1
GENETIC VARIATION IN THE FRENCH CANADIAN POPULATIONS
OF THE SAGUENAY-LAC ST. JEAN AND CHARLEVOIX REGIONS
By
MICHELLE ROSS
Department of Surgery
Division of Surgical Research
McGill University, Montreal
March 1991
A thesis submittl!d to the Faculty of Graduate Studies and Research in partial fulfillment
of the requirements for the degree of
MASTER OF SCIENCE
(c)
Michelle Ross - 1991
\
\
1
.
ABSTRACT
The Saguenay-Lac St. Jean and Charlevoix regions of Quebec are characterized by
high incidences of hereditary diseases such as vitamin D-dependent rickets type 1 (VOD1),
cystic fibrosis, and tyrosinemia. This phenomenon may be due to founder effect or
genetic drift. Therefore, one may also detect differences in allele frequencies of neutral
polymorphisms between these populations and the general population. An assessment of
genetic variation was perfonned utilizing genetic markers spanning chromosome 12q14.
ABele frequencies for the genetic markers in the sample populations did not differ
significantly from those published for other Caucasian populations. One haplotype,
observed in strong linkage disequilibrium with the VDD1 mutation, was found to be
infrequent on nonnal chromosomes. This suggests that all of the VDD1 mutant alleles
cou Id be identical by descent, in keeping with a founder effect for VOD 1 in northeastern
Quebec.
1
r
"
11
1
Résumé
Au Québec, les régions du SaglLenay-Lac St-Jean et du Charlevoix sont
caractérisées par une forte incidence de maladies héréditaires telle que le rachitisme
vitamino-dépendant de type 1 (VDDl), la fibrose kystique, et la tyrosinémie.
Cc
phénomène peut être dû soit à un effet fondateur, soit à une dérive génétique. Par
conséquent, il est possible qu'il existe entre ces populations et la population québécoise en
général des différences de fréquences alléliques pour certains polymorphismes neutres.
L'évaluation de cette variation génétique a été réalisée en utilisant des marqueurs localisés
sur le chromosome 12q14. Les fréqnences alléliques pour ces marqueurs dans les
populations étudiées, n'étaient pas significativement différentes de celles publiées pour
d'autres populations caucasiennes. Par ailleurs, un haplotype donné qui avait été
démontré être fortement lié à la mutation VDDI a été trouvé de manière très peu fréquente
sur les chromosomes normaux. Ceci suggère que tous les allèles mutants VOD 1
pourraient être identiques et supporte le concept d'un effet fondateur pour V001 dans le
nord-est québécois.
1
üi
ACKNOWLEDGEMENTS
The experiments presented in this thesis were perfonned at the Ger.etics Unit of
the Shriners Hospital for Crippled Children, Montreal, Quebec.
1 am deeply indebted to my supervisor Dr. Francis Glorieux, Director of the
Genetic!o. Unit who offered me the opportunity to work in his department. His interest and
constant support were an encouragement in the realization of this thesis.
My special gratitude goes to Dr. Kenneth Morgan, Professor of the Department of
Epidemiology and Biostatistics at McGiIl Universl,ty. This work was conducted at his
suggestion. Through his stimulation, Dr. Morgan has taught me a special interest in
population genetics, and more specifically, a palrticular interest in the historieal
demography and genetics of the French Canadian \')opulation.
1 thank him for his
continued guidance, the many thoughtful discussions and for his critical suggestions for
the manuscript.
1 owe special gratitude to Dr. Margaret Labuda, re'''learch assistant at the Genetics
Unit of the Shriners Hospital, for offering her competent t,echnical guidance and advice
throughout this project. The much appreciated technical assistance and encouragment
provided by the members of the Genetics Unit, in particular, Ms. Judy Grover, are
gratefully acknowledged.
1 am thankful to Ms. Mary Fujiwara, Ms. Judy Grover, Dr. Peter Roughley,
Professor of the Department of S urgical Research, and Dr. Margaret Labuda for their
encouragement and critical reading of the manuscript.
The expert preparation of the illustrations and photographie material by Jane
Wishart was highly appreciated.
1 would like to thank the Shriners of North America, the Natural Sciences and
Engineering Research Council of Canada, and the Programme d'Actions Structurantes of
the Ministère de l'Enseignement Superieur et de la Science du Québec for their financial
support throughout this project.
IV
ORIGINAL CONTRIBUTIONS
1
1.
A more precise estimate of the frequency of hap!otype A. the haplotYPl'
constructed from alleles of the following genetic marker systems:
PYNH 15/Msp I, and p 11-1-7/Msp 1
10
pEFD3.t:!/M~p
1.
a normal sam pie of chromosomes t'mm the
Saguenay-Lac St. Jean region of Quebec was determincd. This haplotypc was ohservc:d
in strong !inkage disequilibrium with chromosomes bearing the
D Dependent Rickets Type 1 (VOn 1) and the reslIlts of these
di~case
allele for Vllamin
expcnmcnt~
support the
hypothesis of founder effect as an explanation for the elevated frcqllcncy of VDD 1 III
northeastem Quebec.
2.
The eharacterization of allele and haplotype frequencies within two suh-
populations of the French Canadian population, the Saguenay-Lac St. Jean ë.nd
Charlevoix regions, for the following genetic marker systems and the chromosomal loci
they represent: pKEV4/Pvu II (COL2A 1), pKEV4/Hind III (COL2A 1), pWEAV214/l-linf
1 (COL2A!), pcXPI3/Ta\.1 1 (ELA 1), pCMM1.2/Taq 1 (012S15), p9Fll/Taq (DI2S4),
pEFD33.2/Msp 1 (D12S14), pYNH15/Msp 1 (D12S17) and pll-1-7/Msp 1 (01256).
3.
From the above data, the genetie similarity between the Saguenay- Lac St. Jean and
Charlevoix populations of northeastern Quebee was determined and provides genetic:
support for the historieal demographie data of these populations which states essentially
that these populations can be considered as one.
4.
An assessment of genetic variation within and between the Saguenay-Lac St. Jean
and Charlevoix populations and with published data on the general
Cauca~ian
population
for the genetic marker systems studied.
5.
The establishment of the amount of genetic divergence between thcsc two
populations and published data for the general Caueasian population for the genetic marker
systems studied.
v
TABLE OF CONTENTS
PAGE
ABSTRACT
Résumé
ii
ACKNOWLEDGEMENTS
111
ORIGINAL CONTRIBUTIONS
iv
TABLE OF CONTENTS
v
LIST OF FIGURES
vii
LIST OF TABLES
viii
INTRODUCTION
1
L
BASIC PRINCIPLES OF POPULATION GENETICS
1.
Estimation of Gene Frequencies in Population
2.
Factors Which Cao Alter Gene Frequency
i.
Founder Effect and Genetic Drift
iL
Natural Selection
iii.
Mutation
iv.
Gene Flc,w and Migration
3
5
6
6
II.
DNA MARKERS AND THE DETECflON OF GENETIC
V ARIATION IN HUMAN POPULATIONS
7
III.
THE POPULATIONS OF NORTHEASTERN QUEBEC
1.
The Growth and Expansion of Charlevoix
2.
The Settlement of the Saguenay-Lac St. Jean
Region
3.
Hereditary Disorders in Northeastern Ql1ebec
4.
Social Factors Influencing These Populations
12
12
IV.
VITAMIN D DEPENDENT RICKETS TYPE 1
18
V.
GENETIC LINKAUE MAP FOR HUMAN CHROMOSOME
12q14 REGION
20
14
14
16
AIMS OF THE STUDY
24
MATERIALS AND METHODS
25
1.
MATERIALS
25
II.
ME1ll0DS
26
26
26
1.
l
1
1
3
Populations Studied
i.
Saguenay-Lac St. Jean Population Sample
ii.
Charlevoix Population Sarnple
iii.
Published Data
27
28
,
\'1
.....,
1
3.
4.
5.
6.
7.
8.
9.
10.
Il.
12.
III.
DNA Isolation
Measuring DNA Concentration
RestrictiC\n Enzyme Digestion of DNA
Agarose Gel Electrophoresis
Southem Transfer
Detection of Restriction Fragment Length
Polymorphisms
Large Scale Plasmid Isolation
Isolation of Inserts
Oligolabelling
Hybridization
Stripping and Reuse of Membmnes
STATISTICAL METiiODS
Detection of Allele Frequency
1.
Construc .lOn of Haplotypes
2.
Genotype Analysis
3.
Obse~ved Heterozygosity
4.
Tests of Significance
5.
Linkage Disequilibrium Parameœr: D
6.
Genetic Distance Parameter
7.
RESULTS
29
30
30
.:n
33
35
36
3~
39
39
39
39
40
40
41
41
42
42
45
1.
ALLELE FREQUENCY VARIATION
45
II.
OBSERVED HETEROZYGOSITIES FOR GENETIC MARKER
SYSTEMS STUDIED
45
III.
GENETIC VARIATION WITHIN THE COL2A 1 GENE
50
IV ..
VARIATION IN HAPLOTYPES CONSTRUCfED
FROM THE LOCI: D12S17, D12S 14, D12S6
59
V.
GENETIC DIVERGENCE BE1WEEN POPULATIONS
61
DISCUSSION
1
2~
64
I..
FOUNDER EFFECTFOR VITAMIN D DEPENDENT RICKETS
TYPE 1 IN THE SAGUENAY-LAC ST. JEAN POPULATION
64
II.
GENETIC VARIATION WITHIN THE SAGUENAY-LAC
ST. JEAN AND CHARLEVOIX POPULATION
68
CONCLlTSIONS
78
LIST OF PUBLICATIONS
79
REFERENCES
80
L
vii
LIST OF FIGURES
PAGE
FIG.
1
The Saguenay-Lac St. Jean and Charlevoix Regions of
13
Northeastem Quebec
FIG. 2
Genetic Linkage Map of Human Chronlosome 12q14 Region
15
FIG. 3
Measurement of DNA Migration After Gr' Electrophoresis
32
FIG. 4
Representative Autoradiographs For: a) COL2A 1 Locus
46
(pWEAV214/HinfI) and b) D12S15 Locus (pCMM1.2rraq 1)
FIG. 5
Representative Autoradiographs For: a) D 12S 17 Locus
47
(pYNHI5/Msp 1) and b) COL2A 1 Locus (pKEV4/Hind In)
FIG. 6
Representative Autoradiographs For The COL2A 1 Locus
(pKEV4/Pvu II)
l
48
-------------------
----
----------
----
\'1\1
1
LIST OF TABLES
PA(.E
Table 1
Birth Prevalences of Several Hereditary Diseases Comlllon
15
in Northeastem Quebec
Table 2
Chromosome 12 Loci and Genetic Marker Systems Stlldied
Table 3
Isolation of Probes from DNA Plasmids
.'1
Table 4
Allele Frequencies Amongst the Saguenay-Lac St. Jean and
49
Charlevoix Populations and Published Data for the Genetil'
Marker Systems Studied
Table 5
Observed and Expected Heterozygosities for Loci Stlldied
51
Amongst the Saguenay-Lac St. Jean and Charlevoix Populations
Table 6
Genotypic Association Between the RFLPs Detected by pKEV4
52
with Pvull and Hind III within the COL2A 1 Gene in the
Saguenay-Lac St. Jean and Charlevoix Populations
Table 7
Maximum Linkage Disequilibrium Values for Two RFLPs withlll
54
the COL2Al Locus: pKEV4/Hind III and pKEV4!Pvu Il wlthin
the Saguenay-Lac St. Jean Population
Table 8
Maximum Linkage Disequilibrium Values forTwo RFLPs within
the COL1Al Locus: pKEV4/Hind III and pKEV4!Pvu Il within
J
the Charlevoix Population
55
lX
1
Table ')
Allele Frequencies for the RFLPs within the COL2A 1 Gene
57
Amongst Various Populations
Table 10
Number of Observed COL2A 1 Haplotypes
58
(pKEV4/Pvu II, pKEV4/Hind III, and pWEAV214/Hinf 1)
Table Il
Number of Obscrved Haplotypes Constructed 4]-om Three Loci:
60
D12S14, D12S17, 012S6, within the Saguenay-Lac St. Jean
Population
Table 12
Nonnalized Genetic Identity (1) u..''!ci Standard Genetic Distance CD)
Between Populations
63
INTRODUCTION
1. BASIC PRINCIPLES OF POPULATION GENETICS
The highly polymorphic nature of the human species is apparent l'rom the man y
different persons who exhibit remarkable variation in a large nllmber of illheritcd
characteristics. The discipline of population genetics is concerned with detcnllllllng how
much genetic variation exists within and between populations, undcrstandmg the natllle
and the source of these inherited differences, with predicting changes that may nccur
111
phenotypic frequency and with determining conditions undcr which thc'ie frcqllcncics
were obtained (Cavalli-Sforza and Bodmer, 1971).
In order to achieve this goal,
population geneticists study the types and frequencies of genes within and betwecll
populations and the factors which influence these distributions.
A brief introduction to sorne basic princip les of population genetics and the factors
which influence gene frequency distribution between population groups wIll he presenlcd.
1. ESTIMATION OF GENE FREQUENCIES IN POPULATIONS
The beginnings of human population genetics can be placed with the derivation of
the Hardy-Weinberg Equilibrium principle. Developed in 1908 by a.H. Hardy and W.
Weinberg, the Hardy-Weinberg principle describes the relationship between allelc
frequencies and genotype frequencies at any locus in any population (}-Iartl and Clark,
1989; Cavalli-Sforza and Bodmer, 1971). This principle
provide~
the fOllndation for
many investigations in population genetics. Predicting genotype freqllencies from the
kn0wledge of allele frequencies is straightforward provided that several
assllmpti()n~
met. Since genotype frequencies are determincd, in part, by mating patterns,
a~
arc
long
a~
mating is at random within a population, the chance an individu al will mate with anothcr
individu ai of a particular genotype is equivalent to the frequency of the genotype in the
population (HartI and Clark, 1989).
Genotype frequencies are abo influl'.!l1ccd hy
2
evolutionary forces such as migration, natural selection and the size of the population.
Gene frequencies within small populations are more readily affected by chance
fluctuations, whereas within large populations these effects become negligble (CavalliSforza and Bodmer, 1971). Therefore in order to accurately predict genotype frequencies,
the Hardy-Weinberg principle is based on several assumptions: the population under
consideration is large, mating is at random, the effects of evolutionary forces such as
migration, mutation and selection are negligble (Cavalli-Sforza and Bodmer, 1971; Hard
and Clark, 1989).
The principle may he fonnulated as follows: if there are twO alleles, A and a, for a
gene, the possible genotypes are AA, Aa and aa. The frequencies of these genotypes are
p2, 2pq, and q2 respectively where p and q are the specific aUde frequencies: p for aUele
A, and q for allele a and p+q =1.
From this calculation, the Hardy-Weinberg principle can be used to de termine gene
frequencies in populations. If one knows the birth prevalence of a trait, the aUele and
heterozygote carrier frequencies can easily he detennined. The Hardy-Weinberg princip le
is, at best, only an approximation and is only reliable ü the basic assumptions are met. If
these assumptions are met, the populations is said to be in Hardy-Weinberg equilibrium.
Deviations from any of the assumptions for which the principle is based can alter gene
frequencies in a population.
One of the basic assumptions of the Hardy-Weinberg principle is that mating is at
random , however, in human populations, mating is often random with respect to some
traits but not with respect to other traits. For example, in humans, mating is often not at
mndom with respect to skin colour and height (Hartl and Clark, 1989). Statures of
husband and wife are known to be highly correlated (Nei, 1987). This type of mating is
known as assortative mating. Another form of non-random mating is consanguinity, or
mating among close relatives. This type of mating has a greater risk of producing
offspring homozygous for a deleterious recessive gene (HartI and Clark, 1989). This risk
3
is proportional to the degree of relationship between the parents and can be quantified by
l
Wright's coefticient of inbreeding (HartI and Clark, 1989; Cavalli-Sforza and Bodmer,
1971; Nei, 1987). The coefficient of inbreeding is the probability that an individual has
inherited (from each parenl) an identical copy of an allele at a given locus (Vogel and
Motulsky, 1986; Cavalli-Sforza and Bodmer, 1971; Harti and Clark, 1989). This is
closely related to the coefficient of kinship or coancestry which is defined as the
probability that two unrelated individuals have inherited an allele identical by descent
(Vogel and Motulsky, 1986; Cavalli-Sforza and Bodmer, 1971; Hartl and Clark, 1989).
In terms of the Hardy-Weinberg principle, genotype frequencies of inbred children
do not occur in Hardy-Weinberg proportions since the degree of heterozygosity is
diminished throughout the whole genome tfor caleulation see Cavalli-Sforza and Bodmer,
1971; Vogel and Motulsky, 1986). Inbreeding, like assortative mating, increases the
frequencies of homozygotes. If the number of consanguineous marri ages is elevated, one
may observe an increase in autosomal recessive conditions due to the increased probability
that two individuals heterozygous for the same mutant allele will mate (Vogel and
Motulsky, 1986; Bowen, 1985; McKusick et al. 1964). Due to the deerease in inbreeding
in modern populations, the number of homozygotes for recessive disease has decreased
(Vogel and Motulsky, 1986).
2. FACTORS WHICH CAN ALTER GENE FREQUENCY
i.....EQunder Effect and Genetic Drift
A founder effeet oceurs when a small number of individuals, that by chance carry
only a small fraction of the total genetic variation of the parental population, establish a
new population (Kelly et al. 1975; Cavalli-Sforza and Bodmer, 1971; Diamond and
Rotter, 1987). Due to this statistical sampling process and the element of chance,
differences in allele frequeneies between the new sub-population and the parental
population can ocellr and endow the new population with gene frequencies that arc not
representative of the parental popuiation (Gelehrter and Collins, 1990).
In small
4
«
populations, genotype frequencies are more likely to be affected by chance statistical
fluctuations from generation to generation. In this manner, sorne alleles may, by chance,
be passed on more often than other alleles which may eventually disappear from the
population. This phenomenon is known as random genetic drift (Cavalli-Sforza and
Bodmer, 1971; Hartl and Clark, 1989). Recessive deleterious alleles introduced into a
population by the founder effect, may spread, becoming elevated in the population by
genetic drift (Cavalli-Sforza and Bodmer, 1971).
It has been postulated that founder effect and genetic drift are responsible for the
increased frequency of rare, recessive disorders in several human populations. To
establish founder effect as the basis for inequality of gene frequencies, Kelly et al. (1975)
postulated that one must show a c1early identified population, a marked difference in gene
frequency for that population as compared to the parental population and a common
ancestral origin of heterozygotes by pedigree analysis. The Oid Order Amish, an example
of a closed population due to religious, social and cultural isolation (McKusick et al.
1964) have an increased prevalence of specifie rare hereditary disorders inc1uding Ellisvan Creveld syndrome (gene frequency of .07), pyruvate kinase deficiency (gene
frequency of .1) and albinism (gene frequency of .11) (McKusick, 1978). For the Old
Order Amish populations, founder effect has been established by genealogical
reconstructions which reveal potential "founder" individuals for each disorder. For
example, aIl cases of Ellis - van Creveld syndrome have been traced to Samuel King who
immigrated to Lancaster county in 1744 (McKusick et al. 1964), and all cases ofpyruvate
kinase deficiency have been traced to Jacob Yoder, who immigrateJ to Mifflin county in
1742 (McKusick et al. 1964).
The Afrikaner population of South Africa is
al~n
characterized by an elevation in
frequency of several hereditary disorders: porphyria variegata, Huntington disease, lipoid
proteinosis, familial hypercholesterolemia, Gaucher disease and cystic fibrosis (Botha and
(
Beighton, 1983a). Each disorder can be traced to a common ancestor by genealogical
5
reconstructions of heterozygotes, demonstrating a founder effect for these diseuses in the
white South African population (Botha and Beighton. 1983b).
The use of molecular biology techniques can aiso provide evidence that a founder
effect for a hereditary disease may have occurred in a population. When a new mutation
occurs or is introduced into a population and subsequently spreads throughout the
population, it tends to be initially inherited with other aile les al nearby loci that were
present on the "founding" or originating chromosome (Lan der and Botstein, 1986). This
association is known as linkage disequilibrium and is therefore indicative of a founder
effect.
iL Natural Selection
Natural Selection represents the action of environmental forces on a genotype.
Depending on the biological fitness of an individual, the consequences of these forces will
either be negative or positive. Individuals with genotypes which confer a greater degree
of fitness will, on average, leave more offspring than less (Hartl and Clark, 1989). If a
deleterious allele decreases the fitness of an individual by causing non-fertility, the
frequency of the deleterious allele in the next generation will be diminished. If an allele is
beneficial for survival, only those individuals with the allele will survive and reproduce to
pass on the beneficial effects of the allele to the next generation (Vogel and Motulsky.
1986). In sorne populations where deleterious alleles have attained high frequencies, the
disease allele may confer a selective compensating advantage to individuals who arc
carriers of the allele. Several types of evidence suggest that the high frequency of Tay
Sach rlisease in Ashkenazi Jews may be the result of a benefit to the resistance to
tuberculosis in heterozygous individuals. Tuberculosis may have, in fact, acted as a
strong selective agent on the Ashkenazi Jewish population (Rotter and Diamond, 19X7).
ln areas of Eastern Europe where there was an increased frequency of tuberculosis, the
Ashkenazi Jewish population had a lower number of deaths due to tuberculosis than other
Jewish populations residing in the same area (Rotter and Diamond, 1987). Peterson ct al.
6
(1983) reported that areas with the highest incidence of Tay Sachs disease in Ashkenazi
(
Jews, were also the areas of the highest incidence of tuberculosis, the putative selective
agent. Therefore, those individuals who were homozygous carriers would die, while the
numerous heterozygous carriers wou Id survive and resist tuberculosis better than normal
individuals. In this manner, the Tay Sachs disease gene was perpetuated in regions with
high levels of tuberculosis.
Hi. Mutation
Mutation is the ultimate source of variation and as such, provides the raw material
for évolution. Mutation refers to the spontaneous changes in the genetic material. These
changes can represent single nucleotide base sequence substitutions, deletions, insertions
and errors in DNA repair mechanisms (Vogel and Motulsky, 1986). Mutations can also
represent chromosomal translocations, deletions, insertions, and non-disjunction events.
Environmental causes of mutations in humans include ionizing radiation and chemical
mutagens (Vogel and Motulsky, 1986). The mutation rate at a particular locus is
expressed as the average number of rt"::· . . tions per locus per generation. The average
estimate for mutation rates within genes for autosomal dominant, recessive and sex-linked
disorders is 3 x 10 -5 (Cavalli-Sforza and Bodmer, 1971).
IV. Gene Flow and Miwtion
Movements of individuals from one region
1O
another will lead to subsequent
matings with individuals from other regions. This phenomenon mixes the gene pools of
different groups and by doing so, migration and subsequent matings together tend to
reduce genetic differences between groups (Rogers and Jorde, 1987). If two populations,
in Hardy-Weinberg genotypic proportions, with different alle!e frequencies are pooled, the
resulting population will have an allele frequency that is the average of the two initial
populations (Hard and Clark, 1989). After one generation of random mating, the pooled
populatioll will
hl','II.,
l-I:'A"dy-Weinberg genotypic proportions. However, the frequency of
homozygotes wi II he smaller than the average frequency of homozygotes in the two non-
,
,
7
pooled populations. This is known as Wahlunds principle (Hartl and Clark, 1989;
Cavalli-Sforza and Bodmer, 1971). In human populations, the main effect of isolate
breaking, by an influx of immigrants, is to decrease the frequency of children born wlth
hereditary diseases resulting from homozygous recessive genes (Hartl and Clark, 1(89).
However, the Wahlund effect is based on the assumption that emigrants leaving a group
constitute a random sample of the individuals born there. This
I~
not always the case.
Migration may be "kin-structured", that is, involving groups of relatives rather than
individuals. Fix (1979) revealed how the formation of new populations based on kill
migration can have biased samplings of the original population, reflected by differenccs in
allele and genotype frequencies between the two populaions. Rogers and Jorde (1987)
assert that, based on statistical simulations, kin migration can cause decreases in lhe
genetic variance of a population as compared to genetic variances predicted under the
assumption of random migration.
It is now apparent that the evolutionary forces of migration, mutation, nalural
selection and genetic drift all determine the differing degrees of genetic variation that exist
within a population. Therefore, measurements of genetic variation are central to
population genetics theory and practice.
II. DNA MARKERS AND THE DETECTION OF GENETIC VARIATION
IN HUMAN POPULATIONS
Until now, the discussion has focused primarily on genetic variation in the context
of rare, mutant alleles at ger."tic loci that contribute to hereditary disease, and the
mechanisms that elevate these alleles in human populations. E.nphasis is placed on the
overall rarity of these alleles since the vast majority of individuals do not carry
thc~c
mutant alleles, but rather the normal alleles. However one of the main objectives of
population genetics is to describe the amount of genetic variation al ail loci wilhin
populations and study the mechanisms of maintenance of genetic variation. To achicvc
this goal, the analysis of genetic variation between populations is studied al
varioll~
8
struL:tural loci across the genome as weIl as at disease loci. This variation is described as
(
the difference in genetic polymorphisms and their frequency within and between
population groups.
Genetic polymorphism is defined as the occurrence in the same population of two
or more aile les at one locus, each with appreciable frequency (Cavalli-Sforza and Bodmer,
1971). The locus is generally tenned to be polymorphie when the frequency of the most
common allele is equal to or less than 0.99 (Nei, 1987; Vogel and Motulsky, 1986). It is
important to differentiate between the many disease loci which have multiple polymorphie
alleles - in this instance referring to disease-conferring alleles, rather than neutral
polymorphisms which have no known phenotypic effect (Kimura, 1989).
The human genome has, at best estimates, 50,000 to 100,000 structural loci
(McKusick and Ruddle, 1977). Obviously in order to detennine the exact amount of
genetic variation that exists between populations, all of these loci must be studied.
However, for practical purposes only a sample of aIl genes are studied. To extrapolate
these results to the whole genome of a population is still question able since no one really
knows if the limited number of genes studied is truly re presentative of the whole genome
(Hartl and Clark, 1989).
Genetic variation of a population is often measured by the number of polymorphie
loci and the average heterozygosity per locus (Nei, 1987). In 1972, an electrophoretic
survey on genetic enzyme polymorphisms using 71 different loci determining enzyme
structure was conducted by Harris and Hopkinson (1972). This survey on European
populations determined that, for allioci studied, the average heterozygosity was 6.7% per
locus (Harris and Hopkinson, 1972)
However, because these polymorphisms result
from differences in substitutions of neutral amine acids, it is estimated that only one-third
of ail possible mutations resulting in amine aeid substitutions would cause alterations in
the net charge of the corresponding enzyme or protein. This implies that only one-third of
all polymorphisms would be detectable via expected changes in electrophoretic mobility
9
(Vogel and Kopun, 1977; Harris and Hopkinson, 1972). Therefore. the average
heterozygosity or frequency of genetically determined polymorphisms may be as high as
20%. This high level of genetic heterogeneity has led many to believc that Ilot ail
mutations in DNA are for adaptive purposes and are maintained by !o.ome form of
balancing selection, as would be suggested by Darwin (1859). The neulral lheory of
molecular eV'.Jlution (Kimum, 1989) asserts that most of the intraspecific vanabi lit y al Ihl'
molecular leve1 is essentially neutral.
To date, more than one-third of all human loci studied have been found to be
polymorphie (Gelehrter and Collins, 1990). This is illustrated by infomlation on 1,300
genetic systems, where over 3400 alleles or variants are known (Salzano. 1976). The
most commonly studied genetic systems are protein polymorphisms such as the ARO
blood groups, Rh factors and serum protein polymorphisms (Vogel and MOlulsky, 1986).
However, total genetie variation must be studied at the DNA level as well because a large
proportion of the genome does not appear to be involved in expressed gene produets or
gene regulation.
With the advent of modern genetic molecular biology techniques, DNA analysis,
via determination of DNA sequence and restriction enzyme analysis, has contributcd a
great deal of information on genetic polymorphisms in humans. With the diseovery of
DNA sequencing technology, it is now possible to sequence DNA for a large number of
individuals and assess variation. Jeffreys (1979) has estimated that one in every one
hundred base pairs in the ~ globin gene varies polymorphically. It remains unclear
whether data on genetic variability within coding regions can be extrapolated to the rcst of
the genome (Cooper et al. 1985). Assessing sequence variation is, however, a very time
consuming process. With the discovery of restriction enzymes, enzymes isolated [rom
bacteria which recognize specifie DNA sequences and cleave the DNA only at those
specifie sites, it is now possible to study DNA variation in terms of variability in the ~izc
of DNA fragments generated by the restriction enzyme. If a mutational event
III
the
1
10
restriction enzyme DNA recognition sequence occurs, the restriction enzyme will no
longer cleave the DNA at this site. Fragments of DNA of different lengths result from
either the creation or destruction of a restriction enzyme cleavage site. The creation of a
~lte
would cause the enzyme to c1eave DNA at that site and result in a sm aller size
fragment, whereas the loss of a site wou Id result in a much larger size fragment. When
the fragmt'nts or 'alleles' as they have come to be known are polymorphie - when the
freq uency of the most common allele is eq ual to or less than 0.99 (Nei. 1987) - they are
called Restriction Fragment Length Polymorphisms (RFLPs). first described by Botstein
ct al. (1980). These RFLPs can be detected in any region of DNA - introns, exons or
intergenic sequences. If one is interested in studying polymorphisms along an extended
region or segment of DNA. haplotypes cao be constructed from multiple, closely-linked
polymorphisms. Genetic variation of a population can therefore be studied by examining
the frequencies of the various size fragments or alleles and the haplotypes generated.
To detect an RFLP, a probe is used that is complementary to the DNA near the
restriction enzyme cutting site. A segment of DNA is chosen from a library of cloned
DNA fragments representing the full human genome. It is denatured. made radioactive
and hybridized to a southern blot of DNA digested with multiple restriction enzymes. If
various size fragments are seen the cloned DNA had detected a variable cutting pattern and
this probe, al0ng with the restriction enzyme is known as a genetic marker system (White
et al. 1988).
Using 15 different RFLPs detected in the human genome from 19 cloned DNA
segments, Cooper et al. (1985) estimated thm 1 in every 200 to 1 in every 300 base pairs
within the human genome is polymorphie. RFLPs are detected at an increased frequeilcy
in restriction enzyme sites which contain a CpG. Due to high levels of DNA methylation
at this site, 5-methyl cysteine is subject to frequent replacement by thymidine because of
de-amimltion of the methylated base (Barker et al., 1984). Therefore more polymorphie
variation is observed with RFLPs detected by Msp 1 (CCGG) and Taq 1 (TCGA)
t1
restriction enzymes. Indeed, Cooper et al. (1985) confimlcd that restrktion enzymes sudl
J
as Msp 1 and Taq 1 detect more polymorphie variation than other enzymes which do not
contain a CpG.
The ultimate goal of RFLPs is to construct a genetic linkage mup and to utilizc this
information for mRpping genetic diseases. Since these RFLPs behave as co-dominant
Mendelian alleles, it is possible to identify RFLPs in close proximity with a diseuse gcne
and observe the segregation of the RFLP with the disease gene within an affected family
pedigree (Botstein et al. 1980). The marker and disease loci are said to be linkcd if the
RFLP co-segregates with the disease allele in the affected offspring from carrier parents
(White and Lalouel, 1988). The probability of the transmission or co-segregation of the
two closely-linked loci is dependent on the distance between them and how often they
would he separated by a cross-over event occurring between homologous chromosomes
during meiosis (White and Lalouel, 1988). If the RFLP is localized to a particular
chromosomallocation, the location of the disease gene wou Id also be determined. This
procedure is useful in instances where the bioehemical defect of the diseuse is not known.
Critical for this methodology is the availability of large families with two or more affected
children (Botstein et al. 1980). The most successful use of this approach to date has heen
the localization, and later cloning and characterization of the defective gene for cystic
fibrosis (Kerem et al. 1989; Riordan et al. 1989; Rommens et al. 1989).
Until RFLP markers became available, the number of DNA loci thm could he
analyzed between populations was limited. There are now probably weil over t ,000
polymorphie probes available (Willard et al. 1985).
As weil as RFLPs, other
polymorphie DNA markers have been discovered. These are known as variable numher
of tandem repeats (VNTRs), which are created by the presence of short sequences which
are repeated multiple limes in tandem, ranging from a few to over 100 copies. Restriction
enzymes eut outside the tandem r~peats and, depending on the number of repeat~, vanou~
fragments of different lengths can result (White and Lalouel, 1988).
12
When two or more loci in close proximity along a chromosome are eonsidered
(
togcther in a randomly mating pcpulation, the genotype frequeneies are not always those
of the products of the gene frequencies of the alleles at each locus (Nei, 1987). Some
combinations of linked genes occur more often than expected. This phenomenon has
become known as linkage disequilibrium. For polymorphie loci, linkage disequilibrium,
or non-random association of the polymorphie restriction sites among themselves or
nelghboring loci, has been observed (Vogel and Motulsky, 1986).
ABele, genotype, and haplotype frequencies of polymorphie markers, as well as
the assessment of the average observed heterozygosities per locus and evidence for the
occurrence of linkage disequilibrium between markers, are a11 means of describing
differences in DNA polymorpilisms at coding and non-coding loci between populations.
The amount of genetic divergence between populations (defined as the standard genetic
distance measure) can he determined as a function of allele frequency differences between
populations (N'ei, 1987; Cavalli-Sforza and Bodmer, 1971; Reynolds, 1973).
With
th~
revolutionary molecular biology techniques introduced in recent years -
gene cloning, DNA sequencing, restriction enzyme methodology- genetic differentiation
hetween populations can now he more aptly described.
III. THE POPULATIONS OF NORTHEASTERN QUEBEC
The French Canadian population of North America consists of approximately 6 - 7
million individuals and is located primarily in the province of Quebec. This population is
descended from approximately 8500 immigrants from France who came to Quebec before
the English Conquest of 1759 (Charbonneau and Robert, 1987). Predominantly of the
Catholic faith, the population has increased primarily due to natural fertility (Laberge,
1976).
1. THE GRQWTH AND EXPANSION OF CHARLEYOIX COUNTY
The region of Quebec known as Charlevoix is situated along the north shores of
the Saint Lawrence River between Baie St. Paul and Tadoussac (Figure 1). From 1675 to
1
................................................
SAGUENAY - LAC ST-JEAN
GASPÉ
Figure 1 : The Saguenay-Lac St. Jean and Charlevoix Regions of Northeastern
Quebec
l'
14
1X50, 5X9 indivlduals immigrated to Charlevoix, 51 of whom came to the region from
1
out~ide
of Quebec (Jetté et al. 1990). Hcwever, the vast majority (80%) of these
immigrants to the region came from the Quebec City area (Jetté et al. 1990). In the
absence of any significant immigration to the region after the English Conquest, the
population grew from 1,000 to 8400 between 1759 and 1831 (Jetté et al. 1990) and
cvcntually reached 13.000
In
1852 (Gauvreau et al. 1990). This growth led to an
overpopulation of farms along the shores of the Saint Lawrence River and encouraged an
exodus of the population to the shores of the Saguenay River and into an area now known
as the
Saguenay~Lac
St. Jean region (Figure 1). Today Charlevoix's population is
approxlmately 30,000 individuals.
2, THE SETTLEMENT OF THE SAGUENAY-LAC ST. JEAN REGION
This region was first settled in 1838 by white settlers who came primarily from the
Charlevoix region (Gauvreau et al. 1987). In 1852 at the dme of the first census,
approximately 5,000 individuals lived in this region, ris,ing to 50,000 in 1911 and
eventually reaching its present-day population of 300,000 (Gauvreau and Bourque,
1988). This rapid population growth is primarily due to immigration to the region and a
high fertility rate (Gauvreau and Bourque, 1988).
Between 1838 and 1911, 28,700
individuals migrated to the region even though net migration was positive only before
1870 (Gauvreau et al. 1987). Approximately 80% of the se immigrants before 1872 came
from Charlevoix (Gaureau and Bourque, 1988).
After 1872, the proportion of
immigrants from Charlevoix decreased considerably. Between 1872-1891,60% of the
immigrants to the region were from Charlevoix, and between 1892-1911
le~s
than fort y
percent were from Charlevoix (Gauvreau ..md Bourque, 1988). Immigrants to the region
of non-Charlevoix origin came mainly from the lower Saint Lawrence areas and other
regions of Quebec (Gmdie and Gauvreau, 1987).
3, HEREDITARY DISEASES IN NORTHEASTERN OUEBEC
1S
1
Disorder
Birth Prevalence
Saguenay-Lac St. Jean
Myotonic Dystrophy
1/514
Cystic Fibrosis
1/926
Vitamin D-dependent Rickets Type 1
1/2358
Tyrosinemia (Type 1)
1/1979
Spastic Ataxia-Charlevoix -Saguenay Type
1/1436
Familial Hypercholesterolemia
1/150
Table 1 : Birth Prevalences of Several Hereditary Diseases Common in Northeastern Quebec
Estimates of birth prevalences of hereditary diseases within the Saguenay-Lac St.
Jean are reprinted with permission from DeBraekeleer (in press).
16
(
ln the Saguenay-Lac St. Jean region of Quebec, the birth prevalence of several
heredhary disorders is elevated as compared to the general Quebec population. Since
1969 when the first discovery of a cluster of hereditary tyrosinemia in Chicoutimi was
reported (Laberge. 1969), several other hereditary diseases have been reported to be
elevated in frequency in this region (De Braekeleer, in press). These disorders include
vitamin D dependent rlckets type 1, spastic ataxia of the Charlevoix-Saguenay type,
familial hypocholesterolemia, myotonic dystrophy and cystic fibrosis (Table 1). While
sorne hereditary disorders have an increased prevalence in this population, others are not
known to occur elsewhere. These include spastic ataxia of the Charlevoix-Saguenay type,
sensory-motor polyneuropathy and agenesis of the corpus collosum (De Braekeleer,
1990a). This population has an elevated prevalence of five autosomal recessive disorders:
senl'r')ry-motor polyneuropathy, hemochrornatosis, spastic ataxia of the CharlevoixSaguenay type, tyrosinemia, cystic fibrosis and vitamin D dependent rickets type 1 (De
Braekeleer, 1990a). These autosomal recessive disorders account for one in every 207
live births in the region; it is estimated that one individual in seven is a carrier of one or
more of these diseases (Ue Braekeleer, in press).
This population has becorne of great interest to many researchers from a variety of
backgrounds.
In 1972, an inter-university research consortium between McGill
University, the University of Laval and the University of Quebec at Chicoutimi was
established. The group called SOREP is centered in Chicoutimi. SOREP maintains a
large data base composed of 660,000 baptisrns, marriages and burlals which occurred in
the Saguenay-Lac St. Jean region between 1842 - 1971. From such data it is possible to
reconstruct farnily histories to determine familial orlgins and migration patterns of the
individuals who migrated to the region (Bouchard et al. 1985).
4. SOCIAL FA CfORS INFLVENCING THESE POPULATIONS
17
"
Although localized in a geographically isolated region of northeastern QlIebec,
historical demographic data reveals that the Saguenay-Lac St. Jean region has had a large
influx of immigrants to the region between the time it was first settled in 1838 to 1911. Of
over 28,000 immigrants to the region during this time, Charlevoix maintained strong
representation in the population of the Saguenay-Lac St. Jean region (Gallvreau and
Bourque, 1988).
After 1911, migration to the region became considerably more
diversified (Gradie and Gauvreall, 1987; De Braekeleer, in press). However, even in
light of the diversification of the immigrants to the region after 1911, a strong Charlevoix
component to the population was maintained. Families from Charlevoix experienced a
higher fertility rate than others from outside the region and were more likely to settle
permanently in the area (Roy et al. 1988). As weU, when individuals from Charlevoix
migrated to the region they were more likely to migrate in larger family sizes, including
not only husband, wife and children, but also brothers, sisters, grandparents and other
close relatives (Gauvreau and Bourque, 1988; Gradie and Gauvreau, 1987).
This seemingly kin-structured migration pattern of the Charlevoix immigrants has
led many researchers to believe that the increased prevalence of hereditary diseases in the
Saguenay-Lac St. Jean population may have its origins in the Charlevoix population. A
founder effect for these diseases may have occurred in the Charlevoix region and been
transferred to the Saguenay-Lac St. Jean region in the form of an elevated number of
carriers of the diseases migrating to the region (Gradie et al. 1988; De Braekeleer, in
press). Thus, the effect of kin-migration to the Saguenay-Lac St. Jean region may have
endowed the Saguenay population with a biased sampling of the originating population as
would be suggested by Fix (1979). This phenomenon has also led man y to infer that
the se populations were genetically homogeneous, as seemingly evidenced by the increase
in autosomal recessive hereditary disorders (Laberge, 1976, Gradie and Gauvreau, 1987,
Gauvreau and Bourque, 1988, Bouchard and De Braekeleer, in press). However, recent
studies on several sub-populations of the French Canadian population reveal that these
18
populations are more genetically diversified than previously thought (Kaplan et al. 1989;
(
John et al. 1990; Hetchman et al. 1990; Rozen et al. 1990; De Braekeleer, 1990b).
IV. YITAMIN D DEPENDENT RICKETS TYPE 1
Vitamin D Dependent Riekets, Type 1 (VOD1) or pseudo-vitamin D deficiency
rickets al it was first defined by Prader (1961) refers to a rare, autosomal recessive
hereditary disorder caused by an inbom error of vitamin 0 metabolism.
Patients with VOD 1 appear healthy at birth but the early onset of symptoms such
as muscular hypotonia, weakness, retarded growth and development, pathologie fractures
and convulsions (Prader et al. 1976) may occur within the frrst months of life. Clinical
examinations of patients reveal severe rickets, similar to the rickets observed from vitamin
o deficiency.
Other characteristics of the disease phenol.ype include hypocalcemia and
hypophosphatemia (Delvin et al. 1981).
Fraser et al. (1973) first proposed the basic defect of the disorder to be caused by
the impaired synthesis of the la, 25-dihydroxyvitamin D, the active metabolite of the
vitamin. This is apparent since serum levels of la, 25-dihydroxyvitamin 0 are low
(Delvin et al. 1981). The daily administration of the metabolite, or one of its analogues, to
its normal physiologie eirculating levds restores the disease phenotype to nonna1 (Delvin
et al. 1981). Therefore, the term Vitamin D Dependeney used to characterize the disease,
based on early observations of the correction of the disease phenotype with large amounts
of vitamin D, may not adequately depict the disease mutation.
Since this disorder is directly related to the inadequate synthesis of the la ,25dihydroxyvitamin D, it is believed the defect resides in the renal1a hydroxylase enzyme
(Prader et al. 1976; Fraser et al. 1973). This enzyme is responsible for the synthesis of
la, 25-dihydroxyvitamin 0 from 25 hydroxyvitamin D. The renal la hydroxylase is a
multi-component system composed of three subunits: ferredoxin, ferredoxin reductase
and cytochrome p450 (Ghazarian et al. 1974; Ghazarian and Deluca, 1974; Pedersen et al.
1976). Thus a mutation within the la hydroxylase could affect any of the three subunits.
19
However, Morel et al. (1988) demonstrated that ferredoxin, an iron/sulfur protein,
which serves as an electron-transport intermeuiate for ail mitochondrial forms of
cytochrome p450, is encoded by a single-copy gene located on chromosome 11'1 l3-qter.
Similar rt:sults for ferredoxin reductase reveal that this component of the electron-tmnsfcr
system is also encoded by a single copy gene located on chromosome 17cen-q25 (Solish
et al. 1988). Thus, if a defect must reside within the la hydroxylase multi-component
system, the cytochrome p450 subunit is the most likely candidate since VDDt patients
express no evident deficiencies in the activity of other cytochrome p450 systems.
However, since available evidence suggests that apparent deficient activity of the renal 1ex
hydroxylase only partially impairs the synthesis of 1ex, 25-dihydroxyvitamin D. one ean
not discount a mutation in one of the modulators of the enzyme activity expression
(Rasmusen and Anast, 1983).
Although rare elsewhere, YDDI is unusually frequent in the Saguenay-Lac St.
Jean and Charlevoix regions of northeastem Quebec. The estimated frequency of the gene
is .02 (Bouchard et al. 1985; De Braekeleer, in press). In a more recent study on the
frequency of hereditary disorders in the Saguenay-Lac St. Jean region, estimates of the
carrier frequency for YDD 1 were 1 in every 26 individuals with a birth prevalenee of
VDDI in lof every 2358 individuals (De Braekeleer, in press).
In an attempt to approach the primary defect of the disease usillg linkage analysis,
Labuda et al. (1990) established linkage between the YDDI disease locus and RFLP
markers located on the long arm of chromosome 12. The polymorphie loci that were
found to he segregating with the disease consisted of both coding (COL2A 1, ELA t) and
anonymous DNA loci (DI2S14, D12S17, D12S6, D12S15, D12S4). The three markers:
D12S14, D12S17 and D12S6 are tightly linked and segregate as a haplotype (Lahuda ('t
al. 1990). Of 13 haplotypes observed in 76 individuals, the YDDI mutation was round to
he linked with the same three marker haplotype (A) in 69% of the cases. Among
chromosomes from the Saguenay-Lac St. Jean and Charlevoix regions, the linkage
20
disequilbrium was even stronger: 85% (Labuda et al. 1990). These results, an excess of
(
haplotype A on VDDI bearing chromosomes, seem to indicate a founder effect for VDDt
in northeastern Quebec. This phenomenon can occur when a new mutation spreads
throughout a population. The mutation tends, initially, to be inherited with specific alleles
located close to the disease locus that were originally present on the original chromosome
(Lander and Botstein, 1986) and a linkage disequilibrium effect occurs. However, since
only a small sample of VDDI chrom,)somes were studied (n=16) and a small sample of
normal chromosomes (n=15), it was unknown whether this haplotype is frequent in the
general Saguenay-Lac St. Jean population.
V. GENETIC LINKAGE MAr FOR HUMAN CHROMOSOME J2QJ4
Of the over 3,000 known genetic diseases (White and Lalouel, 1988), the
chromosomal • JCation of 1,300 of these genes is now known (Scriver et al. 1989). The
assignment of a gene locus to a specifie chromosome is based on a variety of methods (for
review see McKusick and Ruddle, 1977). However the ability to locate genes relative to
each other has largely gained momentum in the past decade due to the discovery of highly
polymorphie markers, namely RFLPs and VNTRs (Botstein et al. 1980; White and
Lalouel, 1988).
By following the inheritance of many RFLPs in healthy families, the map
positions of the RFLPs ean be plotted relative to each other and mapped onto the physical
framework of the chromosome. This basic strategy, known as linkage analysis, is now a
popular tool of classical genetics. If two genes are in close proximity to each other on a
chromosome, they will not assort independently but will co-segregate, that is, will be
transmitted to the same gamete as a function of the distance between the two genes. This
distance is described in terms of genetic
distancl~
or recombination frequency (9) or in
centimorgans (cM). If the genetic distance between two loci is 10 cM, there is a ten
percent chance of a recombination event occurring between the two genes (8=0.10). This
represents the probability that the two loci will not segregate together. If the genetic
21
distance is less than 50%, the two loci are said to be linked. If 6 > 0.50, the two genl!s
are far apart and are not considered to be linked, even though they may physically residc
on the same chromosome. It has been estimated that the whole human genome entails
over 3300 cM, with approximately 150-200 cM per chromosome (McKusick, 19HX).
The goal of family linkage studies is to trace the inheritance of a specifie trait or
disease allele segregating in a family with specifie genetic markers. If the marker and the
trait co-segregate, the two loci are said to he linked. Basically, if the diseuse locus is close
to the marker locus, one can predict the status (affected, carrier, non-carrier) of each
family member by following the inheritance pattern of the marker locus. The accuracy of
diagnosis is, however, dependent on how far apart the marker and disease locus are. The
farther apart, the more likely a recombination event will occur and the less reliable the
prediction of the disease status in specifie individuals.
Human linkage analysis is interpreted with the use of statistical analysis, more
specifically via the use of the method of likelihood ratios (Fisher, 1935; Haldane and
Smith, 1947). One determines the probability that a given set of pedigree data would have
been observed under a hypothesis of a specifie recombination distance, versus the nutl
hypothesis that there is no linkage and the recombination distance is greater than 50%.
The ratio of these two probabilities is known as the ODOS ratio. In practice this ratio is
computed for several recombination values and described in terms of the log 10 of the odds
ratio, called the LOD score (Z) for convenience (Lander and Botstein, 1986). Computer
programs are now widely available to calculate these values. The most widely used
program was designed by Ott (1976). Values of LOD scores from independent families
are added until the LOO score reaches a total of r3 (evidence of linkage) or until the LOD
score fans to -2 (evidence of non-linkage). To achieve a postive LOD score, a large
number of families with two or more affected individuals are needed to observe the
segregation of the disease locus and the genetic markers. As well, the use of highly
informative markers is needed. This is Important because the segregation of the marker in
22
the families can only be followed when parents are heterozygotes (Lander and Botstein,
1986).
Figure 2 represents a chromosome 12 linkage map of the loci involved in this
slUdy: COL2Al, D12514, D12S15, D125l7, 01256, 01258, 01254 and BLAl based
on linkage studies conducted by O'ConneIl et al. (1987) and Labuda et al. (1990). The
dustered D 12517, D125 14 and D1256 loci are tightly linked: 012S17 to D12S14, e = 0,
Z
= 11.23,
and to D12S6,
e = 0.19, Z = 4.7, and can be considered as
a haplotype
(Labuda et al. 1990). In Figure 2, the COL2Aliocus was placed at 12q14.3, however a
discrepancy between map locations exists in the literature. The human type II collagen
gene (COL2AI) was mapped to chromosome 12q13.1 to 13.2 by in-situ hybridization
(Takahashi et al. 1989) and to 12q14.3 by southern hybridization analysis (Law et al.
1986). However, new data from non-isotopie in situ hybridization with biotinyated DNA
probes places the COL2A 1 gene at l2q 13.11 (Takahashi et al. 1990).
23
J
....
VDD1
ELA
C0L2A1
D12815
4.5
+
01284
5.0
012814J
012817
01286
2.5
3 Marker
haplotype
(cM)
V--"""
KRAS2
012S32
012S8
Figure 2: Genetic Linkage Map of Human Chromosome 12q14 Region
Sex averaged genetic map distances are given in centimorgans (cM) between
selected DNA marker loci: C0L2A1, ELA, 012515,01254,012517, D1286 as
reported by O'Connel! et al. (1987).012514 and V001 assigned by Labuda et al.
(1990). KRA5 2 and 01288 assigned by O'Connel! et al. (1987). 012532 assigned
by i:l situ hybridization (Oberle et al. 1988).
24
AIMS OF THE STUDY
The goal of the research project presented is to study genetic variation within
families from two sub-populations of the French Canadian population: the Saguenay-Lac
St. Jean and Charlevoix populations of northeastem Quebec.
1.
The Saguenay-Lac St. Jean and Charlevoix populations of northeastern Quebec are
eharacterized by an elevated incidence of several hereditary disorders. This phenomenon
may be due to founder effect and/or genetic drift. If so, differences in frequencies of
neutral polymorphisms may also he detectable. To test this hypothesis, genetic variation
was studied within and between samples from both sub-populations utilizing highly
polymorphie genetic marker systems for seven loci localized on chromosome 12:
COL2AI, ELA 1, D1284, D1286, D12814, D12S15 and D12817. Allele and haplotype
frequencies for these loci provide the basis for assessing the genetic similarity of these
populations to published data.
2.
Vitamin D Dependent Rickets type 1 (VOD1) was recently mapped to chromosome
12q 14 by Labuda et al. (1990). 8ignificant linkage disequilibrium was observed betw.!en
the VDDI disease locus and a three locus haplotype defined by the following probes and
the loci they represent: pEFD33.21D12S14, pYNHI5/D12S17 and pll-I-17!D12S6.
This linkage disequilibrium is indicative of a founder effect for VDDt in the populations
of northeastem Quebec. One aim of this thesis project is to estimate the frequency of this
haplotype in families from this region known not to have VDD1. This could provide
support for the hypothesis of founder effect as an explanation of the elevated incidence of
VODI in northeastern Quebec.
25
MATE RIALS AND METHODS
1. MATERIALS
The following probes utilized in this study, p9Fll. pll-1-7. pCMM1.2.
pYNHI5, and pEFD33.2 were kindly provided by Dr. Y. Nakamura of the Japanese
Foundation for Cancer Research in Tokyo, Japan. pKEV4 was provided by Drs. L.
Peltonen and E. Vuorio of the University of Turku, Finland, pcXP13 by Dr. R.
MacDonald of the University of Texas in Dallas and pWEAV 214 by Dr. E. Weaver of
Thomas Jefferson University. of Philadelphia, Pennsylvannia. The 20 x 20 ugarose gel
electrophoresis apparatus and Sephadex a-50 were obtained from Phurmacia Fine
Chemicals. The 21 x 24 agarose gel electrophoreis apparatus used to run a larger nllmœr
of samples was purchased from International Biotechnologies Inc.
recirculation pumps used were from Sargent Welch.
Power
supplie~;
MinistaItic
for the gel
apparatus, bromophenol blue, and oligonucleotide random primers (pdN6) were from
Bio-Rad. Hybond nylon membrane and [ap32] dCTP were obtained from Amersham Lire
Science Products. Whatman 3MM and 17 chr chromatcgraph)' paper used dllring the
transfer procedure was purchased from Fisher Scientific. The UV Stratalinker (Model
1800) is a product of Stratagene. Blots were hybridised to radioactive labelled probes in
a hybridisation oven obtained from Bachofer in West Germany. X-Omat AR films,
exposure holders and formamide are products of Eastman Kodak.
Gels werc
photographed with Polaroid PolaPan 4 x 5 instant sheet film (Type 52).
Gel
electrophoresis molecular weight markers and agarose (electrophoresis grade) wcrc
obtained from Bethesda Research Laboratories. AlI restriction enzymes and buffers,
pronase, herring sperm DNA, RNase A and Klenow enzyme (labelling grade) werc
ordered from Boehringer Mannheim. Bacto-agar, bactotryptone, and yeast extract used in
plasmid isolation procedures were obtained from Difco Laboratories.
Tris
(Tris(hydroxymethyl)aminomethane), lysosyme, ethidium bromide, ampicillin,
tetracycline, bovine serum albumin, polyvinylpyrolidine and ficol were ordered from
Sigma Chemical Company. AlI other chemicals used were molecular biology grade
,
,;
26
(Fisher Scientific Company). Laboratory glassware was ordered from Fisher Scientific
(
Company. Polypropylene tubes used for DNA isolation were obtained from Sarstedt.
[f.
MEIHOPS
1. POPULATIONS STUDIED
i. Safluenay-Lac St. Jean Population Sample
The tota] Saguenay-Lac St. Jean sample consisted of t":enty "control" families, 16
nonnal chromosomes from obligate VDD 1 heterozygotes and 2 families with autosomal
recessive spastic ataxia of the Charlevoix-Saguenay type (ARSACS). Since preliminary
results from linkage analysis data reveal that the ARSACS locus is not localized to
chromosome 12q (Dr. A Richter and M. Ross, unpublished observations), these
individuals were assumed to he nonnal controls for allele and haplotype frequencies for
genetic markers systems representing loci on chromosome 12q.
DNA was isolated from whole blood collected from 76 individuals representing
the 20 "control" families. These families consisted of 5 one child families, 14 families
with two children and 1 family with two children where blood was available from 3
gmndparents (2 paternal grandparents and 1 maternaI grand parent). These famUies were
collected and made available to this researcher by Dr. M. De Braekeleer (SOREP,
University of Quebec at Chicoutimi, Quebec) and Dr. C. Scriver (deBelle Laboratory for
8iochemical Genetics, McGill University-Montreal Childrens Hospital Research Institute,
Montreal, Quebec). AIl families are established residents of the Saguenay-Lac St. Jean
region (Chicoutimi-Jonquiere areas) and ail parents were born within the Saguenay-Lac
St. Jean region (established with the use of the SOREP population register to determine
birthplaces of parents courtesy of Dr. M. De Braekeleer). No family has a history of an
inherited disease nor consanguinity closer th an second degree.
Allele and haplotype data from 16 normal chromosomes of VDDt obligate
heterozygotes born within the Saguenay-Lac St. Jean region, were al 50 included in this
population sample. To establish the birthplace and regions of origins for these VDD1
27
1
parents, individuals were contacted directly by telephone. These data were obtained with
permission from Dr. F. H. Glorieux (Director of Research, Shriners Hospital for Crippled
Children, Montreal, Quebec) and represent experimental reslilts obtained by Dr. M.
Labuda (Genetics Unit, Shriners Hospital for Crippled Children, Montreal, Qucbec).
DNA samples were also obtained ft'Om two families with spastic ataxh. of the
Charlevoix-Saguenay type (ARSACS), cOllrtesy of Dr. Andrea Richter (Rcsean:h
Associate, Ste. Justine Hospital, Montreal, Quebec) and Dr. Ser:::e Melancon (Rescan.'h
Director, Service de Genetique Medicale, Ste. Justine Hospital, Montreal, QlIcbec).
These two families represent a total of 16 individllals: 1 family with Il children and 1
family with 1 child. Infonnation on birthplaces and residences of these families were
obtained from genealogical reconstructions courtesy of Dr. Andrea
Richtt·~·.
These
families are currently residents of the Saguenay-Lac St. Jean region and both sets of
parents were bom within the region. Due to limited DNA availability for these samples,
data from aU families was not available to complete haplotype information. Ail parental
chromosomes were typed for each polymorphism to establish allele freqllencies where
DNA was available.
iL Charlevoix PQpulation Sample
This sample included data obtained from 14 normal chromosomes of obligate
VDDt heterozygotes born in the Charlevoix region and ten ARSACS families of
Charlevoix origin. The ten ARSACS families represent a total of 88 indiviùlIals.
Information on birthplaces and residences for these families was obtained from
geneological reconstructions courtesy of Dr. Andrea Richter. This information established
that aIl individuals from the ten families were born within the Charlevoix region of
northeastern Quebec. Due to limited DNA availability, samples from some individuals
within a family were not typed. Therefore haplotype analysis was limited for this sample.
However, where available, parents were typed for ail markers studied. The data from the
14 VDD! heterozygotes represents results obtained by Dr. M. Labuda.
28
ii j, Published pata
(
ABele frequencies for each polymorphie locus studied were obtained from
published data listed in Human Gene Mapping 10 (1989). The data represents allele
frequeney studies conducted by researchers on unrelated Caucasian individuuls. Table 2
represents the polymorphie loci studied and the literature references for eaeh
polymorphism are included. For the pWEAV214/Hinf 1 genetic marker system within the
COLUd gene, allele frequeneies were detennined from 36 Caucasians (Weaver and
Knowlton, 1989). The allele frequencies of the pKEV4 polymorphisms deteeted by Hind
If. and Pvu II were detennined from 100 Finnish Caucasians (Vaisanen et al. 1988) and
48 Caucasians respectively (Eng and Strom, 1985). For the genetic marker system pl1-17/Msp l, allele frequeneies were determined from 43 individuals (Buroker et al. 1986), for
pYNH 15/Msp 1,84 Caucasians were typed (Nakamura et al. 1988b), and for p9Flirraq
l, 9 Caucasians were typed (Skolnick et al. 1984). Martin et al. (1988) typed 100
Caueasians to determine allele frequencies for pCMM1.2rraq l, and for pcXP13rraq l,58
individuals were typed (O'ConneIl et al. 1987). Allele frequencies for the genetic marker
system pEFD33.3/Msp 1 were ascertained from 57 Caucasians (Nakamura et al. 1988a).
However, a diserepancy exits between the allele frequencies for allele 1 and allele 4
published from this reference and the respective frequencies given by Ameriean Type
Tissue Culture where the probe is deposited. The allele frequencies whieh were assumed
to be the correct frequencies for each allele were verified by laboratory experiments
performed by this author on 30 unrelated CaucasLn individuals. The allele sizes and their
respective frequencies are eurrently being verified by the Howard Hughes Medical
Institute, at the University of Utah in Salt Lake City (Dr. K. Morgan, personal
communication).
2. DNA ISOLATION
DNA was isolated from 10 ml offrozen peripheral human blood according to the
procedure of Gustafson et al. (1987). First the blood was thawed in a room temperature
29
water bath and transferred to a 30 ml polypropylene tube. 10 ml of PBS was added and
f
i
mixed by inversing the tube which was then centrifuged at 4000 rpm for 15 min in a SS3.l
rotor. The supernatant was poured off and the sedimented ceUs were resuspended in 13
mIs TE-8 buffer (lOmM Tris, 50mM EDTA pH=8). Once aIl the clumps were dispersed.
350 ul of pronase (:!Omg/ml) and 700 ul of 10% SDS were added and lllixed by gentIl'
inversion. This solution was then incubated at 37 degrees celcius from 2-20 hours. Once
the ce Us were lysed, a phenol-chlorofonn extraction procedure was perfomled to remove
residual proteins from the solution. This procedure consisted of adding an equal volume
of phenol (equilibrated with TE-8) to the DNA solution and mixing gently for 3 minutes.
This solution was then spun in a clinical centrifuge at 3000 rpm for 10 minutes. The
phenol phase was removed and the aqueous phase was mixed with an equal volume of
phenol-chiorofonn (1: 1) for 3 min. Again the solution was spun for 10 minutes at 3000
rpm. The aqueous phase was then gently mixed for 3 minutes with an equal volume of
chloroform and spun for 10 minutes at 3000 rpm. NaCI (to a 0.1 M final concentration)
was added to the aqueous phase, and the DNA was precipitated with two volumes of
ethanol. The purified DNA solution was centrifuged for 10 minutes at 5300 rpm (S534
rotor). The DNA pellet was then washed with 20 ml of 70% ethanol to remove any
remaining salt, and spun at 5300 rpm for 5 minutes. The DNA pellet was then let air dry
overnight hefore dissolving in TE-8 buffer (IOmM Tris, ImM EDTA pH=8). The DNA
solution was transferred to a sterile eppendorf and mixed until al! the DNA was dissolved.
3. MEASURING THE CONCENTRATION OF DNA
To quantify the concentration of the DNA solution prepared, a spectrophotometcr
reading was taken to determine the amount of ultraviolet irradiation absorbed by the bases.
This reading was used to determine the concentration of nucleic acids in the sample. A
measurement at A260 allows for the calculation of the DNA concentration directly. An
optical density reading of 1 at A260 (in a 1 cm long cuvet) corre3ponds to 50ug/ml of
double-stranded DNA in solution. By taking another reading at A280, a ratio between the
two readings can he determined (A260/A280). This provides an estimate of the purity of
the sample. Pure preparations of DNA have an OD260/0D280 ratio of 1.8. If the ratio
30
was les s, contamination with protein or phenol had probably occurred and the DNA was
re-precipitated with ethanol to remove any phenol. If required, another phenol-choroform
extraction was performed to remove any proteins that may interfere with determining the
exact concentratIon of DNA in solution or restriction enzyme dig~stions (Sambrook et al.
1989)
4. RESTRICfION ENZYME DIGESTION OF DNA
As previously stated, this research was conducted utilizing restriction enzymes
obtained from Boehringer Mannheim.
For each enzyme, this company supplied
concentrated buffers necessary for optimal reaction conditions. Generally buffers differ
between restriction enzymes according to the salt concentration they contain. Sorne
restriction enzymes cleave the DNA better under high versus lower salt conditions. In
each case, digestions were carried out utilizing the manufacturers buffer solution
according to the following procedure: 3-5 ug of DNA was digested in a total reaction
volume of 25 ul containing a lx final concentration of the restriction enzyme digestion
buffer, 1.6 mM final concentration of spermidine to facilitate the digestion, 2 to 5 units of
the restriction enzyme and sterile water to complete the volume.
According to
mannfacturers specifications, one unit of enzyme is usually defined as the amount required
to digest 1 ug of Â. DNA in 1 hour under recommended conditions (salt concentration and
temperature). However, tbis applies to plasmid or low molecular weight DNA and human
DNA requîres usually higher concentrations of enzyme and prolonged incubation time.
5, AGAROSE GEL ELECTROPHORESIS
Agarose gel electrophoresis is a standard method used to separate DNA fragments
of different sizes. This separation of fragments or fractionation occurs at a neutral pH so
that the DNA is negatively charged. Under these conditions, the DNA sample loaded into
the sample weIl at the cathode end of the gel migrates towards the anode (Sealy et al.
1987). The ability of the DNA to migrate through the gel is dependent on the size of the
DNA fragment (Sealy et al. 1987). The concentration of the agarose in the gel will also
have an effect on the migration rate of the DNA fragment. For ex ample, in the case of a
3% agarose gel, the range of separation of linear DNA molecules is 60 - 5 kb as compared
31
t
to 10 - 0.8 kb if the agarose concentration in the gel is 0.7% (Sambrook et al. 1989). The
amount of voltage applied to the gel also affects the migration of the DNA fragments. It
has been shown that the effective range of separation of agarose gels decreases as the
voltage increases (Sambrook et al. 1989). Thus it is recommended to achieve maximum
resolution of DNA fragments, gels should be run no more than 5V Icm (Sambrook et al.
1989).
For the experiments conducted for this thesis, samples were run on either a 20 x
20 electrophoresis apparatus (maximum of 20 samples) or on a 21 x 24 gel electrophoresis
apparatus (maximum of 30 sarnples) depending on the number of samples that were run.
In ail cases lx Tris-acetate-EDTA (TAE: 0.04 M Tris-aeetate, 0.001 M EDTA) was used
as the electrophoresis buffer. Since the buffering capacity of T AE tends to diminish
during extended electrophoresis, the buffer was constantly recirculated llsing
il
recirculation pump to rnaintain an even cation and anion distribution.
To prepare the agarose gel, 2.0 grams of agarose were dissolved by heating in 225
ml water. 25 mIs of 10 x TAE was added to crea te a final concentration of 0.8%.
Ethidium bromide was added to a final concentration of 0.5 ug/ml from a stock solution of
5mg/ml. The solution was heated to a boiling point three times in a microwave oven and
briefly stirred between heatings to dissolve the agarose. The edges of the gel mold were
sealed with masking tape, and the agarose solution, once cooled to 50° celcius, was
poured into the mold and the comb applied to the mold immediately after. Once the gel
was set (after approximately 45 minutes), enough TAE was added to cover the gel to
il
depth of about 1 mm. Samples were mixed with 1/6 volume of ~onccntrated loading
buffer (0.25% bromophenol blue and 15% Ficoll type 400 in water) and loaded into the
sample weIl. In the first well of each gel, a 1 kb DNA ladder was added as a molecular
weight marker. The 20 x 20 gels were run at 28 volts for a 24 hour time period, while
the 21 x 24 gels were run at 50 volts fûr a 24 hour time period. After the run, the gels
were photographed under ultra-violet illumination with a ruler positioned alongsidc the
molecular weight marker to determine the exact migration of each molecular wcight
fragment (Figure 3).
32
1
1 kb
Ladder
+
DNA Samples
Ruler~
12 kb--.
8kb--.
4kb--.
3kb ....
Figure 3 : Measurement of DNA Migration After Gel Electrophoresis
A ruler was positioned alongside the 1 kb molecular weight marker to determine the
exact migrat.on distance of each molecular weight fragment.
,
33
f
t
1
6. SOUTHERN TRANSFER
Once the DNA fragments have been separated out according to size by gel
electrophoresis, the DNA was transferred to a nylon or nitrocellulose membrane in such a
way that the original pattern was retained. This transfer technique which allows for the
localization of the DNA sequences, was tirst described by Southem (1975). Once the gel
was photographed and unused portions of the gel arc eut away, the gel was soaked in a
denaturing solution (1.5 M NaCI, 0.5 M NaOH) for one hour. After denaturation, the gel
was soaked in a neutralization solution ( 1.5M NaCI, 0.5M Tris, 0.001 M EDTA, pH
=
7.3) for one hour. The transfer apparatus consisted of a plastic or glass tray filled with 10
x SSC blotting buffer (diluted from a 25 x SSC stock: 3.7 M NaCI, 0.375 M Na-citrate)
with a platform in the middle. The platform was covered with Whatman chromatography
paper (17chr) and one sheet of Whatman 3MM, saturated with the blotting buffer. The gel
was flipped over and placed on the platform and covered with a sheet of Hybond
membrane cut to exact size of the gel. Two sheets of Whatman 3MM paper, saturated
with blotting buffer were placed on top of the membrane and a stack of absorbant pa pers
on top of that. A weighted object was placed on the stack of absorbant papers to facilitate
the DNA transfer process. This process was allowed to proceed overllight and the next
morning, the nylon membrane was washed 2 x 15 minutes in 2x SSC solution before UV
cross-linking. To cross-link the ONA to the membrane, the blot was placed DNA side up
and exposed to 1200 kilojoules for thirty seconds in a stratagene UV crosslinker. The
membrane was then washed for thirty minutes at 65° celcius in a .5% SSC and .1 %SDS
solution, and then stored at 4° celcius until used.
7. DETECTION OF RESTRICfION FRAGMENT POLYMORPHISMS
To detect restriction fragment polymorphisms specifie genetic marker/restriction
enzyme systems were used. The systems utilized in this study are described in Table 2.
Ttese RFLP marker systems represent clones of two known genes: ELA 1 (E1a~tase ])
and COL2Al (Collagen type II a 1 chain), as weil as clones of anonymous DNA
sequences (012S14, 012S15, D12S17, D12S4, D12S6, D]2S8). These genetic marker
34
"
Locus
Probe
Restriction
Enzyme
Allele
Designation
Allele Size
(kb)
References
COL2A1
pKEV4
Pvu Il
1
2
3.3
1.7,1.6
Eng and Strom 1985
Hind III
1
2
14.0
7.0
pWEAV214
Hint 1
1
2
3
2.0
1.7
1.0,0.7
Weaver and Knowlton 1989
012814
pEF033.2
Msp 1
1
2
3
4
9.0
7.0
4.3
3.2
Nakumura et al. 1989 (a)
012815
pCMM1.2
Taq 1
1
2
3.5
3.4
O'Connel! et al. 1987
Martin et al. 1988
012817
pYNH15
Msp 1
1
2
3
4.0
3.2
2.6
Nakumura et al. 1988 (b)
ELA 1
pcXP13
Taq 1
1
2
4.3
3.7
O'Connel! et al. 1987
01284
p9F11
Taq 1
1
8.0
3.0
O'Connel! et al. 1987
2
1
2
4.4
3.6
Buroker et al. 1988
""
01286
p11-1-7
Msp 1
Table 2: Chromosome 12 Loci and Genetic Marker Systems Studied
35
systems were selected because of their current use in the laboratory for the VOD 1 family
j
linkage study.
Within the COL2A 1 locus two COL2A 1 probes were used to detect three
polymorphisms. With pKEV4, two RFLPs were detected by Hind III and Pvu Il
(Vaisanen et al. 1988) and with p WEA V 214 (Weaver et al. 1989) an RFLP was detected
by Hinf I. The Hind III polymorphism occurs between exons 20 and 30 (Schwartz et al.
1990) generating either a 14 kb fragment if the Hind III site is absent or a 7 kb fragment if
the Hind III restriction site is present. The Pvu II polymorphie site is located 500 base
pairs 3' to ex on 30 (Schwartz et al. 1990), in the middle of a 3.3 kb fragment. The
presence of the Pvu II polymorphie site results in fragments with sizes of 1.6 and 1.7 kb,
the absence of the Pvu 11 site results in a 3.3 kb fragment. The Hinf 1 polymorphism is
located within the first few exons of the COL2A 1 gene (Schwartz et al. 1990) and
generates a tri-aUelic system: 2.0 kb, 1.7 kb, and 1.0 + 0.7 kb.
AIl of the ONA probes were isolated from specifie ONA plasmids amplified in
bacteria (Table 3).
8. LARGE SCALE PLASMID ISOLATION
A large-scale DNA plasmid isolation procedure was followed te isolate the ONA
plasmids from the bacterial culture. Firstly 250 ml of L Broth (lOg baclotryptone, 5g
yeast ex trac t, and l') g Nael per liter) and the selective antibiotic, either ampicillin or
tetracycline depending on the resistance the plasmid confers to the bacteria, was
innoculated with 1 ml of the ONA plasmid infected bacteria. This was allowed to grow
overnight at 37° celcius to amplify the bacteria containing the ONA plasmid. The next day
the culture was centrifuged at 3200 rpm for 15 minutes and the ceUs resuspended in 15
mIs of a 50 mM glucose, 25 mM Tris pH 8.0, 10 mM EDTA pH 8.0 solution containing 5
mg/ml of lysozyme. This step is necessary to en able the lysis of the peptidoglycan layer
of the bacterial cell wall. At the same time the EOTA prevents nuclease degradation of the
DNA. 30 mIs of a freshly made 0.2M NaOH/l % SOS solution was added to the bactcrial
suspension for 10 minutes on ice. This procedure was followed by a neutralization step in
which 15 mIs of a 3M potassium and 5M aeetate solution was added. The suspension
36
was once again mixed for 10 seconds and let sltand for 10 minutes on ice. The solution
(
was then centrifuged at 5000 rpm for 15 minutes and the supernatant poured through
cheesecloth to a fresh flask. This step removes most of the bacterial genomic DNA. DNA
was recovered from
th~
supernatant by precipilating with 0.6 volume of isopropanol.
After 45-60 minutes at room temperature, the pellet was spun (5000 rpm, 15 minutes)
and resuspended in 3.75 mIs of TE, followed by the addition of 1.25 ml 10M NH4Ac.
After twenty minutes on ice, the protein precipitate was spun (5000 rpm, 15 minutes) and
the DNA was recovered from the supernatant by ethanol precipitation. The DNA pellet
was resuspended in 3.5 mIs of TE. To remove any RNA from the preparation, the DNA
solution was digested with RNase A (lOuglml final concentration) at 37° celcius for 15
minutes. This was followed by the addition of 1.5 mIs of NaCI and one-fourth the
volume of 30% polyethylene glycol (PEG), 1.5 M MaCI solution for 30 minutes on ice.
The plasmid DNA was centrifuged (8700 rpm, 15 minutes) and the pellet resuspended in
200 ul H20, 200 ul2 x proteinase K buffer (.OIM Tris pH 7.8, .005 M EDTA, pH 7.8
and 0.5% SDS) and incllbated at 37° celcius with the proteolytic enzyme proteinase K
(500ug/ml) for 15 minutes. After phenol-chloroform extraction and ethanol precipitation
followed by a 70% ethanol wash, the plasmid DNA was resuspended in 300 ul TE
(Sambrook et al. 1989).
9. ISOLATION OF DNA INSERTS
Once the respective plasmids were isolated, the DNA inserts were released by
digesting the plasmid DNA with specifie restriction enzymes (Table 3).
6 ug of the plasmid DNA was digested with the appropriate restriction enzyme (10
lInits per 6 ~g plasmid DNA) in a 1 x buffered solution (total 25 uls) for two hours at 37°
celcius. The completion of the digestion was checked by running 3 ul of the digestion
solution on a 0.8% agarose mini-gel (42Volts, 2 hours) with a standard molecular weight
marker. The release of the insert from the plasmid vector was verified by confirming the
respective molecular weights. To obtain the insert for labelling, a 0.9% low melting
temperature agarose mini-gel was ron at 17 volts overnight at 4°celcius. The insert was
cut out of the agarose gel and placed in a sterile eppendorf tube. The tube containing the
·f..
37
..1
Locus
Probe
DNA Plasmid
Restriction Enzymes
which release insert
Insert Size
(kb)
COL2A1
pKEV4
pUC8
Barn HI/Pst 1
4.0
COL2A1
pWEAV214
pT2-18
5ph I/Bam HI
3.6
D12514
pEFD33.2
pUC18
Eco RI/Hind III
3.2
012515
pCMM1.2
pBR322
Sail/Eco RV
1.7
D12517
pYNH15
pUC18
Barn HI/Pst 1
3.2
ELA1
pcXP13
p8R322
Hind III
0.92
01254
p9F11
p8R322
Barn HI/Pvu Il
1.0
01256
p11-1-7
p8R322
Hind III/Eco RI
0.5
Table 3 : Isolation of Probes From DNA Plasmids
38
agarose piece with the insert DNA was then weighed and 10 ul of sterile water added per
0.01 grams of agarose-containing insert. This was then boiled for 2-3 minutes and stored
until needed for labelling at -20° celcius.
10. OLIQOLA BELLING
Probes were labelled in agarose with [ap32] dCfP by the use of a hexanucleotideprimed reaction (Feinberg and Volgenstein, 1984). The agarose-containing insert was
first melted at 65° celcius and then 32 ul (0.5 - 2 ngI'A insert DNA) was boiled for three
minutes to denature the DNA and allowed to cool to 37° for 1 minute. This was then
added to
~
ul of 10mg/ml BSA (DNase free), and 10 ul of a prepared stock solution
consisting of: l00ullM Tris, pH 7.5, 12.5 ul lM MgCI2, 1.7 uI2-mercapto-ethanol, 2.5
ul 50mM dATP, 2.5ul 50mM dGTP, 2.5 u150mMdTTP, 250 ul2M Hepes pH 6.6 and
150 ul 90 A260 units/ml oligonucleotides. 50 uCi of [exp32] dCTP (3000Ci/mmol) were
tl.en added with the addition of 2-3 units (1u1) of the Klenow enzyme (large fragment of
DNA polymerase 1). The oligolabelling reaction was allowed to proceed at room
temperature for four hours to overnight. Once the labelling reaction was complete, 150 ul
of water was added and the labelled probe was passed through a Sephadex 0-50 column
to remove unincorporated radioactivity. The probe was then ready for hybridization to
southem blots.
The DI2S14 marker pEFD33.2 requir~d a pre-hybridization step with an excess of
human DNA to prevent sequences homologous to genomic repeats from hybridizing to the
southern blots and obscuring single and low copy bands (Sealy et al. 1985). After aIl
unincorporated radioactivity was removed, 2 ul of sonicated human placental DNA was
added to the pEFD33.2 probe. The DNA was precipitated with 2 volumes of ethanol and
one-tenth the volume of 5M Nael and subsequently spun and vacuum dried. The DNA
was then resuspended in 75 ul of TE, pH 8.0 and 25 ul of 20 x SSC, denatured in boiling
water for three minutes and incubated at 65° celcius to allow re-association between the
human placental DNA and the repetitive sequences within the probe. The probe was then
added to the hybridization solution.
39
11. HYBRIDIZATION
l
5 mIs of hybridization solution (5 x SSC, 1 x Denhardts. O.02M NaP04 pH 6.7.
200ug/ml sonieated denatured herring sperm ONA, 50% fonnamide, in H20) was added
to the blot already placed in the hybridization cylinder. The blot was then prehybridized in
the hybridization oven at 42° Celcius for 2 hours - overnight. Once prehybridization was
complete, the labelled probe was added to the cylinder and allowed to hyhridize ovemight
at 4r Celcius. Following hybridization, the membrane was removed from the cylinder
and washed briefly in 2 x SSC, .1 %SDS at room temperature to remove residual
hybridization solution. The membrane was washed once for 15 minutes in 1 x SSC, 0.1 %
SOS at room temperature, followed by one more room temperature wash for 15 minutes
with 0.1 x SSC, 0.1 % SOS. This was followed by two thirty minute 65 0 celcius washes
with 0.1 x SSC, 0.1 %SOS. The blot was then wrapped in plastic wrap and exposed
ovemight until a maximum of one week, to X-ray film at -700 celcius.
12. STRIPPING AND REUSE OF MEMBRANES
To remove any hybridized probes from membranes, the blots were washed two
times for 15 minutes in a boiling solution of 0.1 x SSC, 0.1 % SOS. The filters were then
able to he hybridized with a new probe or stored at + 4° celcius until further use.
lIT.
STATTSIICAL MEIHODS
1. DETERMINATION OF ALLELE FREQUENCIES
The polymorphie loci chosen for analysis were:
ELA 1, COL2A l, D 12S4,
012S6, 012S 14, 012S15, 012S17. Allele sizes for each genetic marker system were
determined from developed autoradiographs by eomparison of the migration distance of a
given band with that of the ONA markers of known moleeular weight run on the original
gel. Allele frequencies for each genetic marker system were determined by the counting
method. Alleles of one type from ail unrelated individuals were tabulated and divided by
the total number of individuals typed for that particular genetic marker system. For the
chromosome 12 q loci, the following number of chromosomes from unrelated individuals
were typed: ELA!, 99 chromosomes for the Saguenay-Lac St. Jean sample and thirtynine for the Charlevoix sample; 012S 15,98 chromosomes for the Saguenay-Lac St. Jean
40
sample and 48 for the Charlevoix sample; D 12S4, 99 chromosomes for the Saguenay-Lac
St. Jean sample and 46 for the Charlevoix sample; for D12S6, 97 chromosomes were
typed for the Saguenay-Lac St. Jean sample and 44 chromosomes from Charlevoix; for
D12S17, 104 chromosomes of Saguenay-Lac St. Jean origin and 44 from Charlevoix; for
D12S14, 103 chromosomes from the Saguenay-Lac St. Jean and 42 from Charlevoix
were typed. Within the COL2A 1 locus, for the genetic marker system determined by
pWEA V214/Hinf l, 102 chromosomes from the Saguenay sample and 44 from the
Charlevoix sample were typed. For the COL2A 1 genetic marker system pKEV4/Hind III,
104 chromosomes representing the Saguenay-Lac St. Jean sample flnd 32 from
Charlevoix were typed and for the marker system pKEV4/Pvu n, 104 chromosomes from
the Saguenay-Lac St. Jean sarnple and 46 from Charlevoix were typed.
2. CONSTRUcrION OF HAPLQTYPES
Haplotype analysis within the Saguenay-Lac St. Jean population sample was
performed for each of two sets of loci. It was assumed that no cross-over events occurred
between the COL2Al markers and the D12S14, D12S17 and D12S61oci. Due to the lack
of family information caused by low DNA availability for the families representing the
Charlevoix sam pIe, only haplotypes within the COL2A 1 locus were able to be
constructed. Haplotypes were assigned via inspection of family data. Where haplotypes
could he assigned, only unrelated individuals (parental chromosomes) were included in
the analysis.
3. GENOTYPE ANALYSIS
For the COL2AI genetic marker systems, pKEV4/pvu II and pKEV4/Hind III,
genotype analysis between the Saguenay-Lac St. Jean and Charlevoix populations was
performed. Within the Saguenay-Lac St. Jean population sample, from a maximum of 45
unrelated individuals, 42 genotypes were known for these two marker systems, and
within the Charlevoix sample, from a maximum of 20 unrelated individuals, 16 genotypes
were known for both marker systems and were included in the analysis. Genotypic
information for these loci from VDD1 obligate heterozygotes was not included in either
population analysis.
41
1
4. OBSERVED HETEROZYGOSITY
The observed heterozygosities for each polymorphism were calculated by
detennining the number of individuals who were heterozygotes for each genetic marker
system and dividing this by the total number of individu ais screened for the genetic marker
system. Average heterozygosity per population was determined by avemging the
observed heterozygosity over aIl loci studied (Vogel and Motulsky, 1986). The expected
frequencies of heterozygotes for each genetic marker system were determined by
subtracting the sum of the frequency of homozygotes for each marker system by 1. The
homozygote frequencies for each genetic marker system was based on allele frequency
data for each population observed in this study. The formulation is as follows:
Hexpected for each genetic marker system = 1 - r. Pi 2 where Pi represents the
frequency of the ith allele.
For each population, the maximum number of unrelated individuals which could bt
included in this analysis was 45 for the Saguenay-Lac St. Jean population sample and 20
for the Charlevoix population sample. Sample sizes vary for each genetic marker system
due to a number of individuals where genotypic information was unknown. Because of
the differences in sample sizes between populations, the standard errors of the observed
heterozygosity values were also determined. The standard error was calculated by
multiplying the observed heterozygosity by one minus the observed heterozygosity and
dividing by the sample size for each genetic murker system.
5. TESTS OF SIGNIFICANCE
To test allele frequency differences amongst populations, a heterogeneity chisquare test statistic was performed and the P values reported.
To determine the
significance of the observed genotypic association between the COL2A 1 markers,
pKEV4/Pvu II and pKEV4/Hind III, a goodness-of-fit chi-square test statistic was
performed. This test assessed the goodness-of-fit of the observed data to expectcd
genotype frequencies based on Hardy-Weinberg assumptions of random gametic
associations between alleles of different loci (Hartl and Clark, 1989; Sokal and Rohlf,
!,
42
1987; Colton 1974). Expected values of each genotype were obtained by multiplying the
Hardy-Weinberg allele frequencies (from data set) by the sample size.
6. LINKAGE DISEQUILIBRIUM PARAMETER: P
The linkage disequilibrium parame ter, D, gives a quantitative measure of the
amount of linkage disequilibrium or non-random association between aUeles of different
loci or genes. The calculation of the linkage disequilibrium parameter, D , was detennined
for two polymorphisms within the COL2AI gene: pKEV4/Hind III and pKEV4/Pvu II.
Linkage disequilibrium was tested by determining the gametic associations between the
two alleles for each of the two marker systems.
The observed disequilibrium was determined [lfst by calculating the observed
frequencies for the four gametic classes for these two marker systems and subtracting the
product of the heterozygous gametic frequencies from the homozygous gametic frequency
products such that:
D (observed) = Pl1 P22 - P12P21
where PlI = Plq}. Pl2 = Plq2, P21 = P2ql, and P22 = P2q2
and Pl, P2 are alleles 1 and 2 of the pKEV 4/Pvu II genetic marker system and q 1, q2 are
alle1es 1 and 2 of the pKEV4/Hind III genetic marker system.
The theoretical maximum values disequilibrium can attain, given the allele
frequencies for the two marker systems is given by:
D (maximum) = minimum of Pl <12, P2Q 1
and D' =D / Dmaximum where D' is the percentagc the D(observed) is of the maximum
value that the linkage disequilibrium can attain (Hartl and Clark, 1989; Nei, 1987).
7. GENETTC DISTANCE PARAMETERS
The genetic distance measure (D) was calculated using the formulation of Nei
(1987). This measure determines the amount of genetic divergence or genetic distance
between populations by estimating the number of genèS or codon substitutions per locus
per generation due to genetic drift (Nei, 1987). The basis of this measure is the
"nonnalized identity statistic (1)", which is essentially the probability that an allele derived
mndomly from one population will be indistinguishable from an allele derived randomly
43
from another population, relative to the probability that two alleles derived from the same
population will he indistinguishable.
1 is calculated from the following formula:
1 = J xy ! (JxxJyy)i12 where Jxx = 1: Xi 2, where Xi is the frcquency of the ilh
allele in population X (Saguenay-Lac St. Jean); Jyy = 1: Yi 2 , where Yi equals the
frequency of the ith allele in population Y(Charlevoix), and Jxy = 1: xiYi is the probability
that two alleles are indistinguishable when one allele is chosen from population X and the
other Y. When 1 equals 1, the populations are genetically similar.
The standard genetic distance measure is the negative log of the normalized identity
value:
D = -ln (1).
When the geneti,. distance measures equals 0, no genetic divergence has occurred between
the two populations for the loci studied (Nei, 1987; Hartl and Clark, 1989).
To calculate the normalized identity (1) and standard genetic distance (D) for the
ni ne genetic marker systems studied, allele frequencies for each system were obtained
from the data, and the Jxx ' Jyy and Jxy values were averaged across ail loci (Nei, 1(87).
Since the standard genetic distance measure is calculated from samples of populations, the
values of D may vary from one sample to another. The degree to which different samples
produce varying D values is reflected by the number of genes studied and sample size
(Nei, 1987; HartI and Clark, 1989). Due to sample size fluctuation across the genetic
marker systems and the smallness of the Charlevoix sample, D and 1 values were also
calculated using a correction for sampling elTor:
I(cor) = Jxy / (Jxx Jyy )l/2
where corrected values of Jxx and Jyy are tabulated according to :
Jxx = 2nx (1: Xi2 - 1) !2nx - 1
Jyy = 2ny (1: y i2 - 1) !2ny - 1
where 2n = number of genes studied.
To calculate adjusted 1 and D values for sample size variation over ail loci, weighted
averages of Jxx , Jyy and Jxy values based on the number of genes sampled for each
RFLP within each population were determined:
44
Jxx(weighted average) = E Jxxi x #Of genes 1 total number of genes
45
RESULTS
The genotypes for nine RFLPs for individuals from the Saguenay-Lac St. Jean
.
and Charlevoix regions of northeastern Quebec were determined using seven polymorphie
loci on proximal chromosome 12 q. The loci studied were: COL2A l, ELA l, D 12S 15,
D12S4, D12Sl4, D12S17 and D12S6. Figure 2 shows the order of the loci und the
estimated map distances (Q'Connell et al. 1987; Labuda et al. 1990). Representative
autoradiographs for selected genetic marker systems, pKEV4/Hind III (COL2A 1),
pKEV4/Pvu II (COL2Al), pWEAV214/Hinfl (COL2AI), pCMM1.2/Taq 1 (DI2St5),
and p YNH15/Msp 1 (D 12S 17) are shown in Figures 4, 5 and 6.
I, ALLELE FREOUENCY yARIATION
Table 4 presents the allele frequencies for the nine genetic marker systems stlldied
in the Saguenay-Lac St. Jean population, the Charlevoix population lll1d the published
data. The number of chromosomes studied for each locus averaged 99 for the SaguenuyLac St. Jean population sample, 43 for the Charlevoix population sample and 118 for the
literature data on Caucasians. Normal chromosomes from VDDt obligate heterozygolls
parents of Saguenay-Lac St. Jean and Charlevoix origin were included in their respective
population sample. Ali allele frequencies were generated by counting.
The chi-square test of heterogeneity of allele frequencies among the lhrcc
populations did not differ significantly for seven of the nine RFLPs studied. RFLPs al the
loci, ELAl and DI2Sl: were statistically significantly different among the three groups:
p = 0.0143 and p = 0.0001 respectively. The allele frequencies for ELA t and D12S 15
were not statistically different between the Saguenay-Lac St. .Jean and Charlevoix
samples: p = 0.1635 (X 2=1.942, df=}) and p = 0.1466 <X 2 =2.107, df=l) respectively.
Allele frequencies for the seven other RFLPs were also not statistically dlfferent hetween
the Saguenay-Lac St. Jean and Charlevoix population samples.
n,
OBSERYED HETEROZYGOSITIES FOR THE GENETIC MARKEH
SYSTEMS STUDIED
46
a
b
~666666
Indivlduals: 1 2 3 4 5 6 7
2.0kb . . . . '
1.7kb ...
•
1.0kb ...
Constant Sand
O.8kb ...
O.7kb ...
I:UR~
• __ ••• -
Figure 4: Representative Autoradiographs For a) the COL2A1 Locus (pWEAV 214/
Hinf 1) and b) the D12815 Locus (pCMM 1.2ITaq 1)
a) reveals genotypes for the pWEAV 214/Hinf 1 genetic marker system for 7 nonrelated individuals and b) represents genotypes for the pCMM1.2rraq 1 genetic
marker system for an ARSACS pedigree.
47
1
ar:tr<t
-'6-
~6or-T"6-6,..---r-6
b GrQ
--..
14.0kb ....
•
7kb . . . . .
4.0kb~
3.2kb~
2.6kb~
•
_.
_....
-
D-r2.66 - 0
op
~
e ......
....
_...
-
Figure 5: Representative Autoradiographs For a) the D12S17 locus (pYNH 15/
Msp 1) and b) the COL2A11ocus (pKEV 4/Hind III)
a) reveals genotypes for the genetic marker system pYNH 15/Msp 1for an ARSACS
pedigree while b} reveals genotypes forthe genetic maw.er system pKEV 4/Hind III
for two Saguenay-Lac St. Jean "control" families.
48
,
œ-pI
3.3kb .... -
660'Ô •• 6
fil • • • . . . . . .
1.7kb.....
1.6kb.........
•
...
•
~
-" . . . . . .
Figure 6 : Representative Autoradiograph For the COL 2A1 Locus (pKEV4/Pvu Il)
Genotypes are revealed forthe pKEV4/Pvu Il genetic marker system for an extended
ARSACS pedigree. The Pvu Il polymorphie site is loeated near the middle of a 3.3
kb fragment. If the site is preserlt, 1.6 kb and 1.7 kb fragments result (Vaisanen et al.
1988). The RFLP system was interpreted as a di-allelie system (with the 1.6 kb
fragment interpreted as a constant band) aeeording to Vaisanen et al. 1988.
49
Locus
Probe
(Enzyme)
Allele
ELAI
pcXPI3
1
St. Jean
- Salluena~.Lac
Aibre
'01
Fl'lqu.1lC) chromosomes
Charlevoix
Allele
'01
Fl'lqulnclts chromosom..
65
35
99
TT
23
39
2
Publlsh.d Dall
Allale
'of
Fl'lqulnclll chromolOl11"
82
18
x2
among
population.
116
850*
dt.2
012S15
pCMMI2
1
2
30
70
98
42
58
48
15
85
200
1917*
dt.2
01254
p9Fl1
1
2
61
99
70
46
64
36
18
116
dt.2
1
2
3
20
37
44
45
15
72
471
dt-4
54
190
027
dt.2
354
dt.2
COL2AI pWEAV214
30
39
43
102
40
50
50
46
104
53
47
32
57
43
96
38
62
97
30
70
44
36
64
B6
1
2
3
15
67
18
104
18
59
23
44
17
63
168
091
dt.4
1
2
18
103
29
33
42
114
515
dt.6
COL2/1
pKEV4
(Pvu Il)
1
2
.54
COL2Al
pKEV4
(Hmdltl)
1
2
52
01256
p1H-7
1
2
012S17
pYNH 15
012S14
pEF0332
3
4
104
36
32
32
46
48
.34
03
45
02
36
46
098
dl.2
20
15
39
05
41
*Allele frequencies among the populations differed signrflcantly for RFLPs at lOCI ELA1 and
012515: p= 0.0143 and p = 0.0001 respectively.
Table 4: Allele Frequencies Amongst The Saguenay-Lac St. Jean and Charlevoix
Populations and Published Data For The Genetic Marker Systems Studied
Allele frequencies were generated by the gene counting method from non-related
individuals. Allele frequency and sam pie size information for published data represents published information on Caucasians from Human Gene Mapping 10(1990)_
For the Saguenay-Lac St.Jean sample, a maximum of 40 individuals (80 chromosomes) of parental status were available from Saguenay-Lac St. Jean "control"
families, 16 normal chromosomes from obligate VDD1 heterozygotes and 8 chromosomes from four obligate ARSAC heterozygote Individuals. The CharlevoIx
sam pie consisted of a maximum of 54 normal chromosomes: 14 from obligate VDD1
heterozygotes anJ 40 from obligate ARSACS heterozygous individuals. Sample size
variation between genetic marker systems is due to unknown information for specifie
individuals in eaeh population.
50
(
Observed and expected heterozygosities for each RFLP are presented in Table 5. The
proportion of observed heterozygotes for each genetic marker system within the
Charlevoix sample ranges from 0.27 to 0.64. The proportion of observed heterozygotes
for cach genetic marker system within the Saguenay-Lac St. Jean population sample
ranges From 0.41 to 0.80.
Because of a much smaller sample size, the observed
hctcrozygositics for the Charlevoix population sample have a much higher standard error
than the Saguenay-Lac St. Jean population sample. The observed heterozygosities for 7
of the 9 genetic marker sytems studied are elevated in the Saguenay-Lac St. Jean
population sample as compared to the Charlevoix population sample even though the
expected heterozygosity values are similar in both populations.
The average
heterozygosity in the Saguenay-Lac St. Jean population sample is higher, 0.64 as
compared to 0.50 for the Charlevoix population sample. Both these values reflect the high
polymorphie information content of these genetic marker systems and their usefulness for
family linkage studies.
III. GENETIC VARIATION WITHIN THE COLZA) GENE
The RFLPs in the COL2A 1 gene have been useful in analyzing linkage
relationships (Anderson et al. 1990; Labuda et al. 1990). Constructing haplotypes
between the closely-linked RFLPs is often useful to increase the informativeness of
families under study. However within the COL2A 1 gene, several previous reports have
shown significant linkage disequilbrium between the two physically close RFLPS detected
by the probe pKEV4 with Hind III and Pvu II restriction enzymes (Vaisanen et al. 1988
and Schwartz et al. 1990)
In this study significant genotypic association between the RFLPs detected by the
probe pKEV4 and the restriction enzymes, Hind III and Pvu II, was detected both in the
Saguenay-Lac St. Jean and Charlevoix population samples. This genotypic association is
summarized in Table 6. Generally, if an individu al was homozygous or heterozygous for
51
Population
Locus
Probe
Sasuenay.Lae St. Jean
# Individuals H observed
H
typed
±SE
erpecled
# In~=als
Charlevoix
Hobserved
±SE
H
expecled
COL2A1
pKEV4 (PVU Il)
42
0.69 ± 0.08
0.50
16
0.50±0.13
COL2A1
pKEV4 (Hind III)
42
0.57 ± 0.08
0.50
16
0.44±0.13 0.49
COL2A1
pWEAV 214
42
0.76 ±0.08
0.64
13
0.54±0.13 0.68
ELA1
pcXP13
38
0.80 ±0.09
0.46
11
0.64±0.15
D12815
pCMM 1.2
39
0.56 ± 0.08
0.42
16
0.63±0.13 0.50
01284
p9F11
37
0.62 ± 0.08
0.48
16
0.31 ± 0.13
012817
pYNH 15
43
0.58 ± 0.07
0.50
15
0.60±0.13 0.58
012814
pEFO 33.2
41
0.76 ± 0.07
0.65
14
0.57±0.12 0.69
01286
p11·1·7
38
0.41 ± 0.08
0.48
11
0.27±0.17 0.43
0.51
0.36
0.49
Table 5 : Observed and Expected Heterozygosities For Loci Studied Amongst The
Saguenay-Lac St. Jean and Charlevoix Populations
Sample errors are approximates based on a binomial distribL1tion. Sample size
fluctuation between genetic marker systems is due to unknown genotypes for sorne
individuals. Genotypes fram VDD1 obligate heterozygotes were not included in this
analysis for either population.
52
KEV4
e
Pvu Il
Rlna III
1, 1
1, 1
1, 1
1,2
1,2
1,2
2,2
2,2
2,2
1, 1
1,2
2,2
1, 1
1,2
2,2
1, 1
1,2
2,2
Saguenay-Lac St. Jean
Expected
~6servea
6
1
0
6
23
0
0
0
6
-
Total = 42
1.9
4.9
3.3
3.9
10.3
6.8
1.9
4.9
1.9
-
42.0
Charlevoix
Observed Expected
-
3
0
0
0
7
0
0
0
6
16
0.5
1.3
0.9
1.3
3.7
2.7
0.9
2.7
1.9
-16.0
Table 6: Genotypic Association Setween the RFLPs Detected Sy pKEV4 With
Hind III and Pvu Il within the COL2A1 Gene in the Saguenay-Lac St. Jean and
Charlevoix Populations
Significant genotypic association between the RFLPs detected by the probe. pKEV4
with Hind III and Pvu Il restriction enzymes was observed within the Saguenay-Lac
St. Jean population: X2 = 54.47. df =6 (p< 0.001). Significant association was also
observed between these two RFlPs within the Charlevoix population: X2 = 34.09. df
= 6 (p<O.001 ). Genotypes of 16 ARSAC obligate heterozygotes were included in the
Charlevoix sam pie. Genotypes of 38 non-related individuals of parental status from
the Saguenay-lac St. Jean "control" families and obligate heterozygotes from 2
ARSACS families of Saguenay-Lac St. Jean origin comprise the Saguenay-lac St.
Jean population sample.
53
one polymorphism. the individual was also homozygous or heterozygous for tht: other
polymorphism (genotypic data from VOD1 obligate ileterozygotes were Ilot inc1uded in
this analysis for either population). Table 6 compares the obscrved frequencics of the
genotypes versus the expected frequencies in each population. The calculation of expt:cted
genotype frequencies was based on Hardy-Weinberg expectations of random association
between the alleles of each RFLP with each other. Significant genotypic association
between the two polymorphisms was detected when a goodness-of-fit X2 statistical test
between observed and expected values was performed for both the Saguenay-Lac St. Jean
and Charlevoix populations, X2
= 54.47, df =6 (p < 0.001) and X2 = 34.09, df =6 (p <
0.001) respectively.
Since significar.t genotypic association was observed for these two RFLPs within
the COL2AI gene, the amount of linkage disequlibrium (D) existing between thcse two
RFLPs was quantified for both the Saguenay-Lac St. Jean and Charlevoix populations.
Tables 7 and 8 indicate the results of this analysis. Within the Charlevoix population
sample,
con~isting
of 46 phase-known chromosomes (32 from unrelated individuals from
ARSACS families, and 14 normal, phase-known chromosomes from VDD 1 obligate
heterozygotes) an estimated linkage disequilbrium value (D) of 0.23 was obtained. The
maximum value that the linkage disequilibrium parame ter could attain for this set of data is
0.24. Therefore, 98% of the maximum theoretical value the linkage disequilihrillm
parameter could auain was observed in this sample. Within the Saguenay-Lac St. Jean
population sample consisting of 80 phase-known chromosomes (16 normal VDD 1
obligate heterozygotes chromosomes, 60 chromosomes from unrelated individuuls from
the Saguenay-Lac St. Jean control families and 4 chromosomes from unrelated
individual~
from the Saguenay-Lac St. Jean ARSACS families) an estimated D value of 0.21
wa~
attained. This value was 88% of the theoretical maximum value that the linkage
disequilibrium parameter can attain. These results indicate that these two genetic markcr
"
systems within the COL2A 1 gene are not independent of each other.
The
re~lIlt ...
54
Gametle
Ha"'otr,pe
Pvu " H nd III
%
1, 1
1,2
2, 1
2,2
0.53
0.01
0.08
0.39
Frequency
N=80
42
D
D-MAX
Observed
Theoretlcal Maximum
Disequlllbrium
Disequlllbrium
0.21
0.24
Observed
Disequillbrium
as % of Maximum
Disequlllbrium
88%
1
6
31
Table 7 : Maximum Linkage Disequilibrium Values For Two RFLPs Within the
C0L2A1 locus: pKEV4/Pvu Il and pKEV4/Hind III Within The Saguenay-Lac St.
Jean Population Sample
The Linkage Disequilibrium Parameter (0) gives a quantitative measure of the
amount of linkage disequilibrium or non-random gametic association of aile les of
different genes or RFLPs.
55
...
~
Gametic
Ha~lotrrPe
Pvu l, Hnd III
1, 1
1,2
2, 1
2,2
Frequency
%
N ~~46
0.48
0.02
0.02
0.48
22
0
D-MAX
Observed
Theoretlcal Maximum
Disequlllbrium
Disequlllbrium
0.23
0.24
Observed
Disequlllbrium
as % of Maximum
Disequlllbrium
980/0
1
1
22
Table 8 : Maximum Linkage Disequilibrium Values For Two RFLPs Within the
COL2A1Locus: pKEV4/Pvu Il and pKEV4/Hind III Within The Charlevoix Population
Sample
The Linkage Disequilibrium Parameter (0) gives a quantitative measure of the
amount of linkage disequilibrium or non-random gametic association of aile les of
different genes or RFLPs.
L
56
presented here are also consistent with similar results obtained by Schwartz et al. (1990)
and Vaisanen ct al. (1988). Thus, testing individuals with pKEV 4 for both the Pvu n and
Hind III RFLPs is not profitable for providing linkage infonnation.
Among the 58 individuals screened for this genotypic analysis (42 from the
Saguenay-Lac St. Jean sample and 16 from the Charlevoix population sample), 55% of
the îndividuals from the Saguenay-Lac St. Jean region and 44% of the individuals from
Charlevoix, were found to be heterozygous for both the Pvu II and Hind III
polymorphisms (Table 6).
The aJ1e 1e frequencies of the three polymorphisms within the COL2Al gene,
detected by pKEV4 with Hind III, Pvu II and by pWEAV214 with Hinf l, previously
reported for other Caucasian and non-Caucasian populations are presented in Table 9.
The Chi-square test of heterogeneity of allele frequencies for each polymorphism amongst
the populations did not differ significantly: for the pWEAV 214/Hinf 1 genetic marker
system, X2 =4.708, df =4 (p = 0.3186), for pKEV4/Pvu II genetic marker system, '1.2 =
2.234. df = 3 (p = 0.5254), and for pKEV4/Hind III genetic marker system, X2
=7.685,
df = 4 (p = 0.1038).
Haplotypes construcicd from the three polymorphisms are shown in Table 10 for
the Saguenay-Lac St. Jean and Charlevoix populations. Since the polymorphisms have 3,
2, and 2 alleles respectively. the maximum number of haplotypes that can he generated is
12 (2 x 2 x 3). In the Saguenay-Lac St. Jean sample of 73 phase-known chromosomes,
(consisting of 55 chromosomes from parents of Saguenay-Lac St. Jean control families, 2
chromosomes from unrelated Saguenay ARSACS individu ais and 16 normal
chromosomes from Saguenay VDD1 obligate heterozygotes) 8 of the 12 haplotypes were
observed. In the small Charlevoix sample consisting of 39 phase-known chromosomes
(15 nonnal chromosomes from VOD1 heterozygote parents of Charlevoix origin were
included as well as 24 chromosomes from parents of the ARSACS families of Charlevoix
origin), 6 of the 12 haplotypes were observed. A chi-square test of heterogeneity of
57
Probe/Enzyme
Population
pKEV4/HIND III
Sykes et al. (1985)
English
U.S. Whites
Eng and Strom (1985)
Eng and Strom (1985)
U.S. Blacks
This study
Fr. Canadian (Saguenay)
Fr. Canadian (Charlevoix)
pKEV4/PVU Il
Sykes et al. (1985)
English
Valsanen et al. (1988)
Finnish
This study
Fr. Canadian (Saguenay)
Fr. Canadian (Charlevoix)
pWEAV 214/HINF 1
Weaver et al. (1989)
This study
Caucasian
Fr. Canadian (Saguenay)
Fr. Canadian (Charlevoix)
Allele Frequencles
1
0.46
0.65
0.52
n.52
0.53
0.54
0.35
0.48
0.48
0.47
1
0.45
0.54
0.54
0.50
0.55
0.46
0.46
0.50
Numberof
chromosomes studled
2
126
96
46
104
32
2
1
2
0.45
0.43
0.36
0.15
0.20
0.32
72
190
104
46
3
0.40
0.37
0.32
72
102
44
Table 9 : Allele Frequencies For The RFLPs Within The COL2A1 Gene Amongst
Various Populations.
58
SaguenaI-Lac St. Jean
COL 2A1 Ha~lotl~
A Frequency
pKE'r/pvür pREv47Rlna III
pwE~V 21:it/Rlnf 1
2
3
1
1
2
3
2
2
2
1
1
1
2
2
2
1
1
1
1
1
2
2
3
1
1
2
2
2
2
2
1
2
3
1
1
1
12
16,.
24
3
10
0
0
~
a
-
4
1
0
Total =73
0.16
0.22
0.04
0.33
0.04
0.14
0.00
0.00
0.00
0.05
0.02
0.00
-1.00
Charlevoix
A Frequency
12
7
0
13
a
5
1
a
a
1
a
a
-39
0.31
0.18
0.00
0.33
0.00
0.13
0.03
0.00
0.00
0.03
0.00
0.00
-1.00
Table 10 : Number of Observed COL2A 1Haplotypes (pKEV4/Pvu Il, pK EV4/Hind lU,
pWEAV 214/Hinf 1).
Saguenay-Lac St. Jean haplotypes were ascertained tram 55 phase-known parental
chromosomes tram Saguenay "control" tamilies, 2 phase-known chromosomes tram
ARSAC obligate heterozygotes and 16 phase-known normal chromosomes tram
VDD1 obligat9 h~terozygotes. Charlevoix haplotypes were asc6rtained trom 14
pha~e-known normal chromosomes of VDD1 obligate heterozygotes and 25 phaseknown chromosomes from ARSAC obligate heterozygotes. No statistically signiticant difterence in haplotype distribution between the two population samples was
detected : "I!= 8.743, dt = 8 (p=O.3644)
59
haplotype distribution between the two populations did nol differ signitïcantly: X2
8.743, df = 8 (p
=
= 0.3644).
IY. yARIATION IN HAPLOTYPES CONSTRUCTED FROM TIIE LOCI;
012S17. 012S14. AND D12S6.
The three loci, D12S17, 012S14, and 012S6 are tightly linked as revcaled hy
linkage analysis studies. Tight linkage of 012S14 to 012S17 (lod score value of Z =
11.23 , 9
= 0.00) and to
D12S6 (Z
= 4.70, e = 0.019)
was reported by Lahuda et al.
(1990). Therefore, the polymorphisms revealed at these loci by the following genctic
marker systems: pYNH 15/Msp l, pEFD33.2/Msp l, p 11-1-7IMsp 1 carl he considered us
a haplotype. Since the RFLPs have 3,4, and 2 alleles respective1y, the maximum nllmbcr
of haplotypes that can be observed is 24 (Table 11). Labuda et al. (1990) oiJserved
significant linkage disequilibrium between one haplotype (haplotype A) and chromosomes
bearing the VODI disease allele. By contrast, the same study showed that in a sample of
15 normal chromosomes of Saguenay-Lac St. Jean heterozygous parents, only one
chromosome with haplotype A was observed. To determine the frequency of this
haplotype in a larger sample of the Saguenay-Lac St. Jean population, haplotypes
generated by these loci were studied in the 20 Saguenay-Lac St. Jean "control" families.
Unfortunately, phase could not be determined in two families.
Haplotypes were
determined for 74 phase-known chromosomes (representing 17 families where hoth
parental chromosomes were counted and one family where data from 3 grandparents
wa~
available) and 15 phase-knuwn normal chromosomes fromYDDI heterozygolls parents
(Labuda et al. 1990) for a total of 89 chromosomes.
Of the 89 chromosomes, 6
chromosomes (6.7%) carried haplotype A as compared to 13 of 18 (72.2%) chromosomes
with the VDDI disease allele which carried haplotype A (Dr. M. Lahuda, Dr. F. Il.
Glorieux and Dr. K. Morgan, personnal communication). Based on allcle frequcncy data
determined from the Saguenay-Lac St. Jean sam pIe (Table 4) and assuming random
gametic association between alleles of different
gene~,
the expccted frequcncy of thl..,
60
(
012514
pEF033.2
D12517
pYNH15
01256
p11-1-7
B
2
4
2
0
3
E
F
G
H
1
2
2
1
2
2
1
3
2
J
K
2
1
4
1
1
1
1
1,.
t.
1
3
2
1
2
L
2
M
N
P
4
2
1
3
4
4
4
3
3
3
2
3
2
2
1
2
3
3
3
2
1
3
1
2
1
1
2
2
1
2
Haplotype
A
e
1
a
R
S
T
U
V
W
X
y
1
1
1
2
3
3
Saguenay-Lac St. Jean
Chromosomes
1
1
2
1
2
1
1
2
Total =
fJ
~requency
6
25
.07
.28
.03
.01
.12
0
.08
.05
.03
.06
.01
.09
.05
.02
.01
.01
.01
.03
.02
0
0
.01
0
0
3
1
11
0
7
4
3
5
1
8
4
2
1
1
1
3
2
0
0
1
0
0
-89
-
1.00
Sa%Uenay-Lac St. Jean
V DI Chromosom,. ~
R
13
3
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
~requenéy
.72
.17
0
0
.11
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-
0
18
-
1.00
Table 11 : Number of Observed Haplotypes Constructed From Three Loci: 012S14,
012517, 01256 in The Saguenay-Lac St. Jean Population
Observed haplotypes constructed trom RFLPs revealed at chromosome 12 loci :
012514, D12517, 01256, with the tollowing genetic marker systems pEFD 33.2/
Msp l, pYNH 15/Msp l, p11-1-7/Msp 1 respectively. The Saguenay-Lac St. Jean
population sample of 89 chromosomes consisted of 73 phase-known chromosomes,
representing 18 1/2 individuals of parental status from Saguenay-Lac St. Jean
"control" families, and 16 phase-known normal chromosomes from obligate VOD1
heterozygotes. VDD1 chromosomes refer ta chromosomes bearing the VDD1
disease aile le. Haplotype data on VDD1 chromosomes reprinted with permission
from Dr. M. Labuda.
r
l
61
haplotype is 0.019 (0.34 x 0.15 x 0.38). This haplotype was observed in this sample nt a
frequency of 0.07. The most common haplotype observed in the Saguenay-Lac
~t.
Jean
sample was haplotype S, observed with a frequency of 28% (25 / 89). From aHele
frequency data for each of these genetic marker systems ann assuming mndom gmnetic
association between the alleles, S is the most frequent haplotype that is expected to he
observed at a frequency of 0.19. The next most common haplotype observed in this study
was haplotype E with a frequency of 0.12. Three of the eighteen Saguenay-Lac St. kan
chromosomes carrying the YDDl disease aIle le analysed in this study were bearing the
haplotype Sand 2 chromosomes, haplotype E.
Table Il also presents the large diversity of haplotypes that were observed in titis
sample of the Saguenay-Lac St. Jean population. From a maximum of 24 haplotypcs thal
cou Id he constructed with these markers, 19 were observed in a relatively small samplc
size of 89 chromosomes.
V,
GENETIC DIVERGENCE BETWEEN POPULATIONS
The standard genetic distance measure between populations was calculated by
Nei's formula (1987). The normalized identity (1) expresses the probability lhal
il
randomly chosen allele from each of the two different populations will be
indistinguishable, relative to the probability that two randomly chosen
allele~
From lhe
same population will he indistinguishable. If the two populations are identical, 1 = 1. The
standard genetic distance measure (D) measures the amount of genetic divergence bctween
populations. If there is no genetic divergence D =O. Table 12 shows the results of these
calculations. For the loci studied, the normalized identity calculation belwecn ail
populations sampled is very close to 1, indicative of genetic similarity between the
populations. The genetic distance measure, D, ranges from 0.01 to 0.03.
The~e value~
are very close to 0, representing no significant genetic divergence betwcen the
populations. The value of 0 is known to fluctuate From sample to sample based on the
number of genes studied and sample sizes (Nei, 1987). Therefore, new 1 and D value~
62
were recalculated according to Nei's correction for small sample sizes (Nei, 1987). The
(
adJusted values across aIl loci studied gave values of 1 that were equal to 1 and
subsequent standard genetic distance measures of 0 for aU three population comparisons
(results not shawn). These results, for the loci on proximal 12q, indicate the populations
are genetically similar and no genetic differentiation has occurred between them.
63
Population
1. Saguenay-Lac St. Jean - Charlevoix
2. Saguenay-Lac St. Jean - Published Data
3. Charlevoix • Published Data
1
D
0.98
0.99
0.97
0.02
0.01
0.03
Table 12 : Normalized Genetic Identity (1) and Standard Genetic Distance (0)
Between Populations
Standard Genetic Distance (0) and Normalized Genetic Identity (1) measures were
calculated according ta the formulation of Nei (1987) utilising allele frequency data
for ail genetic marker systems studied in each respective population (Table 4).
64
DISCUSSION
I.
FOUNDER EFFECT FOR VITAMIN D DEPENDENT RICKETS TYPE
1 IN THE SAGUENAY-LAC ST . .JEAN POPULATION
The attainment of polymorphie frequencies of greater than 0.01 for diseaseconferring alleles in small human populations, is presumably the result of founder effect
and genetic drift (Cavalli-Sforza and Bodmer, 1971). These small populations or
isolates, are derived from a finite number of ancestors and the subsequent utilization of
genealogical reconstructions has revealed that il is possible for the se populations to attain
polymorphie frequencies of deleterious alleles that presumably occurred in the carrier
state in a few ancestors (Livingstone, 1987). The Old Order Amish, as previously
discussed, provide examples of this phenomenon. The rapid expansion of small
populations also, presumably, contributes to this phenomenon (Livingstone, 1987).
One such population which has historically been characterized by a rapid growth
rate and, more recently, by the elevated prevalence of a number of hereditary diseases (De
Braekeleer, in press) is the French Canadian population of Quebec. This population has
shown remarkable population growth since the initial colonization of Quebec in 1605.
Today's population of over six million are primarily descendants of the the 8,500
immigrants who settled permanently in Quebec between 1605 and 1759 (Charbonneau
and Robert, 1987). After the English Conquest, with little or no significant immigration
to Quebec from France, the French Canadian population grew primarily due to natural
fertility (Laberge, 1976).
The French Canadian populations of northeastem Quebec have anained significant
polymorphie frequencies of several hereditary disorders. Hereditary tyrosinemia has a
frequency of 0.025 in the Chicoutimi district (La berge and Dallaire, 1967), spastic ataxia
of the Charlevoix-Saguenay type a frequency t.f 0.026, and polyneuropathy has a
frequency of 0.02 to name a few (De Braekeleer, in press). The Saguenay-Lac St. Jean
and Charlevoix populations of northeastem Quebec, which are charaeterized by the
65
elevated frequencies of the above mentioned hereditary disorders and others (De
.
Braekeleer, in press), are both populations which have experienced rapid population
growth and expansion since the establishment of white settlements in the regions (Roy et
al. 1988; De Braekeleer, in press). TIle elevated frequencies of hereditary d\sorders
1Il
the se populations may be the result of founder effect, in which the gene was introduccd
intI) the population, and the subsequent spreading in the population, either the result of
genetic drift, natural fertility or both (Cavalli-Sforza and Bodmer, 1971: Livingstone,
1987).
Of relevance to this study is the elevated gene frequency of the Vitamin D
Dependent Rickets Type 1 (VDD1) disease allele in northeastern Quebec.
The
polymorphie frequeney in this population for VDDl is 0.02 (De Braekeleer, in press;
Bouchard et al. 1985). From aIl indications, the elevated frequency of VDD 1 in this
region is theoretically the result offounder effeet and genetic drift. On the basis of recent
evidence From molecular biology studies, this hypothesis seems to he supported.
The mapping of VDDI to chromosome 12q 14 by Labuda et al. (1990) revealed
evidence for significant association between a three marker haplotype, haplotype A, and
chromosomes bearing the VDD1 disease allele. When a new mutation is introdllced and
subsequently spreads throughout a population, the mutation is initially inherited with
other alleles at nearby loci that were present on the originating chromosome (Lander and
Botstein, 1986). Therefore, the elevated frequency of haplotype A on
chromosome~
earrying the VDDI mutation supports the hypothesis of a founder who introduced the
mutation into northeastern Quebec. However, in the study of Labllda et al. (1990), lhe
estimate of the frequency of this haplotype was based on a relatively small sample of
normal chromosomes (n=15) and a small sam pie of chromosomes hearing lhe
disca~c
mutation (n=16). This study presented results of the estimation of the frequency of thh
haplotype in a sample of the resident population of the Saguenay-Lac St. Jean region.
66
The expected frequency of haplotype A on nonnal chromosomes is 0.019 based
(
on allele frequency data for each of the genetic marker systems which comprise the
haplotype (allele frequencies estimated from Table 4 for the Saguenay-Lac St. Jean
population) and Hardy-Weinberg expectations of random gametic association between
alleles of different genes (HartI and Clark, 1989). The results of the present study, in
which 89 nomlal chromosomes from individuals residing in the Saguenay-Lac St. Jean
were analyzed, established the observed frequency of haplotype A in this population to be
0.07 (6/89), confinning that this haplotype is infrequent on nonnal chromosomes in the
general population of the Saguenay-Lac St. Jean region. Therefore, the strong
association seen between chromosomes bearing the VDDI mutation and haplotype A, and
the low frequency of this haplotype in the general population, supports the hypothesis of
a founder who introduced the mutation ioto the Saguenay-Lac St. Jean population. The
results presented here provide suppon for the hypothesis of a founder effect for VDDI to
explain the presence of VDDI in northeastem Quebec.
The recombination distance between the VDDllocus and the loci constituting the
three marker haplotype is estimated to be approximately 5.3 cM based on sex-averaged
distances (Q'Connell et al. 1987; Labuda et al. 1990). Thus, Labuda et al. (1990)
proposed that the non-A haplotypes on chromosomes carrying the disease mutation may
be the result of meiotic recombination events between the mutant chromosome and
normal chromosomes. However, the presence of non-A haplotypes on chromosomes
bearing the mutant aIlele (Labuda et al. 1990) may suggest more than one founding
chromosome and more than one mutation for this disorder. From knowledge of the basic
defect of the disease, reflected by the low levels of circulating la, 25 dihydroxyvitamin
D, more than one mutation for VDDt can not he excluded. The basic defect of the
disease could be indicative of either a mutation in one of the modulators of 1
Cl
hydroxylase, the enzyme responsibte for synthesis of the active metabolite, or a mutation
{
67
within the gene eoding for one of the components of the enzyme, more spedtkally, the
cytochrome p450 subunit.
The higher frequency of haplotype A observed in this study within the SaguenayLac St. Jean sample population of 0.07, as compared to the expected frequency of 0.019,
may be consistent with the elevated carrier frequency of the YDD 1 disease ullele in this
region: one in every 26 individuals is predicted to he a carrier of this disease allele in the
Saguenay-Lac St. Jean population (De Braekeleer, in press). ln a samplc size of H9
chromosomes, 2 chromosomes would he expected to be observed hearing haplotype A
based on the expected frequency of 0.019 in the Saguenay-Lac St. Jean population
sampie. However, in a random sample of Saguenay-Lac St. Jean residents one would
also expect to detect individuals who were carriers of the YODI mutation; the VDDI
disease aIle le is as frequent as 0.02 in this population (De Braekeleer, in press).
Therefore, if one aiso aceounts for the elevated carrier frequency of the VDD 1 disease
allele and the strong association of the VDD1 mutation with haplotype A, the observed
frequency of 0.07 (6/89) is not signifieantly different than one would expect.
The molecular genetic results obtained by Labuda et al. (1990) and this study also
support previous demographic analyses postulating a founder effeet for VOD 1 in
northeastern Quehec. A study of the mean inbreeding levels (the probability that an
indi vidual has inherited two identical copies of an allele at particular locus from each
parent) for families segregating VDD1 from the Saguenay-Lac St. Jean region, as
ascertained from genealogical reconstructions, revealed that consanguinity levels are low,
F= 1.5 x 10- 4 (De Braekeleer, 1990a). However, when the kinship coefficient is
tabulated (the probability that two unrelated individuals have inherited an allele identical
by descent at a particular locus) for the VDDI families, the value is three times greater
than the value observed in control families from the region: 8.9 x 10-4 ver!lu!l 3.0 x 10- 4
respectively. De Braekeleer (l990a) suggested that these results are indicative of a
founder effeet for VDD1 in the northeastern Quebec.
68
(
In a historieal eontext, demographie data regarding the seulement of the
Saguenay-Lac St. Jean region in northeastern Quebec indicates a strong contribution of
immigrants frf)m Charlevoix to the Saguenay-Lac St. Jean population (Gauvreau and
Bourque, 1988). This was especially notable between 1838-1872, when over 80% of
the immigrants to the Saguenay-Lac St. Jean region came from Charlevoix. After 1872,
the proportion of immigrants from Charlevoix decreased to 60%, and between 18921911, less th an 40% of the immigrants were of Charlevoix origin (Gauvreau and
Bourque, 1988). Consequently, the founder effect for VODI may have occurred in
Charlevoix and due to migration, been subsequently transferred to the Saguenay-Lac St.
Jean region (De Braekeleer, 1990a; De Braekeleer in press; Grac'..ie et al. 1988).
The founder effeet of VDDI in the Saguenay-Lac St. Jean region may reflect the
consequence of the familial type migration patterns of the Charlevoix immigrants (Roy et
al. 1988; Gauvreau and Bourque, 1988) which served to maintain a strong Charlevoix
compone nt within the Saguenay-Lac St. Jean population. Immigrants from Charlevoix
migrated to the Saguenay-Lac St. Jean region in larger family units than immigrants to the
Saguenay-Lac St. Jean originating from other regions of Quebec (Roy et al. 1988;
Gauvreau and Bourque, 1988). On average, familial immigration from Charlevoix to the
Saguenay-Lac St. Jean region consisted of a larger number of individuals, averaging 6.8
individuals per family, as compared to 3.3 individuals per family from immigrants
coming to the Saguenay-Lac St. Jean from other regions of Quebec (Gradie and
Gauvreau, 1987; Gauvreau and Bourque, 1988). Therefore, the VOO 1 mutation may
have been introduced into the Saguenay-Lac St. Jean population not once, but many
times. by closely related individuals who were carriers of the disease (De Braekeleer, i '1
press; Bouchard et al. 1984).
Il.
GENEIIC yARIATION WITHIN IHE SAGUENAY-LAC ST. JEAN
AND CHARLEYOIX POPlJLATIONS
69
On the basis of the data presented, no significant genetic differentiation for nine
neutral polymorphisms located on proximal chromosome 12 q, between the SagucnayLac St. Jean and Charlevoix sample populations was observed. The data indicates the
genetic variability of the Saguenay-Lac St. Jean and Charlevoix populations both in the
distribution of allele and haplotype frequencies. These data reveal thm these
IWO
suh-
populations of the French Canadian population do not differ significantly from othcr
Caucasian populations for the loci studied on chromosome 12q.
The bulk of the immigrants who were founders of the present-day Charlevoix and
Saguenay-Lac St. Jean populations were descendants or part of the founding population
of the present-day French Canadian population. The ancestral population, which
consisted of over 8500 individuals (Charbonneau and Robert, 1987) originated from over
38 regions of France (Charbonneau et al. 1987), the large majority of whom came t'rom
the northwestem regions of France, more specifically, the Paris area, Nonnandy, Poitou,
Aunis, Bretagne, Saintongé and Perche (De Braekeleer, 1990b).
Most of these
immigrants came to the New World as unmarried individuals rather than as rnarried
individuals with families (Charbonneau et al. 1987). One could conclude from the size
and diversity of the French Canadian founding population that considerable genetic
variability existed.
The present-day Charlevoix population is derived from a smaller number of
founders: 589 individuals. These immigrants migrated to the region betwcen 1675-1 H50
(Jetté et al. 1990) primarily from the vicinity of Quebec City. Of the 599 immigrants, 51
were individuals who did not come from any region within Quebec: 21 of whom
immigrated to Charlevoix directly from France, 25 of whom came from other European
countries such as England, Scotland, and Switzerland, and another 5 of whom came
from the United States (Jetté et al. 1990). 49% of the founders of this population had
another relation within the region, whereas over 38% of the founders had no other
apparent relation within the Charlevoix region (Jetté et al. 1990).
70
The Saguenay-Lac St. Jean population in northeastem Quebec is derived from a
(
large founding population. At the time of the first census in 1852, over 5,000 individuals
wcre residents of the region rising to over 50,000 in 1911 (Gauvreau and Bourque,
1988). Between 1838-1911, over 28,700 immigrants came to the region even though net
migration was only positive before 1870 (Gauvreau et al. 1987). Approximately 80% of
these immigrants before 1872 originated from the Charlevoix region, after whict the
proportion of immigrants from Charlevoix decreased considerably (Gauvreau and
Bourque. 1988). Immigrants to the region of non-Charlevoix origin came mainly from
the lower St. Lawrence areas and other regions of Quebec (Gradie and Gauvreau, 1987).
The historical record of these populations and more specifically, the French
Canadian population, indicates that the present day French Canadian population was
derived from a relatively large number of individuals who presumably endowed the
present day French Canadian population with extensive biologie al heterogeneity
considering their numbers, their regions of origin in France and their individual, nonfamilial status. The ancestral population was large enough to introduce multiple copies of
most genes into the population (McLellan et al. 1984). The results presented here agree
weB with the historical demography of the population. The reported resuIts reveal the
genetic similarity between the Charlevoix and Saguenay-Lac St. Jean populations as
predicted by the historical demographic data for the two populations, and their genetic
similarity with the general Caucasian population.
However, due to the increased prevalence of hereditary disorders in northeastern
Quebec, it was originally postulated that considerable genetic homogeneity existed within
these populations. This is from the expectation that the increased prevalence of
autosomal recessive disorders results from an increase in genetic homozygosity within
the population due to isolation and the consequences of elevated inbreeding levels and
genetic drift (Bowen. 1985; McKusick et al. 1964; Vogel and Motulsky; 1986).
«
AIthough these populations can not be considered as genetic isolates, they do however
~
5
71
represent geographica11y isolated regions, where rapid population growth was duc
primarily to high fertility rates (Roy et al. 1988; De Braekeleer, in press).
To investigate if elevated inbreeding levels may explain the situation, the high
prevalence of specifie hereditary disorders within these populations, De Braekeleer and
Ross (in press) investigated levels of inbreeding in the Saguenay-Lac St. Jean population
by ten year intervals from 1842 to 1971 using Catholic Church dispcnsal1ons, a method
first introduced by Moroni (1961). Within the Saguenay-Lac St. Jean region, 83,475
marriages took place during 1842-1971. Of these, a total of 3258, representing 3.9% of
aH marriages, required at least one dispensation for consanguinity during this period.
The values of inbreeding were found to be low for the whole period, reaching its highest
level of 2.3 x 10- 3 between 1902-1911 as refIected by the number of marri ages during
this interval which required dispensation: between 1892-1911, 11.2% of aIl marriages
required dispensation. Although, during this time period only 90 of 6177 marri ages were
of the first cousin relationship accounting for 1.2% of a11 the marriages which required
dispensation. Only Il of the total 83,475 marriages were of the uncle/niece relationship.
These results indicate that the Saguenay-Lac St. Jean population has lower levels of
inbreeding than inbreeding levels observed in European populations during the same time
periods (McCullough and Rourke, 1986). Between 1915-1925 and 1955-1965, the
Saguenay-Lac St. Jean population recorded the lowest mean inbreeding levels for first
and second cousins in Quebec (Laberge, 1967). Inbreeding levels for Charlevoix
calculated from Catholic Church dispensations reveal that between 1R8S-1925 the val ues
of inbreeding were found to be 3.4 x 10-4 and were slightly more elevated during the
periods between 1945-1965: 13 x 10-4 (Laberge, 1976). In both instances the values of
inbreeding are low, therefore, high levels of inbreeding could not explain the
phenomenon of elevated hereditary disorders.
The results of this study provide no evidence for genetic divergence betwecn
these two populations and the general Caucasian population for nine neutral
72
polymorphisms on chromosome 12q. In fact, for the genetic marker systems studied, the
populations were similar. This is based on genetic distance analysis using allele
frequencies of the genetic marker systems for eaeh population studied. The standard
genetic distance
mea~l.1re
ranges from 0.01-0.03; when D equals 0, the populations are
indistinguish.tble. The genetie distance measure was smallest hetween the Saguenay-Lac
St. Jean population sample and the published data: 0.01. However when Nei's standard
geneti~
distance measure is adjusted for sampling errors, an three populations are
indistinguishable. Data from many loci are required for reliable estimates of genetic
distance, particularly when the genetic distance is small (Nei, 1987).
The conclusions drawn from the data presented agree weIl with studies at other
loci. A study of allele and haplotype frequencies of HLA-A and HLA-B loci on normal
chromsomes from 16 families segregating familial hemochromatosis from the SaguenayLac St. Jean region, (chromosomes not carrying the disease-allele were considered to he
replesentative of the normal population) were compared to HLA-A and HLA-B allele and
haplotype frequencies obtained from individuals residing in the Quebec City and
Monteregie regions of the province of Quebec and individuals from France (De
Braekeleer, 1990b).
Despite a small sample size for the Saguenay-Lac St. Jean
population, similar allele and haplotype frequency distributions were observed between
aIl tluee populations. Kaplan et al (1989) observed eight different haplotypes at the
~­
globin gene cluster in 37 normal persons and in 12 ~-thalassemia heterozygotes from six
fnmilies residing in Portneuf County where ~-thalassemia minor oceurs at a 1%
frequency. The distribution of the 5' haplotypes on normal ~A Portneuf chromosomes
was closest to the distributions observed in British subjects based on genetic distance
analysis.
Other molecular biology studies within the French Canadian population also
reveal considerable genetie heterogeneity of mutations within the population. In a French
Canadian study on infantile Tay Sachs disease mutations within the hexosaminidase A
locus, Hechtman et al. (1990) describe a multiplicity of Tay Sachs disease alleles in titis
population including one mutation obselVed in famHies from southeastern Qucbec. one
mutation generally observed in the Ashkenazi Jewish pl'pulation and one mutation still
not described. Clearly, more than one founder accounts for aIl infantile Tay Sachs
disease alleles in the French Canadian population. However, one allele occurring in
southeastern Quebec was found in 18 of 22 individuals and. therefore. one t'olinder may
account for this allele. In an analysis of mutations at the phenyl alanine hydroxylasc
locus in nine French Canadian families with hyperalaninemia, haplolype analysis revealcd
that least six different mutations exist within nine stuJied families (John et al. 1989). In a
sample of affected flmllies with cystic fibrosis, from the Saguenay-Lac St. Jean region,
haplotype analysis and mutation analysis reveal that at least three CF mutations exist in
this population (Rozen et al. 1990). The frequency of the major mutation l\F508, was
significantly lower in the Saguenay-Lac St. Jean sample than in major urban Quehec
families.
In addition, within the Saguenay-Lac St. Jean sample, 86% of CF
chromosomes without the l\F508 mutation had the B haplotype (Rozen et al. 1990). In
studies on North American Caucasians of European ancestry, the majority of l\F508
mutations occur on the B haplotype (Kerem et al. 1989). Another study on
~-thalassemja
in a French Canadian sample from Portneuf Quebec reported the presence of Iwo
mutations commonly associated with
13
thalassemia within populations of the
Mediteranean region (Kaplan et al. 1989). These studies document genetic hcterogencity
of mutations for genetic disease within the French Canadian population.
By comparison, the Old Order Amish and the Hutterite
population~
of North
America are populations with an increased incidence of hereditary disorders but who also
show marked genetic divergence from their ancestral populations. Both populations wcre
founded by a small number of individuals: less than 200 for the Lancaster County OId
Order Amish (McKusick et al. 1964) and 443 individuals for the Hutterite population
(Hostetler, 1985).
74
Due to the closure of these populations to in-migration and the consequence of
increased levels of inbreeding, these populations are charactenzed as genetic isolates
(McKusick et al. 1964; Hostetler, 1985). These two populations exhibit marked genetic
d \vergence
from other Caucasian populations. One is able to detect a decrease in genetic
variability existing in these populations. Morgan et al (1980), comparing the distribution
of HLA-A and HLA-B alleles and haplotypes in Amish and Dariusleut Hutterites
demonstrated restri. Il)n in the number of haplotypes observed in these populations and
the divergence of these populations from eleven other Caucasian populations by genetic
distance analysis using HLA-A and HLA-B aIle le frequencies. The genetic distances
among three Anabaptist isolates, consisting of one Hutterite population and two Amish
groups, is much larger than genetic distances calculated between non-isolates: 0.1480.203 as compared to 0.026-0.075 (Morgan and Holmes, 1982).
The Saguenay-Lac St. Jean and Charlevoix populations, although having an
elevated incidence of hereditary disorders, do not share other features with the Amish and
Hutterite populations of North America. The Saguenay-Lac St. Jean and Charlevoix
populations were not founded by a smaU number of individuals, these populations were
not closed to in-migration and they are not characterized by high levels of inbreeding.
Therefore, the conditions which led to a decrease in genetic variability within the Amish
and Hutterite populations did not prevail in the Saguenay-Lac St. Jean and Charlevoix
regions of Quebec. This is supported by a direct comparison of HLA-A and HLA-B
haplotype distributions between a sample from the Saguenay-Lac St. Jean population
with the Dariusleut Hutterites from Alberta and the
Schrnied~(lleut
Hutterites from South
Dakota. The results of this study revealed that a larger diversity of haplotypes was
observed in the Saguenay-Lac St. Jean population sample than the Hutterite populations
(De Braekeleer, 1990b).
For two of the nine RFLPs presented here, significant statistical differences for
allele frequencies were observed between the Saguenay-Lac St. Jean and Charlevoix
75
population sample and the published data, although no significant difference for allele
frequencies was detected between these two populations of northeastern Qllehec for the
same loci. Due to severallimiting factors of this stlldy, it is unclear whether these reslilts
represent stochastic sample variation based on statistical tluctuations from sample to
sample (HartI and Clark, 1989; Nei, 1987) or if they represent true differences due to
genetic drift. For seven of the nine genetic marker systems studied, no st .. tistically
significant differences in allele frequencies between the two populations and the
published data were detected. The Charlevoix sam pIe .. Iso indicated slightly lowcr
observed heterozygosity levels for each genetic marker system as compared to the
Saguenay-Lac St. Jean population sample. However, because of a small sample size for
the Charlevoix population, the standard errors are high and when accollnted for do not
reveal a signific~nt difference.
The interpretation of the results of this stlldy have sorne limitations. Firstly, the
number of loci stlldied in this sample is small and is representative of only a specifie
region of the genome, namely, the long arm of chromosome 12q. Secondly, the genetic
marker systems studied were chosen on the availability of these markers in the laboratory
for linkage analysis studies and are therefore known for their polymorphie information
content (Botstein et al. 1980). Also, the availability of DNA samples representative of
the Charlevoix population was limited. Ten families were available for stlldy, although
the amount of DNA available was limited and consequently not ail genetic marker
systems could be completed for every individual. The families representative of the
Charlevoix sample were families afflicted with an autosomal reeessive disorder: spastie
ataxia of the Charlevoix-Saguenay type (ARSACS).
ARSACS is prevalent in the
Saguenay-Lac St. Jean and Charlevoix regions and rare outside the reglOn (De
Braekeleer, in press). Families with ARSACS, unlike YOD} families, have higher
inbreeding coefficients as compared to averagè levels observed in control familie"i from
thé same region: 1.3 x 10- 3 as compared to 3.0 x 10- 4 in controls (De Braekcleer,
76
1990a). Even though the values are elevated as compared to the VDDl families, the
values are stil1low and are not representative of highly inbred individuals. Thirdly, the
standard statistical test on large samples for genotypic deviations from Hardy-Weinberg
expectations was not perfonned on these results because of the redueed power to detect
differences due to small sample sizes.
Despite the se limiting factors, the Charlevoix sam pie population for the loci
studied indicates a great degree of genetic diversity. Genetic variability in the fonn of
allele frequencies. the number of observed heterozygotes for each genetic marker system,
and the diversity of haplotype distribution for the loci studied were detected in tbis
population sample. The Saguenay-Lac St. Jean sample used in this study was eollected
specifically for assessing genetic variation in this population at various locations across
the genome. The present study represents just one of many tbat are on-going in this
sample of families. Considerable genetic heterogeneity, based on the same parameters as
the Charlevoix sample, was also observed in this population at the seven polymorphie
loci on chromosome 12q.
However, a11 genetic variation studies are limited to the number of genes that one
can study and their distribution within the genome. Although these studies are thought be
representative of the whole genome, it is uncertain whether this is indeed the case (Hartl
anù Clark, 1989). Therefore, results sueh as those presented, represent a small sample
of the genetic variability that may exist within the population and can not quantify how
mucb genetic variability really is present in these populations. Il is still in question
whether one ean detect signifieant genetie differences between populations. Previous
studies have indicaled that li ttle biological diversity exists between race. Nei (1975)
determined that only 7% of total gene diversity would be attributaule to genetic
differences among races, which corroborated results obtained by Lewontin (1974) who
concluded that 85% of human gene diversity exists between individuals within the same
t'
population, 7.5% between major races and 7.5% between nations within races.
77
Therefore, the ability to detect genetic variability and furthemlOre, to distinguish bctween
population groups is not an easy task. The reliability of an estimate of genetic distance
between populations depends on the number of loci studied and the smaller the gencl1c
distance between populations. the more power needed to detect the differrnces. namely,
the more loci (hat must be studied (Nei, 1987).
This study has focused on an assessment of genetic variability within two French
Canadian sub-populations of northeastern Quebec. The results presented in this text
agree with results from other studies and indicate, at least for the genetic marker systems
studied, considerable biological heterogeneity within the Saguenay-Lac St. Jean and
Charlevoix populations of northeastem Quebec.
The French Canadian population of Quebec. although representing a linguistic
and cultural isolate within North America, is a biologically heterogeneous population for
the loci studied on chromosome 12q.
L
78
CONCLUSIONS
From this study it is conc1uded that:
1.
The establishment of haplotype A as an infrequent haplotype on normal
chromosomes from the Saguenay-Lac St. Jean region supports the hypothesis of a
founder effect for YDD 1 in northeastern Quebec as an explanation for the elevated
frequency of the disease in this region.
2.
An assessment of genetic variation within the Saguenay-Lac St. Jean and
Charlevoix populations of northeastem Quebec and compared to published data on
Caucasians for seven loci on proximal chromosome 12q, indicates the genetic similarity
between the se populations and the general Caucasian population. This study did not
detect any evidence for genetic differentiation of these populations from the general
Caucasian population.
3.
On the basis of the results presented here, the Saguenay-Lac St. Jean and
Charlevoix populations of northeastem Quebec represent biologically heterogeneous
populations for the loci studted on chromosome 12q.
79
LIST OF PUBLICATIONS
De Braekeleer, M., and Ross, M .. (in press). Inbreeding in Saguenay-Lac Jean (Quehec.
Canada): A Study of Catholic Church Dispensations 1842-1971. Human
Heredity.
Ross, M., Labuda, M., Morgan, K., and Glorieux, F.H. (1990). Genetic Variation of
the Saguenay-Lac St. Jean Population of Northeastern Quebec. American Journal
of Human Genetics. 47:A564.
80
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