Download Population Differences in the Polyalanine Domain and 6

Papers in Press. First published October 27, 2005 as doi:10.1373/clinchem.2005.056192
Clinical Chemistry 52:1
000 – 000 (2006)
Molecular Diagnostics
and Genetics
Population Differences in the Polyalanine Domain
and 6 New Mutations in HLXB9 in Patients with
Currarino Syndrome
Mercè Garcia-Barceló,1,2 Man-ting So,1,2 Danny Ko-chun Lau,1 Thomas Yuk-yu Leon,1
Zheng-wei Yuan,1,3 Wei-song Cai,1,3 Vincent Chi-hang Lui,1 Ming Fu,1,4
Jo-Anne Herbrick,5 Emily Gutter,6 Virginia Proud,6 Long Li,4
Jacqueline Pierre-Louis,7 Kirk Aleck,8 Ernest van Heurn,9 Elena Belloni,10
Stephen W. Scherer,5 and Paul Kwong-hang Tam1,2*
Background: The combination of partial absence of the
sacrum, anorectal anomalies, and presacral mass constitutes Currarino syndrome (CS), which is associated with
mutations in HLXB9.
Methods: We analyzed HLXB9 mutations by direct
sequencing in 5 CS families, 6 sporadic cases, and 97
healthy Chinese individuals and potentially pathologic
expansion of HLXB9 GCC repeats in patients, 4 general
populations [Chinese, Japanese, Yoruba, and the Utah
subset of the Centre du Etude Polymorphisme Humain
(CEPH)] from the HapMap project, and 45 Chinese
individuals.
Results: We identified 6 novel mutations, including 2
missense mutations affecting highly conserved residues
(Ser185X, Trp215X, Ala26fs, Ala75fs, Met1Ile, and
Arg273Cys). Allele and genotype distributions showed
marked statistically significant differences. (GCC)11 was
the most common allele overall; its frequency ranged
from 90% in CEPH to 68% in Yoruba and 50% in Chinese
and Japanese populations. (GCC)9 was almost as common as (GCC)11 in Chinese and Japanese populations,
whereas its frequency was <10% in Yoruba and CEPH
populations. The Yoruba population had the highest
frequency of the largest alleles [(GCC)12 and (GCC)13],
which were almost absent in the other groups.
Conclusions: Lack of HLXB9 mutations in some patients and the presence of variable phenotypes suggest
DNA alterations in HLXB9 noncoding regions and/or in
other genes encoding HLXB9 regulatory molecules or
protein partners. If HLXB9, like other homeobox genes,
has a threshold beyond which triplet expansions are
pathologic, those populations enriched with larger alleles would be at a higher risk. The data illustrate the
importance of ethnicity adjustment if these polymorphic markers are to be used in association studies.
1
Department of Surgery, 2 Genome Research Center, The University of
Hong Kong, Pok Fu Lam, Hong Kong SAR, China.
3
Department of Pediatric Surgery, China Medical University, Shenyang,
China.
4
Department of Surgery, Beijing Children’s Hospital, Beijing, China.
5
Program in Genetics and Genomic Biology, The Hospital for Sick
Children, Toronto, Canada.
6
Department of Pediatrics, Children’s Hospital of the King’s Daughters,
Norfolk, VA.
7
Department of Obstetrics and Gynaecology, Mount Sinai Hospital, University of Toronto, Toronto, Canada.
8
St. Joseph’s Hospital, CHC Phoenix Genetics Program, Phoenix, AZ.
9
Surgical Unit, University Hospital, Maastricht, The Netherlands.
10
IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, and IEO,
Istituto Europero di Oncologia, Milan, Italy.
* Address correspondence to this author at: Division of Paediatric Surgery, Department of Surgery, University of Hong Kong Medical Centre, Queen
Mary Hospital, Hong Kong SAR, China. Fax 852-2817-3155; e-mail
[email protected].
Received June 16, 2005; accepted October 10, 2005.
Previously published online at DOI: 10.1373/clinchem.2005.056192
© 2006 American Association for Clinical Chemistry
The Currarino syndrome (CS; OMIM 176450) has been
described as a triad of partial sacral agenesis with intact
first sacral vertebra (sickle-shaped sacrum), presacral
mass, and anorectal malformations (1–3 ). The spectrum of
anorectal malformations ranges from anal stenosis to
imperforate anus with/without anal fistula to the spinal
cord or to the urogenital system. Females are more
frequently affected than males (2, 3 ). Isolated anorectal
malformations have an estimated incidence of 1 in 5000
live births, but the incidence of the complete triad is
unknown.
CS is associated with mutations in the homeobox gene
HLXB9 and may segregate in families as an autosomal
dominant trait, mainly as a result of haploinsufficiency
1
Copyright © 2005 by The American Association for Clinical Chemistry
2
Garcia-Barceló et al.: HLXB9 in Currarino Patients and the General Population
(4, 5 ). The phenotype of the disease is variable (39% of
patients present with a severe phenotype, 29% are clinically apparent, 28% have changes apparent only on x-ray,
and 4% are asymptomatic), indicating that the manifestation and severity of the disease might depend not only on
HLXB9 mutations, but also on the effect of other, as yet
unknown, genes that could act as modifiers. Primarily
because of its great phenotypic variability, CS is frequently misdiagnosed. A combined diagnostic protocol
(6 ) includes a plain anteroposterior radiograph of the
sacrum as the first step. If hemisacrum is present, molecular analysis of the HLXB9 gene and radiologic study of
parents and relatives are indicated to distinguish between
sporadic and familial cases of CS.
HLXB9 has 3 exons encoding a 403-amino acid protein.
The HLXB9 protein functions as a transcription factor
regulating gene expression in both developing and adult
tissues, although little is known about target genes or
protein partners. Structurally, HLXB9 contains a homeodomain, a highly conserved region of 82 amino acids,
and a polyalanine region consisting of 16 alanines (Fig. 1).
Polyalanine tracts, coded by imperfect trinucleotide repeats (GCN) (7, 8 ), contain ⬃20 alanines. Polyalanine
expansions have been described in 9 genes (HOXD13,
RUNX2, ZIC2, HOXA13, FOXL2, SOX3, ARX, PHOX2B,
and PABPN1) as the cause of congenital defects (8, 9 ).
Except for PABPN1, all of these genes code for transcription factors with important roles during development and
differentiation, as is the case for HLXB9 (10 ).
To date, 25 CS-causing HLXB9 heterozygous mutations
have been identified. All missense mutations are clustered
in the homeodomain, whereas nonsense and frameshift
mutations are mostly on the NH2 terminus of the protein
(2, 11 ).
We report HLXB9 mutations found in 5 CS families and
6 sporadic cases. In addition, we present an analysis of the
distribution of the polyalanine tract polymorphisms in 4
different populations [Chinese, Japanese, Yoruba, and
Centre du Etude Polymorphisme Humain (CEPH)] from
the haplotype mapping (HapMap) project.
Participants and Methods
patients and controls
After obtaining informed consent, we studied 31 individuals (18 patients and their relatives). All patients presented with the complete Currarino triad. Six of the
affected individuals had no family history of CS and were
classified as sporadic cases (S1 through S6 in Table 1).
Patient S4 was also affected with Poland syndrome [congenital underdevelopment or absence of the chest muscle
and bone (pectoralis and sternal) on one side of the body
and cutaneous syndactyly of the hand on the same side],
and patient S6 with club foot. Parents of these sporadic CS
patients were also included in the study when available.
In the remaining affected individuals, CS was classified as
familial because characteristics of the CS phenotype had
been observed in some family members. These familial
cases were distributed in 5 families (F1 through F5), all of
Caucasian origin, whose phenotypes and family structures are depicted in Fig. 2. We studied 97 healthy
Chinese individuals from Hong Kong as controls. We also
analyzed the polyalanine region of HLXB9 in 270 samples
representing individuals from 4 different populations.
These samples included a panel of 30 trios from the
Yoruba in Ibadan, Nigeria; a panel of 30 trios from the
CEPH collection (US Utah residents with ancestry from
northern and western Europe); and a panel of 45 unrelated Japanese individuals in Tokyo and 45 unrelated Han
Chinese individuals in Beijing, all represented in the
HapMap project. We measured GCC repeats [(GCC)n] in
48 additional healthy Chinese from Hong Kong.
sequence and data analysis
We used PCR amplification and direct sequencing to
screen for DNA variants in the coding regions and basic
promoter of the HLXB9 gene. Sequencing was performed
with the Big DyeTM Terminator (Ver. 3.0) Cycle Kit
(Applied Biosystems) and an ABI 3100 sequencer (Applied Biosystems). Primers (designed from GenBank sequence NT_007741.12), PCR, and sequencing conditions
are available in the Data Supplement that accompanies
the online version of this article at http://www.clinchem.
Table 1. Phenotype, HLXB9 mutation, and GCC-repeat analyses of the sporadic CS patients.
Mutation/(GCC)n genotype
Patient
S1a
S2a
S3d
S4d
S5a
S6a
a
(M)b
(M)
(F)
(M)
(F)
(M)
Phenotype
Proband
Father
Mother
CS
CS
CS
CS ⫹ Poland syndrome
CS
CS ⫹ club foot
Arg273Cys; (GCC)11/(GCC)11
⫺; (GCC)11/(GCC)11
⫺; (GCC)11/(GCC)11
⫺; (GCC)11/(GCC)11
⫺; (GCC)9/(GCC)11
Ser185X; (GCC)9/(GCC)11
NA
⫺; (GCC)11/(GCC)12
NA
⫺; (GCC)11/(GCC)11
⫺; (GCC)9/(GCC)11
Ser185X; (GCC)9/(GCC)11
⫺;c (GCC)11/(GCC)11
⫺; (GCC)9/(GCC)11
NA
⫺; (GCC)11/(GCC)11
⫺; (GCC)11/(GCC)11
⫺; (GCC)11/(GCC)12
Chinese origin.
M, male; F, female; NA, not available.
c
⫺, no mutation detected.
d
Caucasian origin.
b
3
Clinical Chemistry 52, No. 1, 2006
org/content/vol52/issue1/. Hardy–Weinberg equilibrium was tested according to the procedure described by
Guo and Thompson (12 ).
Results
mutation analysis
We identified 6 novel heterozygous mutations (Fig. 1),
including 2 nonsense (Ser185X and Trp215X), 2 frameshifts (Ala26fs and Ala75fs), and 2 missense mutations
affecting highly conserved amino acids such as the initiating methionine (Met1Ile) and Arg 31 (Arg273Cys) of the
homeodomain region. Met1Ile is the first HLXB9 missense
mutation localized outside the homeodomain. All frameshift and nonsense mutations described predict truncated
proteins that, if stably translated, would lack the homeodomain region, possibly affecting its DNA-binding and
transcription-regulation activities. The mutations described below were detected in 2 of 6 sporadic cases
(Table 1) and in 4 of the 5 CS families (Fig. 2 and Table 2).
These mutations were not observed in 97 Hong Kong
Chinese (this study) or in 87 Caucasian controls (5 ).
sporadic cases
Among the sporadic cases (Table 1), only 2 male patients
(S1 and S6) harbored HLXB9 mutations. S1 had a c815C⬎T
transition leading to a substitution of residue 273 of the
protein (arginine) with a cysteine (Arg273Cys). This missense mutation affects a highly conserved amino acid
(amino acid 31 of the homeodomain R31 within helix 2)
involved in the binding of the HLXB9 protein to the target
DNA (13–15 ). R31 also contributes to the correct packaging of helices II and III by forming a salt bridge with a
conserved glutamate residue at position 42 in helix III
(14, 16 –18 ). Missense mutations in R31 have been found
in the human homeobox genes PITX2, MSXI, MSX2,
LMX1B, and HOXD13 and are associated with 5 different
developmental diseases: iridogoniodysgenesis syndrome,
tooth agenesis, enlarged parietal foramina, nail-patella
syndrome, and several digital anomalies, respectively
(18, 19 ). Studies of these homeobox genes have demonstrated the in vivo relevance of this mutation, which has
even been defined as a “hot spot” for disease (18, 20 ).
Functional analyses revealed that substitution of the R31
residue by a histidine reduces the capability of the protein
to bind DNA and also the ability to activate transcription
(21, 22 ).
Patient S6 harbored a c554C⬎A transversion originating the nonsense mutation Ser185X (TGC changes to stop
codon TGA), which predicts a truncated protein consisting of only 185 amino acids. Interestingly, Ser185X was
also found in the apparently healthy father of S6.
familial cases
Family 2. Three affected members (II2, II3, and III1)
harbored a c3G⬎A transition predicting a methionine
(ATG) to isoleucine (ATA) change at the initiating codon.
Interestingly, Met1Ile is the first HXLB9 missense mutation found that is not clustered in the homeodomain
region (2, 11 ). The c3G nucleotide is conserved 100% in
the initiation codons of all eukaryotes and in most prokaryotes (23 ). A mutation in the initiating codon would
likely lead to loss of the signal for initiation of translation,
suggesting either that no protein is produced or the
translation initiation site moves up- or downstream. In
HLXB9, the next in-frame ATG codon would correspond
to methionine 223. Translation initiation from this internal
ATG site would yield a 180-amino acid protein lacking the
N-terminal domain (which is supposed to be involved in
Fig. 1. Schematic drawing showing the HLXB9 gene
and the HLXB9 protein with the mutations identified
in this study.
Correspondence between HLXB9 coding regions and HLXB9
protein is also represented. Nucleotide positions are defined in relation to the first nucleotide of the start codon,
which is designated position ⫹1, and according to RefSeq
NM_005515.2, which comprises 11 GCC repeats. The
amino acid residues are numbered according to RefSeq
NP_005506, starting at the initiator methionine residue.
4
Garcia-Barceló et al.: HLXB9 in Currarino Patients and the General Population
Table 2. HLXB9 mutation and GCC-repeat analyses of the
familial CS patients.a
Family
Family member(s) analyzed
Mutation/(GCC)n genotype
F1
II2
III7;III8
II2; II3;III1
I1
I2
II1
I1; II3
I2
II3
II4; III1
III2
⫺;b (GCC)9/(GCC)11
⫺; (GCC)11/(GCC)11
Met1Ile; (GCC)11/(GCC)11
⫺; (GCC)9/(GCC)12
Ala26fs; (GCC)11/(GCC)11
Ala26fs; (GCC)11/(GCC)12
Ala75fs; (GCC)11/(GCC)12
(GCC)11/(GCC)11
Trp213X; (GCC)11/(GCC)11
⫺; (GCC)9/(GCC)11
Trp213X; (GCC)9/(GCC)11
F2
F3
F4
F5
a
b
For kinship and phenotypes, see Fig. 2.
⫺, no mutation detected.
stop codon 153 codons away from Ala-75 (Ala75fs). The
predicted truncated protein would be 175 amino acids
shorter than the wild-type HLXB9.
Fig. 2. Pedigrees of the 5 families included in this study.
A, ventricular septal defect; B, neurogenic bladder, azoospermia, and left
unilateral renal hypoplasia with chronic interstitial nephritis; C, neurogenic
bladder and mitral valve prolapse; D, urinary reflux; E, sacral dimple with bony
outgrowth. ⴱ, individual screened for HLBX9 mutations.
protein–protein interactions), whereas translation initiation from ATA would yield smaller amounts of wild-type
HLXB9 protein (24 ). Either scenario could account for the
affected individuals of family 2, who harbored the same
mutation but showed different phenotypes. Remarkably,
the mother (I2) of the affected sisters (II2 and II3) and her
asymptomatic daughter (II1) had no abnormal x-ray findings and no complaints. Unfortunately, DNA of those
asymptomatic family members who seemed to have
transmitted this mutation was not available for analysis.
Family 3. A 77delC was observed in the mother (I2) and
daughter (II1) but was absent in the father (I1). DNA
samples from the rest of the family were not available.
This deletion replaces the alanine of codon 26 with a
glycine and introduces a frameshift that originates a stop
codon 196 codons away from Ala-26 (Ala26fs), predicting
a truncated protein with 222 amino acids instead of 403.
Family 4. Both I1 and II3 harbored a duplication of 17 bp
(c196_212dup) in exon 1 that originates the replacement of
Ala-75 by proline, creating a frameshift that generates a
Family 5. A c638G⬎A transition was detected in 2 family
members (II3 and III2). This transition originates the
nonsense mutation Trp213X (TGG changes to stop codon
TGA), which predicts a truncated protein consisting of
only 213 amino acids. The severity of the phenotype
differed considerably between the mother (II3) and son
(III2). Interestingly, family member III1, who did not carry
Trp213X, presented with a phenotype (sacral dimple with
no abnormal magnetic resonance imaging findings) almost identical to that of her brother (sacral dimple with a
bony outgrowth and no abnormal magnetic resonance
imaging findings).
Chromosomal microdeletions affecting the HLXB9
gene, which have been described in some Currarino
patients (4 ), can be detected by fluorescence in situ
hybridization or copy number analysis, which were not
performed in this study. Thus, patients with no detected
mutation may have had a deletion affecting either completely or partially one HLXB9 allele.
analysis of the length of the polyalanine tracts
(GCC)11 was the most common allele among all individuals analyzed, although the (GCC)8, (GCC)9, and (GCC)12
alleles were also observed (Tables 1 and 2). We could not
establish any correlation between the number of GCC
repeats in patients and the presence of disease or with the
variable penetrance of the HLXB9 mutations. A larger
number of patients is required, however, to draw any
conclusion regarding association of any given GCC allele
with the disease. All (GCC)n alleles present in patients
were also present in the controls. Surprisingly, statistically
significant differences in the global (GCC)n allele and
genotype distributions were observed between the healthy
Chinese analyzed in this study and the Caucasian controls
described previously by Belloni et al. (5 ). Comparison
Clinical Chemistry 52, No. 1, 2006
(Table 3) revealed highly statistically significant differences for alleles (GCC)9, (GCC)11, and (GCC)12 and most
of the genotypes including them. Most strikingly, in the
Caucasian population, the frequency of (GCC)11 was 90%,
but the frequency of this allele in Chinese was considerably lower (50%). These differences in allele frequencies
are reflected in the genotype composition of the populations.
We then investigated the (GCC)n allele and genotype
distributions in other populations. A summary of the
population genetic characteristics of HLXB9 is presented
in Fig. 3. No deviation from Hardy–Weinberg equilibrium
was observed in any of the populations. The (GCC)n allele
and genotype distributions of the Hong Kong Chinese
population matched those of Japanese and Chinese populations, the population described by Belloni et al. (5 ),
and the CEPH individuals.
In the Chinese and Japanese individuals, (GCC)9 was
almost as common as (GCC)11, whereas its frequency was
⬎10% in the Yoruba and CEPH populations. The Yoruba
population had the highest frequencies of the largest
alleles [(GCC)12, 26.6%; (GCC)13, 2.7%], which were almost absent elsewhere. Whereas 80% of the CEPH individuals were represented by 1 main genotype [(GCC)11/
(GCC)11], genotype diversity was increased in the Yoruba
population, in which (GCC)11/(GCC)11 and (GCC)11/
(GCC)12 were equally represented, and was highest in the
Chinese and Japanese populations.
Discussion
The CS phenotype related to HLXB9 mutations is attributed to haploinsufficiency, whereby the wild-type allele
Table 3. Allele and genotype distributions of the GCC
repeats at the HLXB9 locus in Chinese (this study) and
Caucasian (5 ) populations.
Frequency (%)
Alleles
(GCC)8
(GCC)9
(GCC)11
(GCC)12
(GCC)13
Genotypes
(GCC)9/(GCC)9
(GCC)11/(GCC)11
(GCC)12/(GCC)12
(GCC)8/(GCC)11
(GCC)9/(GCC)11
(GCC)9/(GCC)12
(GCC)11/(GCC)12
(GCC)12/(GCC)13
a
b
Chinese
populationa
Caucasian
populationb
␹2 (P)
0.4
33.0
50.0
16.2
0.4
0.6
7.5
90.2
1.7
0.0
0.13 (0.71)
39.75 (⬍0.000001)
77.45 (⬍0.000001)
23.72 (⬍0.000001)
0.60 (0.64)
12.4
26.2
2.8
0.7
31.0
10.3
15.9
0.7
1.2
81.6
0.0
1.2
12.6
0.0
3.4
0.0
9.17 (0.0024)
67.00 (⬍0.000001)
2.44 (0.20)
0.13 (0.72)
10.04 (0.0015)
9.62 (0.0019)
8.42 (0.0037)
0.60 (0.64)
This study; n ⫽ 145 individuals.
Belloni et al. (5 ); n ⫽ 87 individuals.
5
Fig. 3. Allele (top) and genotype (bottom) distributions of the GCC
repeats at the HLXB9 locus in 4 human populations.
cannot provide sufficient HLXB9 protein (4 ). Thus, a
mutation in one allele that leads to a ⬎50% decrease in
gene product may cause symptoms. The phenotypic variability observed among family members carrying the
same mutation and the unpredictability of the phenotype,
however, can best be explained by the effects of other
genes (4, 5, 11 ). Sensitivity to modifier genes is inherent to
gene products whose correct functioning depends on
relative amounts of interacting products, a characteristic
intrinsic to homeobox genes. The transcription factors
encoded by homeobox genes are expressed in different
cell types and regulate the expression of many target
genes, controlling the formation of organs and body
structures during development. The specificity of these
processes is achieved only through their interaction with
cell- or tissue-specific protein partners (25 ). HXLB9 mutations may hamper the interaction of the HXLB9 protein
with its tissue-specific partners, and as a result, multiple
body structures would be affected to a different degree
and with different phenotypic consequences. In addition,
mutations in genes encoding HLXB9 protein partners or
transcriptional regulators may influence HLXB9 expression, increasing the chances of phenotypic variability and
accounting for patients with no HLXB9 mutations. Conversely, the normal phenotype of healthy individuals
harboring mutations may be related to variations in
expression of the nonmutated HLXB9 allele in different
tissues and at different developmental stages. The CS
phenotype caused by mutations in HLXB9 may therefore
be sensitive to modifications elsewhere in the genome
affecting HLXB9 protein partners or transcriptional regulators.
6
Garcia-Barceló et al.: HLXB9 in Currarino Patients and the General Population
The mutations described in this study are likely to lead
to a 50% decrease in protein function, although because of
the lack of direct functional evidence their pathogenicity
can only be inferred. No correlation between type or
location of the mutation in the protein and severity of the
phenotype could be established. As in previous studies,
HLXB9 mutations were found in nearly all patients with
familial CS and in only 30% of the patients with sporadic
CS (2–5, 11, 26 )). Those sporadic patients with HLXB9
mutations may be descendents of asymptomatic individuals in whom signs of the condition can be detected only
by radiologic analysis. Anorectal, pelvic ultrasound, and
pelvic x-ray examinations should be conducted on relatives of patients with the sporadic form of CS to distinguish between familial and sporadic CS. Such examinations may have been revelatory in the case of patient S6
and his asymptomatic father (both harboring Ser185X), on
whom no radiologic examination was conducted.
Although both pathologic and nonpathologic polyalanine polymorphisms for several genes have been found in
the general population, there is little information regarding their distribution in different ethnic populations
(8, 27–31 ). To our knowledge, ethnic differences in polyalanine polymorphism frequencies have been described
only for the RPL14 and the MICA genes (27, 28 ). It is
tempting to speculate that if there was a threshold beyond
which expansions are pathologic, those populations enriched with alleles with higher number of repeats would
be at higher risk. In the PABPN1 gene (encoding an
mRNA polyadenylation factor), the wild-type allele consists of 6 triplets (GCG6), and expansions of 2–7 triplets
are pathologic and associated with oculopharyngeal
muscular dystrophy (10 ). One triplet expansion (GCG7)
acts as a modifier of the dominant disease phenotype
caused by expansion of more than 1 triplet (GCG7/
GCG⬎7) or, in homozygosis, (GCG7/GCG7) as a mutation.
Most interestingly, and perhaps relevant to the ethnic
variety in the distribution of polyalanine polymorphisms,
is that in the PABPN1 gene GCG7 is also found in 2% of
healthy Caucasians in combination with GCG6. In this
instance, there is a clear cutoff between wild-type and
mutant alleles that clearly shows how a different ethnic
distribution of this allele could translate into an ethnicityspecific disease risk. Because rare polymorphisms exist in
4 of the 9 genes in which alanine expansions cause
disease, it is most important to screen similar sequences in
other pathologies (8 ). Our analysis of the HLXB9 triplets
indicates that alleles (GCC)8, (GCC)12, and (GCC)13 are the
rarest in the populations studied. Given that (GCC)12 and
(GCC)13 alleles represent a short expansion of the most
common allele and that only expansions appear to be
pathologic, it would be interesting to investigate a larger
cohort of patients to determine whether these larger
alleles have a role similar to that of GCG7 of the PABPN1
gene and how this relates to its different ethnic distribution. Our data emphasize the importance of group strat-
ification if these polymorphic markers are used in association studies.
We extend our gratitude to all those who participated in
the study. This work was supported by research grants
from the Hong Kong Research Grants Council (HKU
7509/05M). S.W.S. is a Scientist of the Canadian Institutes
of Health Research and an International Scholar of the
Howard Hughes Medical Institute.
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