conversion of two moles of ADP to one mole of
ATP and one mole of AMP. This reaction can proceed in both directions (20) and occurs
within red blood cells, skeletal muscles, and other tissues (35). PFKM (E.C.2.7.1.11) is a
~
key regulatory enzyme of glycolysis. In the first step of glycolysis, PFKM catalyses the
phosphorylation of fructose-6-phosphate to fructose -1-6 diphosphate. COXVa is one of the
two isofonns of nuclear encoded subunit V of cytochrome c oxidase (COX; E.C.1.9.3.1),
a protein of central imponance in oxidative respiratory system (19). Although the oxidative
potential is developed in trained subjects, the ratio of glycolytic over oxidative potential is
of interest. Glycolytic potential can decrease at the detriment of an increase in oxidative
potential thus favouring oxidation over glycogen catabolism. These 3 enzymes are thus
involved in the skeletal muscle ATP regeneration process. AKIM, PFKM, and COXVa
genes were shown to exhibit 2 alleles polymorphism using the TaqI (2), KpnI (13), and SstI
(1) restriction enzyme, respectively.
Therefore, the purpose of this study was to investigate: 1) the association between
DNA polymorphism of AKIM, COXVa, and PFKM genes and aerobic perfonnance of
athletes and 2) the response of V0 max to endurance training in sedentary subjects given
2
an endurance training program.

---

62
SUBJECTS AND METHODS
Ten sedentary male subjects from Quebec City, aged 17 to 27 years, and 28
,
sedentary men from Tempe, Arizona aged 24 to 29 years, took pan in this study. They had
no history of regular panicipation in sports nor previous engagement in an exercise training
program. The group of endurance athletes consisted of seventeen highly trained male
cyclists (22 to 35 years old) from Canada and eight male South-Africa Caucasian marathon
runners (18 to 32 years old). These athletes had been engaged in regular training for several
years. Both groups of subjects provided written informed consent, prior to their panicipation
in this study.
Training and \\'0 max of the sedentary subjects. The sedentary subjects from
2
Quebec were trained on a cycle ergometer for 20 weeks, 3 times increasing to 5 times per
week, for a maximum of 40 to 45 min per session, at an intensity reaching 70% to 80% of
the maximal heart rate reserve. The sedentary subjects from Arizona were trained on a
cycle ergometer for 12 weeks, 3 times per week, for a maximum of 40 min per session, at
a heart rate associated with each person's onset of blood lactate accumulation (OB LA), Le.,
4 mM
blood lactate. The
V0
max of these
subjects
was determined on an
2
electromagnetically braked ergocycle. After an initial 3 min period at 50 W, the power
output in Quebec was increased every 3 min by 25 W until volitional exhaustion; rpm was
maintained at about 60 throughout the test. In Arizona, the test for V0 max was initiated
2
at 80 W, with increments of 20 to 30 W every 2 min until exhaustion. Respiratory
parameters (Ve, V02, VCO~ were continuously monitored using automated open-circuit
systems (Beckman Metabolic Measurement Can). Heart rate was monitored with an ECG.
\\'0 max of the endurance athletes. The V0
2
2 max of the French-Canadian athletes
was determined on a cycle ergometer (Monark-Crescent, Varberg, Sweden) beginning at
100 Wand increasing the load by 25 W each 3 minutes for a maximum of 14 to 17 min.
The measurement of V0 max of the South-African athletes was done on a treadmill
2
(Quinton Insttuments). These athletes began running at a velocity of 17krn/h. with

---

· ",.,
63
increments of 0.5 kmlh every 30 s until exhaustion. If the peak speed of the treadmill,
which in this case was 25km/h, could be maintained for 30 s, the treadmill slope was then
increased by one degree every 60 s.
DNA extraction and RFLPs analysis. Total DNA extraction and RFLP analysis
were perfonned as previously described (12). Five IJ.g of DNA from each individual was
digested with 20 U of the endonucleases TaqI for AK1M, KpnI for PFKM, and SstI for
COXVa. The digested fragments were separated on agarose gels (0.8% to 1.2%) in TEA
buffer, pH 8.5 (40 mM Tris, 20 mM Na acetate, 20 mM NaCI, and 2 mM EDTA),
denatured in an alkaline solution, and transferred by Southern blotting (32) to
a
nitrocellulose membrane (Schleicher & Schuell). Hybridyzation was perfonned at 42° C in
50% fonnamide as appropriate with specific probes labelled with p32 by random priming
(14) to a specific activity of 1-2 x109 cpm per IJ.g. Filters were then washed and exposed
to X-ray films for 5 days. Autoradiograms were interpreted using phage lambda digested
with Hind III and Eco RI as size standards. The probes used for this study
were:
hAK1B3.25 for AK1, derived from a human genomic clone (lambda hAK1-l) (2); pcox1.11
containing a full length cDNA corresponding to the nuclear encoded subunit Va of human
cytochrome c oxidase (E.C.1.9.3.1) (28); and PCHPFKM1, a 2.86-kb cDNA clone,
containing 76 bp of 5' -unrranslated sequence, 2340bp encoding human muscle PFK, and
399 bp of 3'untranslated sequence plus a poly (A) tract (13). These probes detected 2
alleles RFLPs of 6.4 and 5.5 kb for TaqI-AKIM, 17 and 11 kb for KpnI-PFKM, and 20.7
and 16.8 kb for SstI-COXVa. Allele frequencies were calculated by direct allele counting
for each group and the different genotypes are described in tenns of DNA fragment
restriction size in kb. Subjects for which DNA pattern on autoradiogram was not clearly
identified were excluded. Therefore, the number of subjects for some genes ranged from
33 to 38 for sedentary subjects and from 23 to 25 for athletes.
Statistical analysis. For sedentary subjects, t-tests were perfonned to evaluate the
training effect on V0 max. The \\'0 max training response was adjusted for difference in
2
2
pre-training V02 max and training sites by a multiple regression procedure. A chi-square

---

64
procedure was used to compare the allelic and genotype frequencies between high and low
responders, and athletes and sedentary subjects. An analysis of variance was used to
determine whether the different genotypes observed were associated with the response to
training.
.....,.....
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---

65
RESULTS
The sedentary subjects were the same as those who participated in the study of
Dionne et al. (12). The pre-training V0 max and the effect of the training program on V0
2
2
"
max are shown in table 1. Although the average increases in V0 max were 0.5±O.3 Vmin
2
or 9 ±4 mVmin·kg· 1 but large interindividual differences in the response were observed
ranging from 0.01 to 1.0 Vmin or from 1 to 17 mVmin·kg· 1• All training changes were
significant at P<O.01. The V0 max of the 17 French-Canadian Caucasian cyclists and that
2
of the 8 South-African Caucasian marathon runners were 79±4 and 71±5 mVmin·kg· 1
respectively.
Table 1 about here
The allele frequencies of the 3 genes were then compared between the sedentary
subjects and the athletes (Table 2). No differences were observed in allele frequencies of
the 3 genes between both groups, even though the frequency of the 17 kb KpnI-PFKM
allele was slightly higher among sedentary subjects than in athletes.
Table 2 about here
Table 3 shows genotype frequencies observed in sedentary subjects and athletes. The
3 expected AK1M genotypes were observed in the athletes while there was no subject
homozygotes for the rare 5.5 kb allele in the sedentary group. Genotype distributions for
KpnI-PFKM were not significantly different between both groups. The SstI-COXVa
genotype distributions are noteworthy. Despite comparable allele frequencies, 78% of the
athletes were heterozygous for the 16.8 and 20.7 kb alleles and only 9% were homozygous

---

66
for the 20.7 kb allele, while genotypes were almost equally distributed in the sedentary
subjects. Those distributions are significantly different (P<O.05) between groups.
Table 3 about here
The effect of the genotypes for these 3 genes onpretraining \\102 max and its
response to training are shown in Table 4. V0 max, either in pretraining or in its response
2
to training, did not vary significantly according to the genotypes. In the same way, no
difference was seen between genotypes and \\102 max for athletes (results not shown).
Table 4 about here
To better analyse the contribution of alleles and genotypes to the sensitivity to
training, sedentary subjects were divided in 2 subgroups according to their response to
training adjusted for the pretraining level of V0 max and the training site. Those having
2
improved from 0.5 to 1.0 1/min were designed as high responders and those having
improved less than 0.4 1/min as low responders. The average gain in V0 max with training
2
of low responders was 0.3±O.1 1/min while it was 0.7±O.2 1/min (P<O.OO 1) in
the high
responders. High and low responders were compared on the basis of their allele and
genotype frequency distributions at the 3 loci studied. In Figure 1 panel A, It can be seen
that the 17kb KpnI-PFKM allele was more frequent among the low responders (79% vs
64%) as was the 20.7 kb SstI-COXYa allele (61% vs 50%); these differences were not
significant. As seen in panel B, for each gene, the low responders had a higher frequency
of subjects homozygous for the more frequent allele, while high responders exhibited a
higher frequency of heterozygous for AK1M (47% vs 37%). A higher number of subjects
" ,
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---

67
homozygous for the rare allele of PFKM (17% vs 5%) and for COXVa (27% vs 22%),
although these differences were not significant.
FIGURE 1 about here
The genotype frequencies observed in this study did not differ significantly from the
values expected from the Hardy-Weinberg equilibrium, except for those of athletes at the
COXVa locus which departed strikingly from expected values (Table 2; P<O.OO5).
...
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---

68
DISCUSSION
It is now well documented that the response to endurance training and endurance
,
perfonnance are related to genotype heterogeneity among individuals. The contribution of
genetic components to variation in aerobic perfonnance (8) and response to training (7) has
been an area of investigation in our laboratory over the last decade. Since analyses of
genetic heterogeneity at the protein level did not allow the identification of loci significantly
associated with variability in \\10 max or its trainability and with the status of elite athletes
2
(5,8,9), studies were undertaken to identify directly at the DNA level sequence variations
associated with these phenotypes. The candidate gene approach was selected for this study
(22,37), i.e., an association is established between phenotypes of interest and DNA sequence
variations at gene loci are selected for their potential role on the phenotypes studied.
Results are reported regarding the association between trainability or performance and DNA
sequence polymorphism at 3 gene loci of ATP regeneration pathways.
Enhanced capacity to produce ATP results from muscular adaptations to exercise.
This can be partially attributed to modifications of the activity of enzymes implicated in
different metabolic pathways. The activity of the 3 enzymes selected for this study were
shown to adapt to endurance training. Long term, low frequency electrical stimulation
(which mimics endurance training) induces a reduction in AK activity in rabbit muscle
(16,26). PFK activity decreases (18,36) after endurance training. It seems that exercises of
a greater intensity are required to increase the biosynthesis of some glycolytic enzymes such
as PFK (15,31). Finally, COX exhibits a significant increase in its activity after an
endurance training program (17,39) and after a chronic low frequency electrical stimulation
(34).
Previous studies estimated the heritability of numerous skeletal muscle enzyme
activities. Using data on detennination of heritability for PFKM activity in brothers,
dizygotic twins, and monozygotic sibships (4), it was suggested that genetic factor..v
appeared to account for 25-50% of the total phenotypic variation in the activity of PFKM,

---

69
and of the glycolytic to oxidative activity ratio (PFKJOGDH). To our knowledge, no data
are available on the heritability of AK and COX. However, a very significant familial
ressemblance was observed for oxidative potential, as determined in the variation of the
,
glycolytic to oxidative activity ratio (PFK/OGDH ratio) (4). The heritability was below
50%.
In this study, we looked for the contribution of DNA sequence variations at the gene
loci of the 3 enzymes to the genetically determined interindividual variance observed in
trainability and to the status of endurance athletes. For this purpose, we used sedentary
subjects ex1)ibiting large interindividual differences in their response to endurance training
(27) and endurance athletes of relatively high level based on their V0 max (10,24,25,29).
2
No variation in the AKIM allele and genotype distributions was seen between
sedentary subjects and athletes, and no association was observed between AKIM genotypes
and the V0 max response to training. Based on these results and on previous observations
2
indicating that the AK 1 inherited charge variants (6) did not contribute significantly to
performance and trainability in 295 unrelated sedentary individuals submitted to an
endurance training program, the AKl gene can be excluded as a candidate gene potentially
involved with these 2 phenotypes.
A difference in genotype frequencies for COXVa was found between sedentary
subjects and athletes, suggesting that this marker may be associated with genetic
components which are determinants in the capacity to reach a high performance level.
COXV is believed to be involved in the coupling of electron transpon and in the proton
pumping action (19). The specific function of each subunit is not yet known. COXVa RFLP
may be of physiological significance for the sensitivity to training. Alternatively, however,
this effect may be due to another closely linked gene.
To further analyse the contribution of alleles and of genotypes to performance and
sensitivity to training, allele and genotype distributions were compared in high and low
•
• • • ,~ ~ Or· .:"7 •

---

70
responders to training and in sedentary subjects and athletes. When looking at PFKM-KpnI
allele
distribution, we noticed that the sedentary subjects carrying the 17 kb allele are
mainly found among the low responders; Interestingly enough, the allele frequency observed
~
in athletes was more comparable to the one of the high responders with comparatively
fewer subjects carrying the 17 kb allele. For COXVa, the genotype distributions tended to
be different between high and low responders. Forty six percent of the high respondres were
heterozygotes compared to 33 % for the low responders. Curiously, such a discrepancy was
even more striking when comparing sedentary subjects with athletes, in which group 78%
of the subjects were heterozygotes despite comparable allele frequencies. Thus, COXVa-SstI
genotype distributions for high responders and athletes suggest an advantage of
heterozygocity at this locus for short and long-tenn adaptations to training.
In this study, no association was found between the genotype of these 3 genes and
the pretraining \\'0 max or its response to training. So far, few studies have reported such
2
associations, and the mitochondrial genome could potentially be associated with the
response to training (12). As for nuclear genes, an association was detected between the
muscle creatine kinase locus and the response to training, even though very few subjects
were studied (11).
In conclusion, the results of the present study suggest that heterozygotes for the
COXVa-SstI alleles have an advantage in endurance perfonnance. The present data make
this marker a potential locus for closer analyses on the genetic detenninant of response to
training and athletes status. More studies are also needed to understand the potential role
of the PfKJ.\\.f locus.
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---

71
ACKNOWLEDGMENTS: Probes were generous
donations from Or A. Nakazawa,
University of Calgary and Alberta Children Hospital, Canada, for AK1M; Or A.
McLachlan, Scripps Clinic and Research Foundation, Lajolla, California, for PFK; and E.A
,
Schon, Colombia University, New York for CQXVa. This research was supported by a
grant from NSERC (Canada).
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72
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. " ..-, ..... ..
-:

---

TABLE I:
\\'0 max OF SEDENTARY SUBJECTS AND ENDURANCE ATHLETES
2
PRE-TRAINING
INCREASE IN \\'0 MAX
2
\\'0 max
AFTER TRAINING
2
SUBJECTS
-
-
X ± SD
X ± SD
min
max
SEDENTARY
SUBJECTS
Vmin
3.2 ± 0.2
0.5 ± 0.3§
0.01
1.0
(n=38)
cJ
ml/min -kg-!
43 ± 6
9 ± 4§
1
17
---J
---J
CANADIAN
CYCLISTS
mllmin -kg-!
(n=17)
79 ± 4*
SOUTII-AFRICAN
RUNNERS
ml/kgmin-!
(n=28)
71 ± 5*t
*
Significantly different from sedentary subjects, P ~ 0.05.
t
Significantly different from Canadian cyclists, P ~ 0.05.
§
All training changes are significant at P ~ 0.01.

---

TABLE 2:
COMPARISON OF ALLELE FREQUENCIES BElWEEN SEDENTARY SUBJECTS AND ENDURANCE
ATHLETES
Allele
SEDENTARY
ENDURANCE
Xl.
LOCUS
RFLP
size
SUBJECTS
ATIILETES
(kb)
N
%
N
%
P Level
6.4
60
79
39
78
AKIM
TaqI
0.890 NS
5.5
16
21
11
22
........
(X)
17
53
72
28
58
PFKM
KpnI
0.129 NS
11
21
28
20
42
20.7
37
56
22
48
COXVa
Sstl
0.391 NS
16.8
29
44
24
52
*
Chi-square between sedentary subjects and endurance athletes for allele distributions; NS = non significant

---

TABLE 3:
COMPARISON OF GENOTYPE FREQUENCIES BETWEEN SEDENTARY SUBJECTS AND ENDURANCE
ATHLETES
SEDENTARY
ENDURANCE
X2t
SUBJECTS
ATHLETES
WCUS
RFLP
Genotypes*
N
%
N
%
P Level
- -
6.4:6.4
22
58
15
60
AKIM
TaqI
5.5:6.4
16
42
09
36
0.433 NS
5.5:5.5
-
-
1
4
.•J
......,
17: 17
20
54
10
42
\\.0
PFKM
KpnI
11: 17
13
35
8
33
0.324 NS
11: 11
4
11
6
25
20.7:20.7
12
36
2
9
COXVa
SstI
16.8:20.7
13
39
18
78
0.013§
16.8: 16.8
8
24
3
13
'"
Genotypes are described in tenn of restriction fragments sizes in kb.
t
Chi-square between sedentary subjects and endurance athletes for genotype distributions.
§
Endurance athletes are significantly different from sedentary subjects by a chi-square test (P<0.05); NS = non significant.

---

TABLE 4:
PRE-TRAINING AND V0 max RESPONSE OF \\'0 max TO ENDURANCE TRAINING IN RELATION TO
2
2
GENOTYPES FOR SEDENTARY SUBJECTS
Pre-training
Response to training
\\'0 max
Imin"
2
LOCUS
RFLP
GENOTYPES'"
mllmin 'kg,J
UNADJUSTED
ADJUSTEDt
-
-
-
X
SO
X
SO
X
SD
P Level
6.4:6.4
43.4
6.3
0.44
0.21
0.46
0.07
AKIM
TaqI
6.4:5.5
43.4
5.6
0.51
0.29
0.48
0.02
NS
5.5:5.5
OJ
0
17:17
45.8
5.5
0.40
0.19
0.42
0.02
PFKM
KpnI
17: 11
41.2
6.6
0.50
0.24
0.51
0.03
NS
11 :11
45.8
6.0
0.57
0.36
0.40
0.09
20.7:20.7
43.4
6.23
0.44
0.24
0.50
0.06
COXVa
SstI
20.7:16.8
45.4
7.13
0.50
0.24
0.42
0.02
NS
16.8:16.8
45.7
4.99
0.42
0.22
0.43
0.01
'"
Genotypes are described in term of restriction fragments sizes in kb.
t
Adjusted for pre-training level and for training sites.
ANOVA between the different genotypes and pre-training and response to training of \\'0 max; NS = non significant
2

---

81
ALLELIC DISTRIBlmONS
GENOTYPIC DISTRIBUTIONS
Cl LOW RESPONDERS
11 HIGH RESPONDERS
6.4
5.5
6.4 /6.4
6.4 /5.5
5.5/5.5
(x 2 = 0.574. NS)
(x 2 =0.511, NS)
TaqI- AK1M ALLELES (kb)
AK1M GENOTYPES (kb)
17
11
17/17
17/11
11/11
(x 2 = 0.129.NS)
(x 2 = 0.326.NS)
KpnI-PFKM ALLELES (kb)
PFKM GENOTYPES (kb)
20.7
16.8
20.7120.7
20.7/16.8
16.8/16.8
(x 2 = 0.365. NS)
(x 2 =0564, NS)
Ssu-COX Va ALLELES (kb)
COX Va GEN01YPES (kb)
Figure 1.
TaqI-AKIM,
KpnI-PFKM
and
SstI-COXVa
allelic
and
genotypic
distributions in sedentary subjects subdivided into low and high responders
to endurance training, NS= non significant.
'"
...... \\ ..;~.:-~.

---

CHAPITRE VI
POLYMORPHISME DE L' ADN MITOCHONDRIAL ET LA \\'0 MAX CHEZ LES
2
ATHLETES D'ENDURANCE.
Le but de cette etude etait de comparer la variation de sequence de l' ADN mitochondrial
(ADNmt) des athletes d'endurance a celle des sujets sedentaires. Soixante dix athletes
composes de 31 hommes et 22 femmes coureurs de longue distance et de 17 cyclistes
canadiens ont pris pan acette etude. Cinq enzymes de restriction (BarnHI, flincH, KpnI,
MspI et NciD ont ete utilisees pour la digestion de l' ADN. Ces enzymes avaient
prealablement servi a l'identification des variants de l' ADNmt associes a une faible ou a
une bonne reponse de la \\'0 max a l'entrainement (Dionne et al., 1991). Les frequences
2
des patrons d' ADN ou morphes des athletes d'endurance on ete comparees a celles de 46
sedentaires. Les variations de sequence de l' ARN de transfert de la threonine detectee avec
l'enzyme MspI (MTT-MspI) et de la sous-unite 5 de la NADH des hydrogenase detectee
avec l'enzyme NciI (MTND5-NciI) etaient observees a une plus haute frequence chez les
athletes (16% vs 6%, P<O.05). Ces variants avaient ete prealablement associes a une \\'02
max de preentrainement elevee. La variation dans la region D-loop de l'ADN mt detectee
avec 1'enzyme KpnI (D-loop-KpnI), prealablement associee a une bonne reponse du \\'02
max, etait 5 fois plus frequent chez les athletes comparativement aux sedentaires (28.5%
vs 6%, P<O.05). Ces resultats suggerent que certains variants de l' ADNmt, prealablement
associes a une \\'0 max de preentrainement elevee et a une benne reponse de la \\'0 max,
2
2
etaient observes a une plus haute frequence chez les athletes d'endurance. Des analyses
complementaires sur un plus grand nombre d'athletes sont presentement en cours. Les
nouvelles donnees seront ajoutees acelles-ei avant publication.

---

83
MITOCHONDRIAL DNA SEQUENCE POLYMORPHISM AND \\TOl max IN
ENDURANCE ATHLETES.
Suzanne A. Adjoa, Marie-Claude Vohl, Pieter Coetze~, Timothy D. Noakes2, Marcel R.
Boulay, lames S. Skinner', Claude Bouchard, and France T: Dionne*
Physical Activity Sciences Laboratory, PEPS, Laval University, Ste-Foy, Quebec, Canada,
G lK 7P4, 2MRC/MCf Bioenergetics of Exercise Research Unit, Deparunent of Physiology,
University of Cape Town, Cape Town, South-Africa, and 3Department of Kinesiology,
Indiana University, Bloomington, IN.
TO BE SUBMITTED TO: Med. Sci. Spons Exerc.
Running title: MtDNA variants and V02 max in athletes.
" .
..' ~........~ '.''';'.

---

· ....... '.",
;~.....
84
ABSTRACT
The purpose of this study was to compare mitochondrial DNA (mtDNA) sequence variation
frequencies of endurance athletes 10 sedentary subjects. Seventy endurance athletes
including 31 male and 22 female long distance runners and 17 male cyclists took part in
this study. Five restriction endonucleases (BamHI, HindI, KpnI, MspI and Ncil) were used
for digestion of purified human genomic DNA. These enzymes were previously shown to
detect mtDNA variants associated with low or high response to training (12). Morph
frequencies of endurance athletes were compared to those of 46 sedentary subjects of
reference. Variation in the tRNA for threonine detected with MspI (MTIT-Mspl) and in
the subunit 5 of NADH dehydrogenase detected with NciI (MTND5-NciI) were observed
in athletes at a higher frequency than in the sedentary subjects (16% vs 6%; P:50.05). These
variants were previously reported to be associated to a high \\'0 max in untrained subjects.
2
A D-loop-KpnI variant, previously reported to be associated with a high response to
training, was 5 times more frequent in athletes (28.5% vs 6%; P:50.05) than in sedentary
subjects. These results suggest that selected mtDNA sequence variants, previously reported
to be associated with high \\'0 max in sedentary people or high response to training, are
2
observed at a higher frequency in endurance athletes.
Analyses are curently underway,with a larger number of athletes and those data will
be added to these before publication.
KEY WORDS: MTDNA VARIANTS, RFLPS, \\'0 max, ATHLETES
2
. ,; ..... ....
'
-..-.:..
.....
' -
:.; .... "":".

---

85
INTRODUCTION
Endurance athletes exhibit "3. high maximal aerobic power. For instance, elite
runnners have a \\'0 max between 70 and 85 mllmin·kg-l (6,26,29). The high \\'0 max
2
2
values of elite athletes partly depend on the potential of mitochondria to use substrates
during exercise for ATP regeneration. Individual differences in \\'0 max and in several
2
skeletal
muscle
enzyme
activities
have
been
shown
to
be
genotype
dependent
(14,19,22,24,25,30). A slight maternal effect on \\'0 max (21,25) has also been reported
2
suggesting a potential role for the mitochondrial DNA genome on \\'0 max.
2
A genetic effect will ultimately imply variation in DNA sequences which may result
in polymorphism of proteins or differences in gene expression. No charge variants were
detected in 11 enzymes of the glycolytic pathway and 9 enzymes of the tricarboxylic acid
cycle of the skeletal muscle (3,23). Similarly, studies on genes and red blood cell genetic
variation (5,7) in Olympic endurance athletes and controls did not reveal any association
between these markers and the status of high performance athletes. However, one
investigation on mtDNA sequence variations (10) suggested an association between some
mtDNA sequence variants and individual differences in \\'0 max and in its response to
2
training in sedentary subjects after endurance training.
" .
, . . . . . , '.~'. 0;- I

---

86
MtDNA codes for subunit I, 11, ITI of cytochrome c oxidase, subunit 6 and 8 of the
ATPase synthetase complex, a subunit of the cytochrome be l-eomplex, seven subunits of
NADH dehydrogenase (38) and for trai'lsfer and ribosomal RNAs. In addition, mtDNA has
a D-loop region which contains the site of replication and transcription and most of its
sequences serve a coding function (1).
In the present study, we tested the hypothesis that mtDNA variants previously
reported to be associated and with a high \\102 max in the untrained state and a high
response to endurance training (10) may also be associated with endurance athlete status.
For this purpose, we compared the frequencies of mtDNA variants between 70 endurance
athletes and 46 sedentary subjects (l0).
.... ..
.....
"~'
,'.: ,
." •• '.~', .~. -;0-;.

---

87
SUBJECTS AND METHODS
Ten sedentary male subjects from Quebec City, aged 17 to 27 years, and 28
sedentary men from Tempe, Arizona aged 24 to 29 years, took pan in this study. They had
no history of regular participation in sports nor previous engagement in an exercise training
program. These subjects are those of the Dionne et al. study (10). The group of endurance
athletes consisted of seventeen highly trained male cyclists (22 to 35 years old) from
Canada and 20 Blacks (2 females and 18 males) and 33 Caucasians (20 females and 13
males) aged 18 to 32 years, from Cape Town, South-Africa (SA). All subjects had been
engaged in regular training for several years, and provided informed written consent, prior
to their participation in this study.
VOz max determination. The \\'Oz max of the sedentary subjects was determined
on an electromagnetically braked ergocycle. After an initial 3 min period at 50 W, the
power output in Quebec was increased every 3 min by 25 W until volitional exhaustion;
rpm was maintained at about 60 throughout the test In Arizona, the test for \\'Oz max was
initiated at 80 W, with increments of 20 to 30 W every 2 min until exhaustion. Respiratory
parameters eve, \\'02, \\,COz) were continuously monitored using automated open-circuit
systems (Beckrnan Metabolic Measurement Cart). Heart rate was monitored with an ECG.
The \\'Oz max of FC athletes was determined on a cycle ergometer (Monark-
Crescent, Varberg, Sweden) with increasing load of 25 W for a maximum of 14 to 17 min.
Q

---

..,
88
The test was initiated at 100 W. Within SA athletes, 8 of them had their \\f02 max
measured while running on a treadmill (Quinton Instruments). They began running at a
velocity of 17km/h, with increments "Of 0.5 km/h every 30 s until exhaustion. If the peak
speed of the treadmill, which in this case was 25 km/h, could be maintained for 30 s, the
treadmill slope was then increased by one degree every 60 s.
The remaining subjects were long-distance runners who participated in the 1990
South Africa National 15 km championship event. Their \\f02 max was estimated through
their personal best times in the range of 43'02 to 01 h 03' 13, which reflected their
respective qualifying time for 15 km. Briefly, the percentage of \\'0 max required for lkm
2
run was calculated from their respective running performance. The corresponding \\'0 max
2
was then predicted from equivalent percent of \\'0 max (20,28).
2
DNA extraction and RFLP analysis. Total DNA extraction and RFLP analysis
were performed as previously described (l0). One g of total DNA was digested with 20
U of the following endonuc1eases: BamHI, HindI, KpnI, MspI, and NciI. These enzymes
were selected because they were shown to be associated with interindividual differences
observed in response to an endurance training (10). The digested fragments were separated
on an agarose gel (1.2% to 2%) in TEA buffer, pH 8.5 (40 mM Tris, 20 mM Na acetate,
20 mM NaCl, and 2 mM EDTA) , denatured in an alkaline solution, and transferred by
Southern blotting (38) to a nitrocellulose membrane (Schleicher & Schuell). Hybridyzations
were performed at 42° C in 50% formamide as appropriate. MtDNA was labelled with p32

---

89
by random priming (12) to a specific activity of 1-2 x109 cpm per IJ.g. Filters were then
washed and exposed to X-ray films for 1 day. Autoradiograms were interpreted using
molecular weight markers (phage lambda HindIIIJEcoRI) as size standards. Assignment of
mtDNA morphs (variants) and the region of mutation was established as reported by
Dionne et al. (10).
Statistical analysis. A t-test was perfonned to compare \\'0 max between groups.
2
A chi square test was used to compare the frequencies of each mtDNA morph between
sedentary subjects and athletes.

---

90
RESULTS AND DISCUSSION
Endurance training program can result in increased \\10 max of 15% or more. Thus,
2
despite prolonged and high-intensity endurance training, average healthy individuals will
never achieve \\10 max values near those of elite athletes (26). Improving \\10 max over
2
2
15% is related to inherited influence (26). In the present study, the \\10 max of female and
2
male athletes (Table 1) was significantly different from that of sedentary subjects who were
endurance trained (PS 0.05). As expected, the \\10 max of males was also greater (P~ 0.05)
2
than that of female athletes. Published data indicate \\10 max values below or equal to 60
2
mUmin·kg-\\ for elite female distance runners (11,15,27,31). In this study, \\10 max values
2
are in the range of those previously reported for elite male (16,26,29) and female
(8,16,24,35,36) endurance athletes.
TABLE 1 HERE
Performance in endurance event such as marathon running, results from individual
\\102 max, the fractional utilisation of \\10 max expressed as percent of \\10 max, and the
2
2
energy cost of running or running economy which determines the efficiency of oxygen
utilization (11). A goexi correlation has been found between \\10 max and performance in
2
marathon running (24)in a heterogenous group. Female and male top runners exhibited
......
.
' . ,
, .. ~.:.;'
.• ",
• • • • ,0\\ .••.:-:",

---

91
similar fractional utilisation of \\10 max (8,24) and this was correlated with perfonnance
2
as was the energy cost of running in marathon and ultramarathon runners. Davies and
Thompson (8) reponed that, the running economy on a motor driven treadmill, when
standardized for body weight, was the same in male and female athletes. Futhennore, no
racial difference was observed in a study of elite runners of different racial groups in
running economy (26). On this basis, for our analysis, data od male and female athletes
were combined even though their \\10 max was different
2
An association between mtDNA variants and exercise-training-induced changes in
\\10 max in sedentary subjects submitted to an endurance training program, have been
2
observed by Dionne et al.(ll). Carriers of the MTND5-BamHI morph 3, MITT-MspI
morph 4, and MTDN5-NciI morph 2 had a higher untrained \\10 max. Those carrying the
2
D-loop-KpnI morph 1 had a higher unadjusted training respose to V0 max. Futhennore,
2
low responders to training were carriers of the MTND5-HincII morph 1. In the present
study, we were interested to compare these results to those found in endurance athletes.
Since our group of athletes was composed of Blacks and Caucasians and of females
and males, we first look at the distribution of the mtDNA variants among our athletes. As
seen in table 2, none of the South-African Blacks were carrying the M1TT-MspI and
NTND5-NciI mtDNA variants. These results were not surprising since it is known that
Blacks carry some mtDNA polymorphism distinct from that of Caucasians (9,13,27). The
MspI morph 3 is rare to absent in Bantu, Senegalese, and Africans (18,32). To our

---

92
knowledge, there is no report of the presence of Ncil morph 2 in SA Blacks. The MTIT-
Mspl and MTND5-Ncil variants were evenly distributed among the other subgroups. As for
the KpnI variant only 6% (3/46) oCFC Caucasians exhibited this variant, while it was
almost equally distributed between SA Blacks (44%; 8118) and SA Caucasians (38%; 9/26).
About 43% (9/21) of the females typed were carrying this mtDNA variant while 34% (8/23)
of the SA males or 23% (9/39) of all males presented it. The MTND5-BamHI morph 3 and
Hindl morph 2 were not found in any of the subgroup of athletes. BamHI morph 3 was
absent in Bantu, Senegalese, and Bushmen (18,32), while it was detected in Caucasians
(18,32) at a frequency of about 6%. There is no report of the presence of Hindl morph 1
in SA Blacks but this morph has been reponed to be rare in Senegalese (32).
Figure 1 presents the frequencies of these mtDNA morphs among sedentary subjects
and endurance-trained athletes. None of the athletes of this study carry the MTND5-BamHI
previously reported to be associated with a high \\'0 max in the untrained state. However,
2
the two remaining morphs, also associated with a high \\'0 max in the untrained state, were
2
found at a higher frequency in athletes. The percentages of MTIT-Mspl and the MTND5-
NciI morphs were significantly higher (16% vs 6% in both cases; P~.05) in endurance
athletes than in control sedentary subjects. Of the 19 athletes carrying these morphs, 42%
of them exhibited both morphs together. None of these morphs were seen in SA Blacks.
Investigations on mtDNA polymorphism in general population, showed a MTIT-Mspl
morph frequencies of 2%, and 8% in Finnish and other Caucasians respectively (37,18). It
.• ". ,. 7'~ ; :;-';":".

---

'..-
-. ~
· · ' / ;
1 ..~.
93
is noteworthy that this morph had a frequency 2 to 8 times higher in athletes than what is
commonly found in Caucasian populations.
Interrestingly, none of the athletes exhibit the MTND5-HincII morph, previously
reported to be associated to a low response to endurance training (Figure 1). This confmns
earlier observation that the MTND5-HincII is associated to a low responder status (10).
The D-loop-KpnI morph previously reported to be associated with high unadjusted
response to training, was almost five times more frequent in athletes than in control
sedentary subjects (28.5% vs 6%; P:$;0.05) (Figure 1).
FIGURE 1 HERE
In athletes, a low \\'0 max can be compensated by a good running economy (33).
2
In this study, the majority of the carriers of the D-loop-KpnI morph were females.
Moreover, three female athletes having \\'0 max in the lower range (58.6, 59.5, and 60
2
mVminokg- l ) were carriers of this morph. This mtDNA variant is located in the D-Loop, a
region containing the site of replication and transcription of the mtDNA genome. The effect
of KpnI variant on mtDNA expression is still unknown. Futher studies will be necessary

---

94
to assess its relation with determinants of marathon running such as the energy cost of
running or running economy.
In conclusion, these results suggest that some mtDNA sequence variations previously
reported to be associated to high untrained VG max and high unadjusted response to
2
training can also be related to the status of athletes.

---

95
AKNOWLEDGMENTS: The authors are grateful to Or Denis Prud'homme and Richard
Chouinard for the elite athletes '10 max estimation.
2
..
i. "~ ; ... ~.:.: •

---

h.'
96
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101
31.
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.,
1989.
32.
Scorazzi, R., A. Torroni, O. Semino, A. Brega, and A.S. Santachiara-Benereecetti.
Genetic studies on the Senegalese population. I Mitochondrial DNA polymorphism.
Am. 1. Hum. Genet. 43:534-544, 1988.
33.
Sj&l.in, B. and J. Svendenhag. Physiology of marathon running.Track Technique.
94: 3004-03009, 1984.
34.
Southern, E.M. Detection of sequence among DNA fragments separated by gel
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Tokmakidis, S. P., A. Tsopanakis, E. Tsarouchas, and V. Klissouras. Physiological
profIle of elite athletes to maximal effort. In: Sport and Elite Perfonners. D. M.
Landers (ed.) Human Kinetics, Champaign, Illinois. 1984, pp.177-184.
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Wilmore, J. H. and C. H. Brown. Physical and physiological profIle of champion
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102
37.
Yilkki, 1., M. L. Savontaus, and E. K. Nikolaisen. Human mitochondrial DNA types
in Finland. Hum. Genet. 80:317-321, 1988.
38.
Wallace, D. C. Repon of the committee on human mitochondrial DNA Cytogenet.
Cell Genet. 51:612-621, 1989.

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103
TABLE 1: V02 MAX FOR SEDENTARY MALE SUBJECTS AND ENDURANCE
ATHLETES
SUBJECTS
-
N
X±SD
SEDENTARY MALE SUBJECfS
46
43.7 ± 6.3
FEMALE ATHLETES
22
63.2 ± 3.7 *
MALE ATHLETES
48
74.3 ± 5.1 *t
*
Significantly different from values of sedentary subjects, P:$O.05
t
Significantly different from values of female athletes, P:$O.05

---

.-' ,....,:...,.:
TABLE 2: MTDNA VARIANT DISTRIBUTIONS AMONG THE ENDURANCE TRAINED ATHLETES
SUBJECTS CARRYING mtDNA VARIANTS
SUBJECTS
MTND5-
MTTT-
MTDN5-
MTND5-
D-loop-
BamHI
MspI
NciI
HinclI
KpnI
n/n *
n/n *
%
n/n *
%
n/n*
n/n *
%
SOUTH AFRICAN
..,
Blacks
(n)
Female
(2)
0/2
0/2
0
0/1
0
o
0/1
2/2
100
-Po
Males
(18)
0/18
0/18
0
0/18
0
0/10
6/16
37.5
Caucasians
Female
(20)
0/20
4/19
21
2/18
11
0/18
7/19
37
Males
(13)
0/13
2/10
20
3/10
30
0/18
2n
28.5
FRENCH CANADIANS
Males
(18)
0/17
4/17
23.5
4/17
23.5
0/17
1/16
6
n/n*:
Number of subjects canying the morph over the number of subjects which could be typed for this variant

---

105
10-_ _
1CONTROL SEDENTARY SUBJECTS
30
__7_ 1ENDURANCE ATHLE~ES
*
25
*
*
MTND5-BarnHI
MTIT-MspI
MfND5-NciI
MrND5-HincII
D-Loop-Kpn I
Figure 1:
Frequencies of mtDNA variants in control sedentary subjects (12) and
athletes (n= 67 for MTND5-BamHI; 61 for MTIT-MspI; 57 for MTND5-
NciI; 57 for MTND5-HincII; and 63 for D-Loop-KpnI). Athletes were
significantly different from sedentary subjects, ~O.05.

---

CHAPITRE VII
RELAnON ENTRE LE POLYMORPHISME DE LA PHOSPHOGLUCOMUTASE-I, LA
1
PERFORMANCE AEROBIE ET LA REPONSE A L'ENTRAINEMENT
La puissance aerobie maximale (\\T0 max) et la performance en endurance (EP) sont
2
caracterisees par une grande variabilite interindividuelle. Cette hererogeneire est en partie
due ades differences genetiques. La disponibilite des substpts dans le muscle squelettique
et dans le sang est un determinant important dans les variations de la \\'0 max et de la EP.
2
11 a ete egalement rapporte que les changements de l'activite des enzymes suite a un
enrrainement sont en partie genetiquement determines. Le but de cette etude etait d'etablir
la relation du polymorphisme de la phosphoglucomutase-l musculaire (PGMl) avec la
performance et la reponse de la \\'0 max al'entrainement. Cinquante huit sujets des deux
2
sexes (16-31 ans) oot ete soumis aun enrrainement de 15 ou 20 semaines. La \\'0 max et
2
la EP evaluee par un test de 90 minutes (Boulay et al., 1984) ont ete testees sur des
ergocycles et les donnees ont ete ajustees pour le niveau de preentrainement, le sexe et les
programmes d'enrrainement. L'homogenat des exO"aits de muscle a ete soumis aux
procedures de la focalisation is06lecrrophoretique (IEF) pour la determination des genotypes
de la PGM 1. Les 2 entrainements ont significativement ameliore de fa90n similaire la '\\'02
max et la EP (P<O.OI). Des associations ont ete observees enrre les genotypes 1-1- ou 1+2-
et une faible EP (P=O.057). De plus, les sujets porteurs des genotypes 1+2- ou 1+2+ de la
PGMl, avaient une bonne reponse de la \\'0 max a l'enrrainement (P<O.OI). Ces resultats
2
suggerent que les differences de genotypes sont reliees aux variations individuelles
observees en reponse a l'enrrainemeot et a la performance.

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107
PHOSPHOGLUCOMUTASE-l POLYMORPHISM AND RELATIONSHIP WITH
AEROBIC PERFORMANCE AND RESPONSE TO EXERCISE TRAINING
Suzanne A. Adjoa, Jean-Aime Simoneau, Monique Chagnon, France T. Dionne, and Claude
Bouchard*.
Physical Activity Sciences Laboratory, PEPS, Laval University, Ste-Foy, Quebec, G1K 7P4,
Canada
*ADDRESS CORRESPONDENCE TO:
Claude Bouchard, Ph.D.
Physical Activity Sciences
Laboratory
PEPS, Lava! University,
Ste-Foy, Quebec, Canada
G1K 7P4
Tel: (418) 656-5174
Fax: (418) 656-3044
Running title: Genetic variation, perfonnance and trainability

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108
ABSTRACT. Maximal aerobic power (V0 max) and endurance performance (EP) are
2
characterized by large interindividual variations. This heterogeneity is in part due to genetic
differences. Substrate availability in muscle and blood is important to consider for the
'1
understanding of variation in V0 max and EP. It has also been reponed that alteration in
2
muscle enzyme activities induced by training is partly genetically determined. The purpose
of this study was to investigate the relationship between genetic variants of skeletal muscle
phosphoglucomutase-l (PGMl) and V0 max and EP. Fifty eight sedentary subjects of both
2
sexes (16-31 years of age) were submitted to 15 weeks or 20 weeks of exercise training.
V0 max and EP were assessed on cycle ergometers and data were adjusted for pre-training
2
level, gender, and training programs. Muscle biopsies were taken from the vastus lateralis
and homogenates were subjected to isoelectric focusing (IEF) procedures and five PGMl
genotypes were visually determined. Both training programs resulted in
significant and
similar improvements (P < 0.01) of V0 max and EP. Associations were found between
2
PGMl 1-1- or 1+2- genotypes and EP (P=0.057). Among the five genotypes recovered in
this study, the carriers of PGMl 1+2- or 1+2+ genotypes had significantly (P < 0.01) higher
V0 max response to training. These results suggest that genotypic differences in the PGMl
2
gene are related to the interindividual variation in V0 max response to training.
2
KEY WORDS: GENETIC VARIATION, TRAINABILITY, ENDURANCE, V0 max.
2
.
..
, ..,•...; ...;.:

---

109
INTRODUCTION
There is evidence that genetic factors contribute to variation in aerobic perfonnance
,
level and its response to training. Subjects submitted to the same standardized training
program do not exhibit the same response to training. Some subjects are high responders
to training while little or no changes are seen in others (3,19,23). This heterogeneity in
trainability has been shown to be related to the genotype (4,8,23) suggesting that the
sensitivity to training is largely inherited. Among the poten~al factors contributing to the
changes in V0 max in response to training are the concomitant changes in skeletal muscle
2
characteristics, particularly those in the level of enzymes regulating different metabolic
pathways. Training induced changes in muscle enzyme activities have also been reponed
to be genetically detennined (16,25). To define the genetic basis of individual differences
in response to training, earlier studies from our laboratory have considered the associations
between inherited variations in proteins (11,12,20) or DNA sequence ( 13,14) for key
enzymes of the energy regeneration pathways and aerobic perfonnance or response to
training. The present study was undertaken to extend this work previously reviewed (11,12).
The PGM1 is expressed in red blood cells (21) and other tissue and is involved in the
process of carbohydrate catabolism. The purpose of this study was to verify the contribution
of the established polymorphism of one glycolytic enzyme, namely PGM1 (E.C.2.7.5.1)
(10,21,27,28) to the variability in the response to training of V0 max and EP.
2

---

110
METHODS
Subjects: Fifty-eight healthy and biologically unrelated sedentary subjects of both
genders (26 women and 32 men) took part in this study. Their age ranged from 16 to 31
years and none of the subjects had history of regular participation in physical activities. All
procedures were approved by the Ethics committee of Laval University and each subject
gave informed consent before participating in the research protocol.
The training programs. Among the 58 subjects, twenty underwent a 15-week
ergocycle training program, as described by Simoneau et al. (25). This program involved
both continuous and interval work patterns. Twenty-five 30-minute sessions of continuous
exercise at 70% of the maximal heart rate reserve, and 19 short- and 16 long-interval
exercise sessions were distributed in the program so that half the continuous sessions were
completed by the 5th week of training. No interval work bout was performed before the 4th
week of the training program. The short- and long-interval sessions consisted of 10 to 15
bouts of 15-30s cycling exercise and or 4 to 5 bouts of 6O-90s cycling exercise with
interspaced resting periods allowing the heart rate to return to 120-130 bts/min. The short
exercise bouts were initially performed at an intensity of 60% of the individual maximal
work output in lOs and the long exercise bouts were started at an intensity of 70% of the
individual maximal work output in 90s. The intensity of the short- and long-term intervals
was increased by 5% every 5 weeks. Each training session was fully monitored by trained
personnel assuring that all subjects had to the same standardized training stimulus.

---

111
The other thirty-eight subjects undetwent 20 weeks of endurance exercise involving
4 times and increasing to 5 times a week. They did 40 to 45 min of cycling exercise
starting at 60% and increasing to 85% of the heart rate reserve (19,23). Again, the training
sessions were supervised so that each subject had the same standardized training stimulus.
The V0 rnax test. The test was conducted on an electromagnetically braked cycle
2
ergometer. The initial power output was 50 W, with increments of 20 W for women and
25 W men were done every 3 min until exhaustion. Expired air was collected and
monitored continuously with an automated open-circuit system (Beckman Metabolic
Measurement Cart) to obtain measures of "02' "C02 and YE (23). These ventilatory
parameters were used to established the ventilatory anaerobic threshold, as described
previously (30). Gas analysers were calibrated before and after each test with a known
mixture of gases. The highest oxygen uptake in one minute during the test was recorded
as the "02 max.
The endurance perfonnance test. EP was measured as the total power output
accomplished during a 9O-min maximal ergocycle test, a test which has proven to be highly
reliable (9). The test was performed on a modified Monark ergocycle (25). A potentiometer
was connected to the work load setting gear and a photoelectric cell computed each
revolution of the flyweel. These were connected to a microprocessor, which integrated both
electric signals and transformed them in total work performed (kJ).
.... , ..... ;.;v.:.:·,

---

112
The starting work load for the EP test was calculated from ventilatory parameters
recorded during the \\10 max test. Each subject had to maintain an exercise intensity
2
eliciting a heart rate approximativeIy 10 beats lower than that observed ar the ventilatory
anaerobic threshold. This work load was adjusted when necessary throughout the test to
maintain the highest intensity sustainable by the subject. Subjects were advised to pedal at
60 rpm and were also verbally encouraged to maintain the highest possible power output.
Muscle biopsy and isoelectrofocusing analysis (IEF). An muscle biopsy was
obtained by the percutaneous needle biopsy technique in the middle of the vastus lateralis,
14 cm above the patella and approximately 2 cm away from the epimysium. Pieces of
tissue were kept at -80 QC until processed. Tissue samples were then homogenized into 30
volumes (w/v) of 50 mM phosphate buffer, pH 7.5 with a Duall grinder. The 1500 x g (30
min, 4 QC) supematants were frozen at -80
QC and used as enzyme extracts for
electromorph determinations. The IEF was performed with an Uln-aphor Elecn-ofocusing
Unit (2217-LKB). Samples were loaded on ultrathin-layer polyacrylamide isoelectric
focusing gels prepared according to Radola (24). Anolyte was 0.5% H P0 and catholyte,
3
4
2% ethylenediamine, respectively, and distance between wiks was 10 cm. Gels were prerun
at 3 W for 35 min at 10 QC, and samples were then applied 1 cm from the anode. PGMl
was focalized for more than 45 min at a maximum of 1826 V in a pH gradient of 4-7. The
fonnazan technique (15) was used for the visualization of PGMl genotypes. At least 5
variants of PGM1 were established according to the nomenclature defined by Sutton (29).

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113
Data analyses. The effect of training on V0 max and BP was assessed by an
2
analysis of variance. Responses to training expressed as changes in V0 max or EP were
2
adjusted for gender, training program~ and pre-training \\'0 max by multiple regression
2
procedures. The V0 max and EP residual scores were calculated by subtracting the
2
predicted scores from the original scores. ANOVA followed by a Duncan Multiple Range
test was used to test for the relationships between PGM 1 genotypes and the residual scores
of \\'0 max and EP.
2

---

114
RESULTS
Table 1 shows the physical"and physiological characteristics of the whole group.
"0
Subjects of the two different training programs were pooled as their
max responses
2
were in the same range. Training significantly improved "0 max but a wide range of
2
change was observed from 0 to 1 IImin in response to training (P ~.OOl). A significant
increase (P ~O.OOl) in EP in response to training was also observed, with training gains
ranging from 0.4 to 8.1 kJ/kg.
TABLE 1 ABOUT HERE
Figure 1 illustrates the schematic representation of muscle PGM1 migration pattern.
This IEF pattern shows the four alleles and the five PGMl genotypes identified in our
study.
FIGURE 1 ABOUT HERE

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115
Changes in EP in relation to PGMl genotypes are shown in figure 2. The response
of EP of PGM 1 1-1- or 1+2- genotypes were significantly different from the other 3 groups
,
(P~.057). The 1-1- or the 1+2- genotype gained respectively 0.4 and 1.6 kJ/kg lower than
the other 3 groups.
FIGURE 2 ABOUT HERE
Figure 3 presents changes in \\10 max in relation to PGM1 genotypes. The PGM1
2
1+2~ and 1+2+ genotypes were significantly different from the 3 other groups (P < 0.01).
They had respective \\10 max gain of about 0.3 Vmin higher than those of the 3 other
2
genotypes. However, subjects with the 1+2- genotype were not significantly different from
those with the 1-2- genotype since there was a \\10 max gain difference of only 0.2 Vmin
2
between them.
FIGURE 3 ABOUT HERE
.""

---

116
DISCUSSION
"02 max and EP are clearly iqfluenced by several weeks of exercise training but
notwithstanding these recognized changes, there is a wide range of individual response to
training (4,19). In the present study, subjects considered as a group significantly improved
their "02 max and EP but the observed range of responses again confirms the heterogeneity
of trainability. This variation appears to be partly due to the genetic endowment of the
individual, even though little is known about the genetic factors inherited (4,19,23).
One study suggested that mitochondrial DNA sequence variation may contribute to
individual differences in "02 max and in its response to training (14). In that particular
study, low responders to training had a sequence variation in the subunit 5 of the
mitochondrial NADH dehydrogenase (MTND5) gene detected with the restriction enzyme
HincI!. Moreover, subjects with sequence variation at the MTND5 gene detected with NciI
and BamHI, and at the threonine tRNA detected with MspI, had a "02 max significantly
higher in the untrained state than non carriers (14).
The activity level of skeletal muscle enzymes of different metabolic pathways can
be significantly altered in response to training (17,18,26) and they are undoubtedly
important detenninants for "02 max and BP (4,18,22,30). Changes in enzyme activities
following exercise training have also been shown to be associated with the genotype
(16,25). Results obtained up to now from earlier investigations on 11 enzymes of the

---

117
glycolytic pathway and 9 of the tricarboxylic acid cycle revealed that variation in these
gene products is very rare and thus unlikely to account for individuality of aerobic
perfonnance or trainability (5,11,lt,20).
In the present study, the contribution of PGMl polymorphism to the response to
training of "02 max and EP was investigated. PGMl is encoded on chromosome Ip31 (2)
and exhibits at least 4 alleles at the protein level (21). Ten genotypes have been described
(1,28). In this study, five of them were recovered and the PGMl 1-1- and 1+2- were
associated with low response to aerobic perfonnance. Moreover, the PGMl 1+2- and 1+2+
genotypes were found to be significantly associated with a high response to training. It has
been reponed that perfonnance is a more trainable phenotype than \\'0 max (4). However,
2
in the present study, carriers of PGMl 1+2- genotype exhibited an higher improvement of
their \\'0 max. When we verified the contribution of each allele to the response to training
2
of \\'0
max, we found that high and low responses to training of \\'0
max were
2
2
respectively associated with the PGM 1 1+ and 1- alleles. Interrestingly, when looking at
figure 3, we can note that the combination of 1+ allele with a 2- or a 2+ allele is associated
with an even higher \\'0 max response. In contrast, the combination of the 1- allele with
2
a 1-, a 2- or a 2+ allele translate into a lower \\'0 max response. These results are
2
compatible with the notion that the PGMl 1+2- and 1+2+ genotypes exhibit a higher \\'02
max trainability. The PGMl is an enzyme that catalyzes the reversible reaction of
phosphate transfer from glucose I-phosphate to glucose 6-phosphate. However, it cannot

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118
be excluded that the associations observed in this study could also be due to other genes
in linkage desequilibrium with the PGMl locus.
In summary, our results suggest that PGM 1 genotypes contribute to adaptability to
training. To our knowledge, this is the fIrst report indicating that variation in gene products
may be used to detect an association with the response to ,training. Further studies on the
role of PGMl enzyme and its genetic variation on response to training are warranted.
/

---

119
AKNOWLEDGEMENTS: This work was supported by a grant from NSERC (Canada).

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120
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125
TABLE 1: PHYSICAL CHARACTERISTICS OF THE SUBJECTS (n=58) BEFORE AND
AFTER TRAINING
VARIABLES
PRE-TRAINING
POST-TRAINING
CHANGES
X±SD
MIN
MAX
X±SD
MIN
MAX
X±SD
MIN
MAX
AGE
(YEARS)
24±4.2
16
31
BODY WEIGHT
(kg)
63±13
45
98
62±11
44
91
-1±3*
-11
4
BMI
(kg/m2 )
22A±3
17
31
22.1±2A
18
29
3±1.0*
-3.7
1.3
V0 max
2
(l/rnin)
2.6±O.7
1.1
3.7
3.1±O.8
2
4.2
0.5±O.3*
0.01
(mlJrnin.kg-t
41±8
22
56
50±6
36
62
9±4*
2
20
)
EP
(kJ)
630±167
343
1014
840±199
537
1161
209±121 *
-25
516
(kJ/kg)
1O.1±2.2
6.1
14.9
13.5±2.3
9.2
21
3.3±1.8*
OA
8.1
* ALL TRAINING CHANGES ARE SIGNIFICANT; P:s 0.001

---

126
+ ANODE
A
2+
e,s
....
2 -
M
1+
t
1 -
A
B
c
o
E
- CATHODE
Figure 1:
Schematic representation of muscle PGM1 migration pattern. A= 1-1-; B=
1-2-; C= 1-2+; D= 1+2-; E= 1+2+

---

127
5
--..
0.0
~
4 -
T
h
*
T
~
-r
' - '
T
0...
3
r.r.:l
*
I=:
.-
T
-
CI.l
2
~
0.0
I=:
C\\l
1-
..c
U
0
1-1-
1-2-
1-2+
1+2-
1+2+
PGM1
(n=23)
(n=15)
(n=6)
(n=4)
(n=4)
Genotypes
Figure 2:
Training response of endurance perfonnance (EP) in relation to PGM 1
genotypes. ANOVA across the 5 genotypes, F=2.47, P=O.057. * Genotypes
1-1- and 1+2- are significantly different from the 3 other groups by Duncan
Multiple Range test (P<O.05 ). Values were adjusted for pre-training level,
gender and training program.
.. .,' . ". ~. ';'.

---

128
*
c::
*t
T
----
0.8
.-S
-
0.7 -
-'-">:: 0.6-
t-r-
--<
~
-r-
0.5
N
0
T
.>
0.4
~
0.3
CI:l
~
0.2
0
Z
--<
0.1 -
~
U
0
1- 1-
1- 2-
1- 2+
1+ 2-
1+ 2+
PGM 1
(n=24)
(n=20)
(n=7)
(n=3)
(n=4)
Genotypes
Figure 3:
Training response of V0 max in relation to PGM 1 genotypes. ANOVA
2
across the 5 PGMl genotypes, F=3.94, P<O.Ol. * Genotypes 1+2- and 1+2+
are significantly different from the 3 other groups by Duncan Multiple
Range test (P<O.Ol); t Genotypes 1-2- and 1+2- are different from the 3
other groups by Duncan Multiple Range test (P<O.01); Values were adjusted
for pre-training level, gender and training program.

---

CHAPITRE VUI
COMPARAISON DES POLYMORPHISMES DE WNGUEUR DE FRAGMENTS DE
RESTRICTION DE L' ADN DE TROIS GENES CHEZ LES CANADIENS-FRANCAIS
CAUCASIENS ET LES CAUCASIENS ET NOmS SUD-AFRICAINS.
Le but de cette etude etait de comparer des Caucasiens Canadiens-Francais (FC), des
Caucasiens Sud-Africains (SA) et des Noirs SA pour la frequence des genotypes des genes
,
de l'adenylate kinase 1 musculaire (AKIM), la phosphofructokinase musculaire (PFKM)
et la sous-unite Va de la cytochrome c oxidase (COXVa), 3 genes qui codent pour les
enzymes du metabolisme energetique. Les variations de sequence aux loci de l'AKIM, la
PFKM et la COXVa ont ete comparees chez 98 Caucasiens FC, 37 Caucasiens SA et 18
Noirs SA. Les echantillons d' ADN ont ete digeres avec les enzymes de restriction TaqI
(AKIM), KpnI (PFKM) et SstI (COXVa). Les fragments d' ADN ont ete separes par
electrophorese, transferes sur un support solide par la technique de Southern puis ont ete
hybrides avec la sonde correspondante. Les resultats revelent une difference significative
des frequences alleliques et genotypiques de l' AKIM-TaqI et de la COXVa-SstI entre les
2 groupes de Caucasiens et les Nom SA (P<O.OO5). De plus, un nouveau fragment de 4.4
kb pour l'AKIM-TaqI a ere identifie chez les Noirs SA, suggerant un polymorphisme de
3 alleles chez ces derniers. Aucune difference n'a ete observee entre les 2 groupes de
Caucasiens pour les frequences des alleles et des genotypes. Ces resultats suggerent en
outre que les Caucasiens et les Noirs portent souvent les memes polymorphismes de l'ADN
mais a des frequences differentes. Dans ce cas cependant, les Noirs SA portent un variant
de sequence d' ADN qui leur est specifique et que 1'on ne retrouve pas chez les groupes de
Caucasiens.
.
, ...,;.; ..:-;",

---

130
COMPARISON OF DNA FRAGMENT LENGTH POLYMORPHISMS IN FRENCH·
CANADIANS AND SOUTH·AFRICAN CAUCASIANS AND BLACKS
Suzanne A. Adjoa1, Marie-Christine Thibaulr2, Pieter Coetzei!, Timothy D. Noakes3, Marcel
R. Boulayt, Claude Bouchard', and France T. Dionne'
1 Physical Activity Sciences Laboratory, PEPS, Laval University, Ste-Foy, Quebec, Canada
G1K 7P4, 2deceased 01/06/1995, and 3MRC/VCT Bioenergetics of Exercise Research Unit,
Department of Physiology, University of Cape Town, Cape, South-Africa
ADDRESS FOR CORRESPONDENCE:
France T. Dionne, Ph.D.
Physical Activity Science Laboratory
Laval University, Ste-Foy,
Quebec, Canada
G1K 7P4
Tel: (418) 656-3696
Fax: (418) 656-3020
Running title: RFLP distributions in Blacks and Caucasians

---

131
Summary
The aim of this study was to establish whether there are differences between French-
Canadian (FC) Caucasians, South-African (SA) Caucasians, and SA Blacks in muscle
adenylate kinase 1 (AKIM), muscle phosphofructokinase (pFK.M) and subunit Va of
cytochrome c oxydase (COXVa) genes which are encoding enzymes associated with energy
metabolism. DNA sequence variations at the AKIM, PFKM, and COXVa loci were
compared in 98 FC Caucasians, 37 SA Caucasians, and 18 SA Blacks. The DNA samples
were digested with TaqI (AKIM), KpnI (PFKM), and SstI (COXVa) and, after Southern
blotting, were hybridized with corresponding cDNA probes. Analysis of restriction fragment
length polymorphism (RFLP) revealed significant differences between both groups of
Caucasians and SA Blacks for TaqI-AKIM and Sstl-COXVa allele (P<O.OO5) and genotype
(P<O.OO5) frequencies. Futhermore, a new 4.4 kb fragment for TaqI-AKIM, was detected
in SA Blacks, suggesting a 3 allele polymorphism in SA Blacks. No difference was found
between the 2 groups of Caucasians for allele and genotype frequencies. These results are
consistent with the conclusion that Caucasians and Blacks often carry the same complement
of DNA polymorphism but at distinct frequencies. In one case, however, the SA Blacks
carried a specific DNA sequence variant not present in the Caucasian groups.
KEY WORDS: MUSCLE ADENYLATE KINASE 1, SUBUNIT Va OF CYTOCHROME
C OXIDASE, MUSCLE PHOSPHOFRUCTOKINASE, RESTRICTION FRAGMENT
LENGTIf POLYMORPHISM, BLACKS, CAUCASIANS.
"
~.
_._: .;. _;0; •

---

132
Introduction
"
In the present study, DNA sequence variation in genes enccxling muscle adenylate
kinase 1 (AK1M), muscle phosphofructokinase (PFKM) and subunit Va of cytochrome c
oxidase (COXVa) were investigated in subjects from three ethnic groups.
Adenylate kinase (AK; EC: 2.7.4.3) plays an important role in the regulation of
energy metabolism by catalysing the reversible reaction of 2 molecules of ADP to 1 ATP
plus 1 of AMP. This reaction occurs within red blood cell, skeletal muscle and other tissues
(Tills et al., 1970). The skeletal muscle form AKIM, also known as myokinase, and the red
blood cell type (AK1) are known to be under different genetic control (Mc Kusick, 1992).
COXVa is one isoform of subunit V of cytochrome c oxidase (COX; EC: 1.9.3.1), a protein
of central importance in the oxidative respiration system (Kharkats and Volkov, 1989).
COXV is believed to be involved in the catalytic function of the mitochondrial subunits of
COX. The last reaction of the oxydative phosphorylation is a process by which ATP is
synthesized as a result of the transfer of electrons from NADH and FADH2 through the
electron transport chain (Kadenbach and Merle, 1981; Kharkats and Volkov, 1989). PFKM
(EC; 2.7.1.11) is a key regulatory enzyme of glycolysis. It acts in the first step of glycolysis
by catalyzing the phosphorylation of fructose-6-phosphate to fructose 1-6-diphosphate
(Leninger, 1970).

---

133
Among the genes coding for the enzymes studied in the present investigation, red
blood cell AKl has been extensively compared between different populations (Fildes and
Harris 1966; Gordon et aL 1966; Bowman at al. 1967; Tills et al. 1970; Kirk et al. 1971)
using starch gel electrophoresis. The data showed distinct AKl phenotype distributions
among different populations. However, to our knowledge, there are no published data
comparing AKIM, PFKM, and COXVa DNA sequence variations among population
groups. Since these genes are already known to be polymorphic (Bech-Hasen et al. 1989;
Dionne et al. 1991; Adjoa et al. 1994), the purpose of the present investigation was to study
variation at the mutated sites in 153 unrelated individuals, including French-Canadian (FC)
Caucasians and South-African (SA) Caucasians and SA Blacks.

---

134
Subjects and methods
Subjects. Three different population samples were investigated: 98 FC Caucasians, 37 SA
Caucasians and 18 SA Blacks. The FC Caucasians were from the Province of Quebec while
the SA subjects were from Cape Town and the surrounding area.
DNA extraction and RFLP analysis. Total DNA extraction and RFLP analysis were
perfonned as previously described (Dionne et al. 1992). Five ~g of DNA were digested
with 20 U of the endonucleases TaqI for AK1M, KpnI for PFKM, and SstI for CQXVa.
The digested fragments were separated on agarose gel (0.8% to 1.2%) in TEA buffer, pH
8.5 (40 mM Tris, 20 mM Na acetate, 20 mM NaCl, and 2 mM EDTA), denatured in an
alkaline solution, and transferred by Southern blotting (Southern, 1975) to a nitrocellulose
membrane (Schleicher & Schuell). Hybridyzarions were perfonned at 42° C in 50%
fonnamide with specific probes: hAK1B3.25 for AKIM, derived from a human genomic
clone lambda hAK1-1 (Bech-Hansen et al. 1989); pcox1.11 containing a full length cDNA
corresponding to the nuclear encoded subunit Va of human cytochrome c oxidase
(E.C.1.9.3.1) (Rizzuto et al. 1988); PCHPFKM1, a 2.86-kb cDNA clone, containing 76 bp
of 5'-untranslated sequence, 2340bp encoding human muscle PFK, and 399bp of
3'untrans1ated sequence (Shanna et al. 1989). These probes were labelled with p32 by
random priming (Feinberg and Volgelstein, 1984) to a specific activity of 1-2 x109 cpm per
~g. Filters were then washed and exposed to X-ray films for 5 days. Autoradiograms were
interpreted using phage lambda digested with Hind III and Eco RI as size standards. TaqI-

---

135
AKIM detected 3 alleles of 6.4, 5.5, and 4.4 kb, while KpnI-PFKM and SstI-COXVa
detected 2 alleles of 17 and 11 kb, and 20.7 and 16.8 kb, respectively. Allele frequencies
were calculated by direct allele counting for each group.
Statistical analysis. The comparison of allele and genotype frequencies was carried out
using a chi-square test.

---

136
Results and Discussion
Table 1 presents allele frequencies for the AK1M, PFKM, and COXVa genes in
the 3 groups of subjects. The distribution of the TaqI-AK1M alleles in SA Blacks was
significantly different (P :s.;; 0.005) from those of FC and SA Caucasians. The completely
inversed AK1M allele frequencies between SA Blacks and the Caucasians are noteworthy.
In SA Blacks, the 6.4 kb allele was about 2.7 times less frequent (28%) than in FC (81%)
and SA Caucasians (78%), while the frequency of the 5.5 kb allele among SA Blacks was
about 3 times more frequent than in FC and SA Caucasians. The two groups of Caucasians
were not significantly different in the terms of TaqI-AK1M allele frequency. The TaqI-
AK1M allele frequencies observed in this study for Caucasians are similar to those reported
by Bech-Hansen et al. (1989).
TABLE 1 ABOUT HERE
It is also interesting to note that two unrelated SA Blacks exhibited a new TaqI 4.4
kb DNA restriction fragment at the AK1M gene (Figure 1), which, to our knowledge, has
never been reponed in the literature. This suggests that TaqI-AK1M exhibits in SA Blacks
a 3 allele polymorphism not present in the two groups of Caucasians. Autosomal co-
dominant segregation of this new allele has however not been confirmed due to difficulties

---

137
in getting blood samples from the families of these two subjects and from other SA Black
families.
FIGURE 1 ABOUT HERE
Cytosolic AK 1 exhibits 2 main electrophoretic variants: AK 1*1, and AK 1*2 (Fildes
and Harris, 1966; Bowman et al., 1967). AK1 *1 represent 95% of AK1 phenotypes (Luz
et aI., 1990). Earlier investigations on AK 1 enzyme were in tenus of its phenotype
distributions. AK1 has been studied as one of the classical markers of red blood cell
enzymes, using the venical starch-gel electrophoresis method (Gordon et al., 1966; Tills et
al., 1970; Kirk et al., 1971; Hitzeroth and Groeneveld, 1990; Saha et al., 1992). AK 1*2, the
rarer phenotype, is mainly found in caucasoid populations where it has a frequency of about
5% (Kirk et al., 1971; Tills et al., 1970, Bonne et al., 1971; Luz et al., 1990). This
phenotype has a very low frequency in American Blacks (Bowman et al., 1967; Kirk et al.,
1971) and in mixed population of the middle East and East Africa (Tills et al., 1970).
AK1 *2 is also rare or absent in West African Blacks of Ghana and Nigeria (Bowman et al.,
1967). Investigations on SA Caucasians, SA Blacks, SA Malays and SA Coloureds revealed
that there was not a great degree of racial difference for AK1 *1 phenotype frequencies
(Gordon et aI., 1966; Tills et al., 1970). However, the AK1 *2 phenotype was found only
in Caucasians in these studies. None of the SA Blacks, Malays, or Coloureds exhibited this

---

138
phenotype. Even though the TaqI polymorphism site on the 5.5 kb allele is still not
localized, we believe from our results that it does not identify the AKl *2 isofonn not found
in Blacks subjects. Indeed, some Blacks subjects were found to be homozygotes for this
allele in our SA Blacks subgroup (Table 2).
The frequency of KpnI-PFKM alleles was comparable in the 3 groups (Table 1). The
KpnI-PFKM 17 kb allele was present at frequencies of 50%, 44%, and 42% in FC
Caucasians, SA Caucasians, and SA Blacks, respectively. The SstI-COXVa allele
distribution for SA Blacks was significantly different (P<0.005) from that seen with both
groups of Caucasians. As for TaqI-AKIM, the allele frequencies detected for SstI-COXVa
in SA Blacks were inversed compared to those detected in Caucasians, the 20.7 kb allele
being the least frequent and the 16.8 kb the most frequent. There were no significant
difference in the allele frequencies for SstI-COXVa between FC and SA Caucasians. No
other population studies have been reported for these 2 enzymes.
The genotype frequencies for TaqI-AKIM, KpnI-PFKM, and SstI-COXVa RFLPs
are presented in Table 2. For AKIM and COXVa, the genotype distributions in SA Blacks
differ significantly (P <0.(05) from that in both group of Caucasians. In SA Blacks, 45%
of subjects were homozygous for the TaqI-AKIM 5.5 kb allele, whereas in both groups of
Caucasians, homozygotes for 5.5 kb allele represented the least prevalent genotype. No
difference for TaqI-AKMl genotype distributions was observed between both groups of
Caucasians. Both SA Blacks subject exhibiting the new 4.4 kb fragment were heterozygous

---

139
for the 5.5 and 4.4 kb alleles. For KpnI-PFKM, the genotype distribution was similar in the
3 groups. For SstI-COXVa, none of the SA Blacks were homozygotes for the 20.7 kb allele
while they represented about 20% of the Caucasians. Finally, in SA Blacks, almost 70%
of the subjects were homozygotes for the 16.8 kb allele, while in Caucasians, this
proportion was seen for the heterozygotes. No difference could be observed between both
groups of Caucasians in the distribution of the SstI-COXVa genotypes.
TABLE 2 ABOUT HERE
The two SA samples studied in this paper are known to be of different origins. In
Cape Town, South-Africa, 4 main racial groups form the population: Cape Coloured (mixed
race), Cape Malay (with Indian gene pool), SA Caucasians, and SA Blacks. SA Caucasians
are mainly of North-Western European descent (Botha and Prichard 1972; Moores et al.
1991), while the majority of SA Blacks at the Cape belong to the Xhosa chiefdom which
comes from the ancestors of the Zulu Black people (May and Du Toit 1989; Hitzeroth
1986). The Zulus have settled along the Southeastern coast of the Cape Province (Hitzeroth
1986; Moores et al. 1991). FC Caucasians, from the French population of the Province of
Quebec, originate primarily from the Northern and Western region of France (Laberge
1967).

---

140
Until now, genetic markers used to find racial differences associated specifically
with SA Caucasians or SA Blacks (Gordon et al. 1966; May and Du Toit 1989; Moores et
al. 1991) were described in tenns of'isoenzyme variants detected by gel electrophoresis for
proteins such us ABO, MNSs, Rh, Kell, Duffy, AK, 6-phosphogluconate dehydrogenase,
phosphoglucomutase, and glucose-6-phosphate dehydrogenase. In this study, DNA sequence
variants were used to study racial differences. Thus, the TaqI-AKIM and SstI-COXVa DNA
sequence variations allowed us to detect differences between Black subjects and the two
Caucasian groups, while the KpnI-PFKM RFLP cannot be used for this purpose. Other
DNA variants found on mitochondrial DNA (mtDNA) were shown to be specific to
Africans (Horai et al. 1993) or present at a higher frequency in Africans (Denaro et al.,
1981; Graham et al., 1991).
TaqI-AKIM and SstI-COXVa polymorphisms, on the other hand did not allow us
to discriminate between the Caucasian sub-samples. This was not surprising considering that
resaerch on mtDNA polymorphism, which is much more polymorphic than the nuclear
DNA, indicated that Caucasians of different origins were similar among themselves (Brown
,1980; Johnson et al., 1983; Horai and Yasaka, 1990; Tiercy et aI., 1992; Horai et al.,
1993).
These results are consistent with the conclusion that Caucasians and Blacks carry
often the same complement of DNA polymorphisms but at distinct frequencies. In one case,

---

141
however, the SA Blacks had a specific DNA sequence variant not present in the Caucasian
subjects.

---

142
AKNOWLEDGMENTS: Probes were generous
donations from Dr A Nakazawa,
University of Calgary and Alberta Children Hospital, Canada, for AKl; Or A McLachlan,
Scripps Clinic and Research Foundation, California, for PFK; and EA Schon, Colombia
University, New York for CQXVa. This research was supponed in pan by a grant from
FCAR-Quebec.

---

143
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.- .. "~' :;-.:•.. ,

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149
TABLE 1: COMPARISON OF ALLELE FREQUENCIES BETWEEN FRENCH-CANADIAN
CAUCASIANS, SOUTH-AFRICAN CAUCASIANS, AND SOUTH-AFRICAN BLACKS FOR
AK1M, PFKM, AND COXVa
TaqI-AK1M
KpnI-PFKM
SstI-COXVa
Population
kb
N
%
kb
N
%
kb
N
%
French-
6.4
147
81
17
72
50
20.7
100
51
Canadian
5.5
35
19
11
72
50
16.8
96
49
Caucasians
South-
6.4
58
78
17
23
44
20.7
39
56
African
5.5
16
22
11
29
56
16.8
31
44
Caucasians
South-
6.4
10
28"
17
11
42
20.7
5
16"
African
5.5
24
66.5
11
15
58
16.8
27
84
Blacks
4.4
2
5.5
Allele distributions of South-African Blacks are significantly different from those of both
group of Caucasians, P<0.005.

---

TABLE~: COMPARISON OF GENOTYPE FREQUENCIES BElWEEN FRENCH-CANADIAN CAUCASIANS, SOUTH-
AFRICAN CAUCASIANS, AND SOUTH-AFRICAN BLACKS FOR AKIM, PFKM, AND COXVa
TaqI-AKIM
KpnI-PFKM
SstI-COXVa
-
Population
Genotypes·
N
%
Genotypes·
N
%
Genotypes·
N
%
French-
6.4:6.4
57
63
17:17
20
27
20.7:20.7
15
16
Canadian
6.4:5.5
33
36
17:11
32
44
20.7:16.8
70
71
Caucasians
5.5:5.5
1
1
11 :11
20
27
16.8:16.8
13
3
.•J
South-
6.4:6.4
24
65
17:17
7
27
20.7:20.7
8
23
- '
U1
African
6.4:5.5
10
27
17: 11
9
35
20.7:16.8
23
66
0
Caucasians
5.5:5.5
3
8
11 :11
10
38
16.8: 16.8
4
11
South~
6.4:6.4
2
11 b
17:17
4
31
20.7:20.7
0
Ob
African
6.4:5.5
6
33
17:11
3
23
. 20.7:16.8
5
31
Blacks
5.5:5.5
8
45
11:11
6
46
16.8: 16.8
11
69
5.5:4.4
2
11
•
Genotypes are described in terms of kb.
b
Genotype distributions of South-African Blacks are significantly different from those of both group of Caucasians,
P<0.OO5.

---

151
Taq I
kb
1.3
AK1M
Figure 1:
Muscle adenylate kinase 1 (AKIM) DNA sequence polymorphism detected
with the enzyme TaqI. The new 4.4 kb fragment carried by the two South-
African Black subjects is shown (i).

---

CHAPITRE IX
DISCUSSION ET CONCLUSIONS
La reponse de la V0 max a un entrai'nement en endurance aussi bien que 1'augmentation
2
,
de 1'actiYire des enzymes sont genetiquement detenninees. La perfonnance lors d'un
exercise physique depend largement de la capacite a resynthetiser l' ATP. Dans la plupan
des conditions d'exercice, la phosphorylation oxydatiYe est la source majeure d'energie pour
la resynthese de l' ATP. Par consequent, les sujets presentant des penurbations du
metabolisme oxydatif, c'est-a-dire une alteration de la disponibilite ou de 1'utilisation des
substrats oxydatifs tels que chez les patients atteints d'une deficience en phosphorylase ou
en PFK ou chez ceux presentant un defaut dans le transpon des electrons dans la
mitochondrie, ont une perfonnance a 1'exercice tres affaiblie. On comprend des lors
1'importance des enzymes pour la regeneration de l' ATP. Plusieurs genes sont sans doute
impliques dans la determination de la reponse de la performance a l'entrainement. Cette
these ayait pour but de contribuer a1'amelioration des connaissances sur l'identification des
marqueurs genetiques qui modulent la reponse a l'entrai'nement ou qui sont associes a la
performance. Les resultats yont etre presentes et discutes par etude.

---

153
9.1 Polymorphisme de longueur de fragments de restriction detectes avec I 'enzyme
de restriction EcoRI dans le gene de la glycogene synthase chez I'humain.
L' ADN porte plusieurs milliers de genes qui cedent pour des proteines differentes.
L'avenement des techniques de biologie moleculaire permet d'etudier la sequence de ces
differents genes a l'aide de sondes moleculaires d' ADN complementaire a l' ARN messager
(ARNm) de ces genes ou d' ADN genomique. Sur l' ADN, le changement d'un seul
nucleotide peut entrainer la perte ou la creation d'un site de reconnaissance pour une
enzyme de restriction. Chaque enzyme de restriction reconnait une sequence bien specifique
sur I' ADN et coupe I'ADN a ce site, ce qui genere des fragments de differentes longueurs
qui peuvent etre separes sur un gel d'agarose en fonction de leur poids moleculaire. Cette
technique est appelee: analyse de polymorphisme de longueurs de fragments de restriction
ou RFLP. Dans ce chapitre, nous avons caracterise le gene de la glycogene synthase (GS)
de I'humain. Deux fragments d' ADN ont ere identifies avec l'enzyme de restriction EcoRI.
Ces fragments sont transmis de fa~on mendelienne, c'est-a-dire que le patron de fragments
des parents est transmis aux enfants. Le gene de la GS fera l'objet d'etudes ulterieures
incluant des sedentaires et des athletes. Pour cette these, la quantit6 d' ADN de sujets
disponibles ne nous permettait pas de le faire. En conclusion, ce RFLP qui identifie 2
alleles de la GS devient par consequent un marqueur genetique important du gene de la GS.

---

154
9.2 Sous-unites de la cytochrome c oxidase codees par le genome nuc1eaire:
poIymorphisme de Iongueur de fragments de restriction detectes avec I'enzyme de
restriction Sst!.
Les sequences d' ADN des sous-unites de la cytochrome c oxidase codees par le genome
mitochondrial sont polymorphiques. Par conrre, aucun polymorphisme de sequence des
sous-unites nucleaires n' avait ete rappone. Dans cette etude, nous avons identifie un
polymorphisme de 2 alleles avec l'enzyme SstI dans le gene de la COXVa. Ce
polymorphisme est transmis de fa90n mendelienne. Par contre nous n'avons pas detecte de
RFLP pour les sous unites IV et Vb. Au moment oil ces etudes ont ete realisees, la
technique d'analyse de RFLP etait la plus utilisee pour identifier les variations de sequences
enrre les individus. Elle n'identifie toutefois que des variations de sequence associees ala
perte ou au gain de sites de restriction. 11 existe maintenant des techniques telles que le
"single strand conformational polymorphism" (Orita et al., 1989) qui permet de detecter des
variations de sequence qui modifient le profil de migration des fragments d' ADN qui ne
different que par une seule paire de base. Ces variations n 'auraient pu etre detectees par
RFLP puisqu'elles ne creent ni ne font disparaitre des sites de restriction. Si cette technique
avait ete appliquee a l'analyse des genes de la COX IV et Vb, elle aurait probablement
permi d'identifier des variants. En conclusion, le RFLP de la COXVa devient egalement
un marqueur genetique important du gene de la COXVa.

---

155
9.3 Relation entre le polymorphisme de sequence de l'adenylate kinase 1
musculaire, la phosphofructokinase musculaire, la sous-unite Va de la cytochrome
c oxidase et la variation de la \\'0
max: et de sa reponse a l'entrainement en
2
endurance.
Dans le but d'identifier les facteurs genetiques qui pourraient influencer les differences
observees dans la perfonnance et la reponse a I'entrainement, les genes de 3 enzymes du
metabolisme energetique, en I'occurrence I'AKIM, la PFKM et la COXVa ont ete
investigues. Aucune association n'a pu etre observee entre le polymorphisme du gene
l' AKIM et la performance ou la reponse de la \\'0 max: a I'entrainement. Les variations
2
isoelectrophoretiques de la proteine de l' AKIM n'ont pu egalement etablir une association
avec ces 2 phenotypes (Bouchard et al., 1989). Ces resultats n' etaient pas tout a fait
inattendus puisque I' AK est une enzyme qui intervient principalement au niveau du
metabolisme anaerobie et que nos sujets ont ete soumis a un entrainement de type aerobie
et nos athletes etaient des athletes d'endurance. Par consequent, le gene de l' AKIM ne se
revele pas etre un gene candidat dans I'adaptation aux entrai'nement en endurance. Le gene
de la PFK non plus, n'etait pas associe aux 2 phenotypes. Seule la disnibution des
genotypes du gene de la COXVa des sedentaires etait significativement differente de celle
des athletes. En effet, les resultats indiquaient I'avantage d'etre heterozygote pour les alleles
de la COXVa dans l'adaptation a un enttainement en endurance. La COX est une enzyme
qui est consideree comme etant l'un des marqueurs de l' activite oxydative de la
mitochondrie et it est connu que I'augmentation de son activite est concomitante a une
-
• • • • ~', '. <'. -;'.

---

156
augmentation de la V0 max chez les sujets entraines. 11 est possible que l'association
2
observee soit due a un gene en desequilibre de liaison avec la COXVa. Comme a notre
connaissance, la localisation chromosomique de la COXVa n'est pas encore connue, il ne
nous est donc pas possible de preciser les genes qui sont dans le voisinage de ce gene. Les
resultats au niveau des "high" et des "low responders" ne nous permettent pas de repondre
au deuxieme objectif de notre etude. Les tendances observees au niveau de la COXVa des
"high responders" etaient similaires acelles des athletes. L'approche des "high" et des "low
responders" se voulait une fac;on d'etudier les sedentaires qui repondaient vraiment peu et
ceux qui avaient une bonne reponse. Il est evident qu' avec aussi peu de sujets, nous avons
dG selectionner un point milieu dans le continuum de reponses. Il aurait ete preferable
d'etudier, disons, le premier et le quatrieme quartile de reponses. Par ailleurs, puisque: 1)
il est bien connu que le potentiel glycolytique pourrait decroitre au detriment de
I' augmentation du potentiel oxydatif, permettant de favoriser ainsi le catabolisme des lipides
et que 2) le rapport PFK/OGDH est genetiquement determine (Bouchard et al., 1986), nous
avons regarde la distribution des haplotypes de la PFK. et de la COXVa chez les sedentaires
et les athletes ou chez les "low" et les "high responders". Cette analyse ne nous a pas
permis d'observer la presence d'haplotypes specifiques a un sous groupe. Encore une fois,
une limite evidente de cette etude est le nombre de sujets. En conclusion, nous n 'avons pas
observe d'associations entre le phenotype V0 max et sa reponse a l'entramement et le
2
polymorphisme de sequence de l' AKIM, la COXVa et la PFK.. Seule une difference dans
la distribution des genotypes de la COXVa a ete observee entre les sedentaires et les
athletes, comme dans l'etude de Raffel et al. (1992) qui rapportait une frequence elevee
.•
<. ~.", .~ - .:": •
-. - ,. -... , .~~.:":'.

---

157
d'homozygotes pour une classe d'alleles chez les diabetiques insulino-<iependant. Ces
resultats ouvrent quand meme la porte a d'autres etudes plus poussees sur le gene de la
COXVa et cette mutation particuliere pour evaluer leur relation avec la performance en
endurance et sa reponse a I'enrrainement
9.4 Polymorphisme de l' ADN mitochondrial et la ,\\1°
max chez les athletes
2
d'endurance.
Dne seule etude a rapporte que les variations au niveau de l' ADNmt pourraient concribuer
aux differences individuelles observees en reponse a l'entrainement (Dionne et al., 1991).
Dans ce chapitre, nous avons compare les resultats de cette etude aux notres. Il en ressort
qu'aucun athlete ne presentait le morphe associe a une faible reponse a l'entrainement
(MTND5-HincII). Par contre les morphes associes a une \\10 max de preentrainement
2
elevee (MTIT-MspI, MTND5-NciI) et a une bonne trainabilite de la \\10 max (D-loop-
2
KpnI), se retrouvaient a une plus haute frequence chez les athletes. 11 faudrait par ailleurs
mentionner le fait que nos sujets sont des Caucasiens Canadiens, des Caucasiens Sud-
Africains (SA) et de Noirs SA. nest bien connu que ces derniers presentent souvent des
morphes ou des frequences de morphes differents des Caucasiens. Dans ce chapitre, les
morphes MTIT-MspI et MTND5-NciI etaient associes au statut d'athlete. Ce n'etait pas
le cas chez les athletes Noirs SA puisqu'aucun de ces derniers ne presentaient ces morphes.
En effet, le MspI morphe 4 est inexistant chez les Bantus, les Senegalais et les Bushmen
(Scozzari et al., 1988). Par contre, a notre connaissance, il n'y a pas de litterature pouvant
'"
...... :.":,,.:0:.•

---

158
nous pennettre de savoir si le morphe HincII 1 est rare ou inexistant chez les Noirs SA. Le
morphe D-loop-KpnI se retrouve a des trequences presque similaires chez les SA
Caucasiens et Noirs comparativeIi'lent a un pourcentage faible chez les Caucasiens
Canadiens. 11 est anoter egalement que la frequence du morphe D-loop-KpnI observee chez
les athletes canadiens est la meme que celle des sede ntaires. Nous ne pouvons par
consequent exclure le fait que ces frequences elevees du variant D-loop-KpnI des SA soit
associee a leur race. En conclusion, les observations de ce chapite indiquent que ces 3
morphes seraient egalement associes au statut d'athlete. Cependant, des etudes incluant un
plus grand nombre d'athletes de chaque groupe racial avec une \\'0 max elevee, seraient
2
necessaires afin de nous permettre de confmner ces observations.
9.5 Relation entre le polymorphisme de la phosphoglucomutase-I, la perfonnance
aerobie et la reponse a l'entrainement.
L'etude de ce chapitre avait pour but de verifier la contribution du polymorphisme de la
PGMl a la performance evaluee par le test de 90 minutes (Boulay et al., 1984) et a la
reponse de la \\'02 max a l'entrainement. L'on sait que la perfonnance aerobie de 90
minutes et la \\'0 max sont influencees par l'entrainement et qu 'en outre, les enzymes du
2
metabolisme energetique sont des detenninants imponants de ces 2 phenotypes.
L'heritabilite de la \\'0
max et de la perfonnance de 90 minutes est respectivement
2
d'environ 25% et 60%. Dans ce present chapitre, les resultats revelent une correlation
significative entre les 5 genotypes de la PGMl identifies dans notre etude et la reponse de
' ..• -._1 ... ~ ...",:,.

---

159
la V0 max a l'entrainement. Les genotypes 1-1- et 1-2+ etaient associes a une faible
2
perfonnance et les genotypes 1+2- et 1+2+, a une bonne trainabilitl5 de la V0 max. Les
2
etudes anr.erieures portant sur la klation entre le polymorphisme des proteines et la
trainabilire n'avaient pu etablir ce lien. Cette etude est la premiere a etablir une relation
entre le polymorphisme d'une proteine et la perfonnance et la reponse a l'entrainement.
Nous ne pouvons exclure la possibilite que l'effet soit du a un gene en ctesequilibre de
liaison avec la PGMl. Le gene de la PGM1, enzyme de la glycolyse et de la glycogenolyse,
est localise pres de
plusieurs enzymes du metabolisme du
glucose:
la
glucose
cteshydrogenase (GDH), la 6-phosphogluconate des hydrogenase (PDH), I'UDP-galactose-4-
epimerase (GALE) et le transporteur 1 de glucose (GLUTl). En conclusion, ces resultats
indiquent que les differences genotypiques du gene de la PGMl sont reliees aux variations
interindividuelles observees en reponse a l'entrainement et a la perfonnance.
9.6 Comparaison des polymorphismes de longueurs de fragments de restriction chez
les Fran<;ais-Canadiens et les Caucasiens et Noirs Sud-Africains.
Dans ce chapitre, nous avons compare les genes de l'AKIM, la PFKM et la COXVa, entre
3 echantillons de populations. Les resultats indiquent que les distributions alleliques et
genotypiques de l' AKIM et de la COXVa des Noirs Sud-Africains sont differentes de celles
des 2 groupes de Caucasiens Canadiens et Sud-Africains. De plus, nous avons identifie chez
2 des sujets Noirs SA, un troisieme fragment de l' AKIM qui n'avaient jamais ete rapporr.e.
A notre connaissance, il n'existe aucune etude comparant les RFLPs de ces 3 enzymes chez
'"
...._, .....;--:.

---

160
differentes popu1ations, nous ne disposons donc pas de donnees qUI pourraient nous
permettre de faire des comparaisons~ Cependant, au niveau de la proreine de l' AKl des
.,
globules rouges, plusieurs etudes ont ete publiees. Des 2 isoformes (AKl * 1 et AKl *2)
ayant fait l' objet de ces etudes, il ressort que l' AKl *2 est tres rare voire absente chez les
Noirs. Par consequent, cette isoforme ne peut etre engendree par le fragment de 5.5 kb de
I' ADN de I' AKl de la presente etude, puisque plusieurs Noirs SA etaient homozygotes pour
ce fragment. En conclusion, les Caucasiens Canadiens et SA sont similaires pour le
polymorphisme de ces 3 genes malgre leurs origines differentes. Par contre, les Noirs SA
sont significativement differents d'eux pour les genes de l' AKIM et de la COXVa.
9.7 Conclusions generales
a) Des variants de sequence de genes d'enzymes associees 11 la production d'energie par les
cellu1es musculaires ont ete identifies.
b) Un genotype que 1'on retrouve le plus souvent chez les athletes, soit celui de
1'heterozygozyte du gene de la COXVa a ete mis en evidence.
c) Certains morphes de I' ADNmt qui influenc;aient la V0 max de preentrainement et sa
2
reponse, se retrouvent a une plus haute frequence chez les athletes.
...
.
: .. -.-", ,~, ":"':

---

161
d) L'etude sur la PGM1 est la premiere aetablir une relation entre le polymorphisme d'une
proreine et la performance aerobie ainsi que la reponse de la V0 max a un entrainement
2
en endurance.
e) Les Caucasiens et les Noirs portent souvent les memes polymorphismes de l' ADN mais
ades frequences differentes.
f) Les Noirs Sud-Africains portent un variant de sequence de l' ADN du gene de l' AK1M
qui leur est specifique et que l'on ne retrouve pas chez les groupes de Caucasiens.
g) Nous avons mentionne dans le chapitre IT que la V0
max est un phenotype
2
multifactoriel, done pouvant etre influence par plusieurs variables. Les etudes de cette these
se sont axees sur des genes d'enzymes impliquees dans le processus de regeneration et de
production de l'energie.
Les travaux de nocre laboratoire portant sur la genetique et la
sensibilite a l'entrainement se poursuivent depuis pres d'une decennie et, a notre
connaissance, il n'y a pas encore de marqueurs genetiques perrnettant de predire la
perfonnance ou la sensibilite d'un individu a l'entrainement. Cependant, les resultats
obtenus jusqu'a present (Dionne et al., 1991) et ceux de cette these (Chapitres V et VII)
sont encourageants. De plus, les sondes, pour la plupan des genes du metabolisme
energetique ainsi que d'autres strategies d'identification de polymorphisme de sequence sont
maintenant disponibles. C'est done en analysant le plus de genes candidats possibles que
1'0n pourra evaluer la contribution des genes sur la variation observee de chaque sous-

---

162
phenotype (Figure 2) associe ala sensibilite aI'entrainement ou aun phenotype tel que la
perfonnance en endurance. Dans cette these, il aurait ete interessant de caracteriser quelques
un des sous-phenotypes de la Vo
max tels que quantifier l'activite des enzymes
2
musculaires apanir de biopsies ou de mesurer le debit cardiaque. Cela n'etait pas possible,
parce que la majorire de nos sujets ont ete recnltes dans d'autres centres. 11 faudrait par
ailleurs souligner que le nombre restreint de sujets ayant fait l'objet des etudes de cette
these constitue une limite a nos conclusions. Par consequent, une banque de donnees
incluant de nombreux sujets avec des athletes d'elite ayant une '\\'0 max tres elevee (plus
2
de 75 mVmin·kg-\\ par exemple) pennettrait de faire des analyses plus etayees. De meme
il serait interessant d'avoir des donnees sur un plus grand nombre de sujets soumis a un
entrainement en endurance.
. -. .. -." ~ ". -..-":".

---

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