Published online before print
January 14, 2003, 10.1101/gr.302003
Vol 13, Issue 2, 173-181, February 2003
Linkage Disequilibrium and Haplotype Diversity in the Genes of the Renin–Angiotensin System: Findings From the Family Blood Pressure Program
Xiaofeng Zhu1,4,5,
Denise Yan2,4,
Richard S. Cooper1,
Amy Luke1,
Morna A. Ikeda2,
Yen-Pei C. Chang2,
Alan Weder3 and
Aravinda Chakravarti2
1Department of Preventive Medicine and Epidemiology,
Loyola Stritch School of Medicine, Maywood, Illinois 60153, USA;2
McKusick–Nathans Institute of Genetic Medicine, Johns
Hopkins University, Baltimore, Maryland 21287, USA; 3Division
of Hypertension, University of Michigan School of Medicine,
Ann Arbor, Michigan 48109, USA
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ABSTRACT
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Association studies of candidate genes with complex traits have
generally used one or a few single nucleotide polymorphisms (SNPs),
although variation in the extent of linkage disequilibrium (LD) within
genes markedly influences the sensitivity and precision of association
studies. The extent of LD and the underlying haplotype structure for
most candidate genes are still unavailable. We sampled 193 blacks
(African-Americans) and 160 whites (European-Americans) and
estimated the intragenic LD and the haplotype structure in four genes
of the renin–angiotensin system. We genotyped 25 SNPs, with all but
one of the pairs spaced between 1 and 20 kb, thus providing resolution
at small scale. The pattern of LD within a gene was very heterogeneous.
Using a robust method to define haplotype blocks, blocks of limited
haplotype diversity were identified at each locus; between these
blocks, LD was lost owing to the history of recombination events. As
anticipated, there was less LD among blacks, the number of haplotypes
was substantially larger, and shorter haplotype segments were found,
compared with whites. These findings have implications for
candidate-gene association studies and indicate that variation between
populations of European and African origin in haplotype diversity is
characteristic of most genes.
[The sequence data described
in this paper are available in GenBank under the following accession
nos: AGT, MIM 106150; Renin, MIM 179820;
ACE, MIM 106180; Angiotensin receptor I, MIM 106165.
Supplementary material is available online at
http://www.genome.org.]
The pattern of linkage disequilibrium (LD) at a
locus has important implications for disease gene mapping using
comparisons of allele frequencies in affected and unaffected
individuals (Lander and Schork 1994 ; Jorde 2000; Risch 2000; Abecasis
et al. 2001). Virtually all association studies conducted for complex
phenotypes use only one marker, either a single nucleotide polymorphism
(SNP) or an Alu element. The adequacy of this design depends
critically on the extent of LD within the gene or region being
investigated, and which SNP is used for analysis. The inability to take
population-genetics architecture into account probably contributes to
the inconsistent and disappointing results seen in complex-trait
association studies.
Estimates of the extent of LD across the human genome vary considerably
(Tishkoff et al. 1996; Kruglyak 1999; Dunning et al. 2000;
Taillon-Miller et al. 2000; Abecasis et al. 2001; Reich et al. 2001).
Substantial heterogeneity has been observed across various regions;
many features, including biological and stochastic factors, likely
contribute to this phenomenon (Taillon-Miller et al. 2000; Abecasis et
al. 2001; Reich et al. 2001). Because LD also reflects the history of a
population in terms of age and the number of founders, it would also be
anticipated that different populations would have different patterns.
In particular, shorter LDs and greater haplotype diversity have been
seen in human African-origin populations (Chakravarti et al. 1984; Zhu
et al. 2000; Reich et al. 2001). Reich et al. (2001) recently examined
19 randomly selected genomic regions spanning 160 kb in a sample of
Swedes and Nigerians. Among Europeans, the half-length of LD, which is
considered a useful minimum for association studies, was 60 kb, whereas
it was only 5 kb for the Africans (Reich et al. 2001). These results
are consistent with the previous reports of greater genetic diversity
among Africans, both at the level of individual polymorphisms and
haplotypes (Cargill et al. 1999; Halushka et al. 1999; Rieder et al.
1999).
Recent investigations of global haplotype patterns demonstrate that
pronounced haplotype structure on "blocks" exists in the human
genome (Daly et al. 2001; Johnson et al. 2001; Patil et al. 2001). By
considering haplotypes as the basic unit, rather than individual SNPs,
Daly et al. (2001) found that LD is clearly a monotonic function of
physical distance and that historical recombination is the major
determinant of the breakdown in LD. This finding has important
implications for association mapping. In a study of Crohn's disease,
Rioux et al. (2001) successfully performed an association analysis
using haplotype blocks and isolated a 250-kb segment harboring the
susceptibility locus from the original 18-cM region.
The renin–angiotensin system (RAS) plays a critical physiological role
in the cardiovascular system. The primary genes that comprise the RAS
have, perhaps not surprisingly, been the focus of an enormous number of
association studies over the last decade. Even though important genetic
influences have been noted for the direct phenotype of some of the RAS
genes, most notably angiotensin I-converting enzyme (ACE; Soubrier et
al. 1994), their role as susceptibility or protective genes for
cardiovascular disease is still unresolved (Fornage et al. 1998;
O'Malley et al. 1999; Keavney et al. 2000; Zhu et al. 2000; Svetky et
al. 2001). It is reasonable to assume that more detailed information
about the organization of genetic variation at these loci will be
required before the full impact of the RAS genes can be appreciated.
The purpose of our study was to determine the LD distribution and
haplotype structure in the RAS genes among individuals within and
between populations by genotyping a set of SNPs in a community-based
sample of U.S. whites (European-Americans) and blacks
(African-Americans).
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RESULTS
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Our original data set includes 193 black families and 160 white
families. Because the numbers of siblings in the two groups were
comparable, we randomly selected one sibling from each family after
excluding the probands. The genotyped siblings, thus selected, included
193 black and 160 white individuals. Five SNPs were genotyped in each
of the four genes, with the exception of ACE, for which 10
SNPs were genotyped. The length of the genomic segments covered in each
gene were, Renin (REN), 14 kb; angiotensin
receptor type I (AGTR1), 45 kb; angiotensinogen
(AGT), 4.5 kb; and ACE, 21 kb. In Table
1 the minor allele frequencies for the SNPs
in the four genes are presented for both populations, along with
p-values for the HWE test. The  c values for all
the SNPs between whites and blacks are also presented in the last
column. The minor allele frequencies were all >10%, with the
exception of REN C-3212T, AGTR1 A44221G, and
ACE A7941G in whites, and AGT C3889T in blacks. No
significant departure from HWE was found, except for ACE
A7941G and ACE C19329T in whites. In general, blacks had a
higher frequency of the alleles that were designated as minor among
whites. To quantify the degree of population diversity,
FST (Wright 1931), which is defined as the genetic
variance among populations divided by the genetic variance within the
total population, was calculated. Our data yielded a value for
FST of 0.084, with a 95% confidence interval of
(0.049, 0.119) obtained by bootstrapping all sites (Weir 1996).
Linkage Disequilibrium
The most informative approach to defining LD would involve
characterization of the haplotypes in each gene. Unfortunately,
genotype information was missing for a number of SNPs, in particular
for AGT. Therefore, we first examined pairwise LD measured by
Lewontin's D‘ (Table 2). Because
some entries in the 2-by-2 tables testing for LD were rare, we used
Fisher’s exact test to test the null hypothesis that
D‘ = 0. The p-values were then adjusted by the
number of SNP pairs in the gene using a Bonferroni correction, because
pairwise LD tests within a gene were not independent. In Table 2 the
shaded area indicates that significant LD exists within each gene after
adjusting for multiple tests. The relevant findings are summarized
separately for each gene below.
View this table:
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Table 2. Linkage Disequilibrium Statistic (D') in RAS Genes in Whites (Lower
Triangle) and Blacks
(Upper Triangle)
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REN
In whites all observed D‘ values were equal to 1, whereas
this was true for only three of the D‘ values among blacks. As
noted, when D‘ = 1, in the absence of intragenic
recombination and repeated mutation, the maximum number of haplotypes
that can be observed for a pair of SNPs is 3. These data therefore
indicate an absence of intragenic recombination and repeated mutation
in whites. In whites, LD among C-4021T, C-3212T, and C10377T was
significant, but not with A4280C or G5795T. These results demonstrate
that the LD test is not necessarily significant even when
D‘ = 1. Except for G5795T, all the markers studied here had
low minor allele frequencies. For SNPs with this distribution the test
of LD may not have much power (Thompson et al. 1988). In blacks
significant LD occurred between C-4021T and C-3212T, and between A4280C
and G5795T, C10377T. Overall, therefore, considerable LD exists in this
gene among whites, but much less occurs among blacks.
AGTR1
In the AGTR1 gene, two LD clusters, separated by 44 kb,
were observed for both whites and blacks. One cluster consisted of the
SNPs A-777T, G-680T, and A-119G, and the other consisted of C43732T and
A44221G. Within these clusters all the D‘ values were equal to
1, but LD was weak between them, indicating frequent recombination
events.
AGT
At the AGT locus the D‘ values were >0.8 for both
the black and the white samples. Seven of the 10 pairwise D‘
estimates in whites and 9 of 10 in blacks were equal to 1, leading us
to conclude neither recombination nor multiple mutations are present in
both whites and blacks. Eight of the 10 and 3 of the 10 pairwise tests
were statistically significant in whites and blacks, respectively.
Significant LD was only observed between neighboring SNPs in blacks. In
both populations C3889T was not in significant LD with any of the other
SNPs, except for A-6G in whites. The minor allele T for C3889T had a
low frequency in both whites and blacks.
ACE
For ACE, all the D‘ values were >0.9 in whites,
yielding significant LD, except for A7941G, which had a minor allele A
frequency of 5% in whites. In blacks, all the D‘ values were
>0.6 except for some associated with A-239T. Significant LD was also
found among all markers except A-239T.
To provide an overall estimate of LD versus distance, we plotted both
D‘ and r2 against distance for the two
groups (Fig. 1). LD decays
as distance increases independent of the measure in blacks. However, LD
decays much slower in whites than that in blacks, probably because of
the short region considered. The pronounced contrast by racial group is
likewise apparent in this graphical format.
Haplotype Analysis
We next defined the haplotypes and estimated their corresponding
frequencies using the computer program PHASE. Stable estimates were
obtained using 10,000 burn-in cycles and 10,000 iterations (see
Supplementary Tables 1–4 available online at http://www.genome.org).
The number of haplotypes among blacks was greater except for
AGT, and haplotypes shared in both populations account for
90% of those occurring in whites, but much less in blacks. The
frequencies of those haplotypes occurring only in whites were 7.1% for
ACE, 1.5% for AGTR1, 8.1% for REN, and
3.3% for AGT. The number of major haplotypes
(frequency  4%) in whites was less than among blacks. To obtain
the global LD for each of the 4 genes we performed simulation tests
(Blanton and Chakravarti 1987; Antonarakis et al. 1988). We estimated
the distribution of the number of different haplotypes under the
assumption of random association of the SNPs within a gene when all of
the observed chromosomes were sampled. Table
3 presents these results based on 1000
replications. The first column represents the candidate genes, and
columns 2 to 5 present the number of chromosomes observed, the number
of different haplotypes inferred by PHASE, the average number of
haplotypes by simulations, and the minimum number of haplotypes by
simulations in whites, respectively. Similar results in blacks appear
in columns 6 through 9. In each of the four genes, the observed number
of haplotypes was less than the minimum number simulated under the
random association assumption. These results imply that the empirical
p-values of global LD tests are <0.001, leading to the
conclusion that strong global LD exists within all four genes.
Haplotype Blocks
Using the definition provided in Methods, we defined the haplotype
blocks in the four genes. Table 4 displays
the haplotypes and the corresponding frequencies in blocks. The solid
lines between two blocks indicate that >4% of all chromosomes are
observed. One haplotype block for REN in whites was defined
with four major haplotypes. To represent them, A4280C, G5795T, and only
one of the three markers (C-4021T, C-3212T, and C10377T), are required,
because the latter set are in complete LD. In contrast, two similar
blocks were defined in blacks, one consisting of C-4021T and C-3212T
and the other consisting of A4280C and G5795T. The SNP C10377T does not
fall into either block. All four SNPs are necessary to represent the
two blocks. Surprisingly, to represent most of the haplotypes
(frequency 4%) in this gene for blacks, all five SNPs are
required, although the genomic segment covered is only 14 kb long. When
we considered each block as a locus and the haplotypes as the
corresponding alleles, we obtained the c values of 0.16
and 0.22 for these two blocks. We also found that the c
value for a block is greater than the c values for the
SNPs within the block.
In the AGTR1 gene, there are two blocks for both whites and
blacks, with one block consisting of A-777T, G-680T, and A-119G, and
the other including markers C43732T and A44221G. For both whites and
blacks, any one of A-777T, G-680T, and A-119G can represent the first
block. In the second block, only C43732T is necessary to represent this
block in whites, but both C43732T and A44221G are required for blacks.
The c values we obtained for these two blocks were 0.17
and 0.29. Similar to the Renin gene, the c value
for a block is not less than the c values for the SNPs
within the block in this gene.
In AGT there is one block that can be defined in both whites
and blacks. AGT C3889T and one SNP from each of the two pairs
C-532T/A-217G or A-6G/C4072T are necessary to represent the four major
haplotypes in whites. However, to represent the five major haplotypes
in blacks, both C-532T and A-217G must be included. Considering this
block as a locus, we obtained the c value 0.43, which is
greater than the c values of all five SNPs.
In the ACE gene, all the SNPs define one block in whites. Only
two SNPs, namely, A-239T and any other SNP except A7941G are required
to represent most haplotypes. In blacks, our definition also identifies
a block including A7941G, C8342T, A10593G, A11599G, A12292G, A15990G,
C17911T, C19329T, and A20060G. However, A-239T does not fall within
this block because the haplotype distribution exceeds the 95%
bootstrap confidence interval after adding this SNP. The four major
haplotypes only account for 76% of the total in this block. Three
SNPs, namely, A7941G, one of A10593G and A11599G, and one of C8342T,
A12292G, and A15990G are required to represent the four major
haplotypes. Considering this block as a locus, we obtained the
c value 0.47, which is greater than the c
values of all nine SNPs.
By defining DNA segments in this fashion, LD within a block is
virtually complete, with historical recombination or repeated mutation
at their margins. We consistently observed that haplotype blocks were
shorter among blacks and consisted of a subset of the SNPs from the
blocks in whites. It is also interesting to note that when the
haplotypes were considered as the alleles there was much more
heterozygosity, and therefore more information derived from the samples
than could be obtained from typing single SNPs. To verify the
consistency of these results we conducted similar analyses for a data
set that included one parent from each family, and the results were
essentially the same.
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DISCUSSION
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Our analysis makes possible a detailed description of the genomic
organization of a sample of important cardiovascular candidate genes in
two populations with contrasting levels of diversity. Although the
number of SNPs we studied is relatively small, the sample size is
large, compared with more recent genomic analyses, and the data
presented here permit several conclusions. The primary source of
heterogeneity in terms of the LD was observed between the two
populations. Among whites very little historical recombination can be
detected in the RAS genes, and only the SNPs at the AGTR1
gene, which were spaced at 45 kb, show LD clusters that defined two
haplotype blocks, with an interval of substantially decreased LD
between these two sections. Among blacks, on the other hand, LD was
weaker than among whites, except for AGT, for which the LD in
blacks and whites was similar. The explanation for the similar LD in
AGT among blacks and whites may be that a small region in this
gene has been examined, with the largest distance between two markers
being <4 kb. Although population admixture may change the LD pattern,
with the small difference in allele frequencies and such a short
distance, it would be unlikely to have a major effect. More haplotypes
are also observed in blacks, with the exception of the AGT
gene, for which the reverse was true. However, in AGT there
are five major haplotypes in blacks, but only four in whites. Among
whites, there was one block observed in ACE, AGT, and
REN, and two in the AGTR1 receptor. However, among
blacks more haplotype blocks could be defined. These findings in whites
are consistent with recent reports (Daly et al. 2001; Patil et al.
2001), which concluded that haplotype blocks range from 10–100 kb in
European-derived populations. Our study also indicates that the African
populations have much shorter haplotype blocks. The clustering of
blocks also indicates local hotspots of recombination (Chakravarti et
al. 1984; Clark et al. 1998; Templeton et al. 2000; Jeffreys et al.
2001). Taken together, these haplotype analyses clearly demonstrate
that blacks have more haplotype diversity at these loci than do persons
of European descent. The pairwise LD analysis is also consistent with
this result. Within a block, three or four haplotypes will usually
account for >90% of the total haplotypes. Based on these findings, we
were able to select fewer SNPs to represent most of the haplotypes
within a block.
We also defined haplotype blocks by using the method of Daly et al.
(2001). The differences of defining haplotype blocks by these two
methods occurred only in the REN and ACE genes in
blacks. In REN, the method by Daly and colleagues defines two
blocks, with the second block including SNPs A4280C, G5795T, and
C10377T; in ACE, it defines two blocks, with the second block
including C19329T and A20060G. However, the score statistics of the
blocks, defined by the ratio of haplotypic heterozygosity and expected
haplotypic heterozygosity (Daly et al. 2001), are not significantly
different from that of blocks defined in our method, indicating that
the differences may be due to the sampling variation. Thus, the
haplotype blocks defined in these two methods are essentially the same.
Confidence in the generalizability of our findings is reinforced by
their congruence with previous reports. Whether based on single genes
(Halushka et al. 1999; Zhu et al. 2000) or random genomic segments
(Reich et al. 2001), the relevant published data are similar in every
respect to our findings. Even though they used very different sampling
methods to choose genomic regions and markers, Reich et al. (2001)
report estimates of LD half-length similar to ours, namely, 5 kb for
blacks. Our findings therefore extend the observation of short LD to
persons of African descent in the Western Hemisphere. Although the
confidence limits on the estimated LD half-lengths have not been well
defined, the similarity of the finding among U.S. blacks and Nigerians
indicates that recent population admixture in the U.S. has not
noticeably increased the extent of LD when we deal with a genomic
distance <45 kb.
Variation in the extent of LD among human populations has obvious
implications for gene mapping (Tishkoff et al. 1996; Wright et al.
1999; Taillon-Miller et al. 2000; Abecasis et al. 2001; Reich et al.
2001). Jorde (2000) has drawn attention to the possibility that a
two-tiered strategy would be most efficient for association studies. If
the aim is to localize a genetic effect within a 50–100-kb range, then
European populations might be targeted; on the other hand, to carry out
fine-mapping at the 1–5-kb range, a study of African-origin groups
would be more productive. We recently demonstrated the practical
significance of this observation in a study localizing the 3' region
within the ACE gene that has the greatest influence on ACE
plasma activity (Zhu et al. 2000). In Jamaicans the effect could be
resolved to a small region; however, in Germans, given the limited
number of recombination events that were available to study,
localization was not possible (Zhu et al. 2000). Similarly, using 13
markers in ACE in a Nigerian sample, we were able to identify
a second 5' region that influenced plasma activity and use this
information to find an association with blood pressure (Zhu et al.
2001). Using all the segregating variability expressed in populations
of African ancestry, Zhu et al. (2001) demonstrated the limitations of
the much-studied Alu motif in ACE as a marker of
susceptibility. This experience, if it can be generalized, indicates
that association studies can become more informative if the appropriate
target population is chosen and the entire store of genetic information
at the target locus is fully exploited.
Our estimates of c values indicate that defining haplotype
blocks could increase the c values, thereby increasing the
power to detect LD in an admixed population. With the availability of
abundant SNPs across the human genome (Sachidanandam et al. 2001), we
should be able to define the necessary haplotype blocks (Daly et al.
2001; Patil et al. 2001). It might, therefore, be possible to select
dense blocks with maximum haplotype frequency differences between two
founder populations to conduct genome-wide admixture mapping.
Several limitations of this study must be acknowledged. The SNPs were
chosen on the basis of frequency and the available published
information, but we have no way to evaluate whether this introduced a
bias. However, with the large sample size and the replication of the
results by selecting one parent from each family, such bias should be
negligible. Although the probands for this study were identified based
on blood pressure near the upper 15%, the age–gender-specific
distribution, our analysis sample excluded all the probands.
Furthermore, our sampling method is unlikely to influence the patterns
of LD, because the variation of blood pressure explained by this region
is very small (Zhu et al. 2001).
It seems reasonable to suggest that the complexity of genomic
organization has been underestimated in most association studies of
complex traits. Multiple variants, both in coding and promoter
sequences, may well have complementary small effects. If this is true
for most genes, more exhaustive search methods will be required than
have been used. Defining the haplotype blocks in the region of interest
may be the necessary first step to avoid the "hit or miss" approach
that characterizes mapping based on single SNPs.
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METHODS
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Selection of Participants
The participants in this study were enrolled in the GenNet
component of the NHLBI-sponsored Family Blood Pressure Program
(Province et al. 2000); the design and sampling procedures are to be
published (B.A. Thiel, A. Chakravarti, R.S. Cooper, A. Luke, S. Lewis,
A. Lynn, H. Tiwari, N.J. Schork, A.B. Weder, unpubl.). In brief,
sibships including persons between the ages of 25 and 40 were enrolled
if their systolic (SBP) or diastolic (DBP) blood pressures were in the
upper 25th and 15th percentile for black or white, respectively.
African-Americans were recruited in Maywood, Illinois, whereas persons
of European descent were enrolled in Tecumseh, Michigan; ethnic
classification was based on self-identification. The protocols were
reviewed and approved by the review boards of the respective
institutions. The original sample included 616 individuals from 201
black families in Maywood, and 618 individuals from 160 white families
in Tecumseh. Our analysis focused on a sibling set consisting of one
sibling randomly selected from each family, after excluding the
proband. This sampling scheme should reduce the bias due to the
ascertainment. The siblings chosen included 193 blacks and 160 whites.
Laboratory Methods
SNPs of the Renin–Angiotensin System
The candidate genes studied were REN (MIM 179820),
AGTR1 (MIM 106165), AGT (MIM 106150), and
ACE (MIM 106180) (http://www.ncbi.nlm.nih.gov/Omim). A total
of 25 SNPs were genotyped. Because the human genomic sequences are
still being refined, the position of an SNP within a gene may be
different from previous publications. We named an SNP based on its
position relative to the first base of exon 1 (according to the NCBI
human genome sequence build 30) and also gave its corresponding
reference SNP identification numbers (rs#; Table 1). The cytogenetic
map, polymorphic sites, and location of the SNPs for the REN,
AGTR1, and AGT genes used in this study are shown in
Table 5. The 10 ACE SNPs were
obtained from Rieder et al. (1999).
DNA Isolation
DNA from buffy coat samples was extracted using the Puregene
commercial kit, which combines cell lysis and protein precipitation
(Gentra System, Inc.). DNA concentration was assessed by a Tecan GENIos
Fluorometer using Picogreen.
Genotyping
The PrimerExpress software was used to design probes and primers,
and a 5' nuclease allelic discrimination Taqman assay was used for SNP
genotyping (Perkin-Elmer Biosystems). The assay includes two
fluorescent Taqman oligonucleotide probes: an allele 1-specific probe
labeled with VIC and an allele 2-specific probe labeled with FAM
(6-carboxyfluorescein). The VIC or FAM reporter dye is covalently
attached to the 5'-terminal base of the probe, and a nonfluorescent
quencher dye, TAMRA (6-carboxy-tetramethylrhodamine), is attached at
the 3' end. Each 25-µL PCR reaction contained 50 ng of genomic DNA,
900 nM each PCR primer (Research Genetics), 200 nM each probe, and
Taqman Universal PCR Master Mix (a solution containing AmpErase
Uracil-N-glycosylase (UNG), deoxyribonucleotides, uridine,
passive reference dye (ROX), TaqGold DNA polymerase, and reaction
buffer (Applied Biosystems P/N 4316033). Amplifications were performed
under the following conditions: 50°C for 2 min for AmpErase UNG
degradation of any carryover DNA, followed by AmpliTaq Gold enzyme
activation at 95°C for 10 min before 40 cycles of 95°C for 15 sec
and 62°C for 1 min in a PTC-225 DNA Engine Tetrad thermal cycler (MJ
Reseach). Fluorescence in each well was measured after PCR using the
ABI Prism 7700 Sequence Detector System (SDS, PE Biosystems).
Statistical Analysis
Allele frequencies for each SNP were calculated by allele counting,
and the Hardy–Weinberg equilibrium was tested using the  2
test with 1 df. Pairwise LD was measured by D‘ (Lowontin 1964)
and r2 (Hill and Robertson 1968) within each ethnic
sample. Pairwise LD was tested by Fisher’s exact test (Chakravarti et
al. 1984). For each gene, haplotypes were reconstructed using the
computer progran PHASE (Stephens et al. 2001). PHASE uses Gibbs
sampling to estimate the posterior probabilities of an individual's
haplotypes given the observed genotypes, and thereby assigns haplotype
phases. A global test of LD was performed according to the simulation
method of Blanton and Chakravarti (1987), in which the observed number
of haplotypes was compared with the simulated number of haplotypes
under the assumption of linkage equilibrium (Antonarakis et al. 1988).
We defined a block as a DNA region in which there was no apparent
historical recombination. To determine these intervals, we first
examined the pairwise D‘ values. A pairwise D‘ value
of 1 indicates that no more than three of the four possible haplotypes
are observed (Leitersdor et al. 1989) and indicates the rarity of
recombination between a pair of SNPs. We first searched for intervals
in which all SNPs had pairwise D‘ > 0.8 and assumed that
they constituted the minimum blocks. These intervals were then expanded
by adding SNPs to the ends to find the longest intervals, as follows:
The estimated haplotypes and their 95% confidence intervals were
bootstrapped before adding an SNP. If the haplotype frequencies after
adding an SNP fell into the corresponding 95% confidence intervals, we
concluded that the added marker belonged to the same block. We repeated
this procedure until we found that adding a marker led to a statistical
change in the haplotype distribution. Based on this definition, we
would anticipate that no apparent recombination events had occurred
within a block.
We also calculated composite c, which is defined as half
of the sum of the absolute value of all allelic frequency differences
at a locus (Shriver et al. 1997):
where fi1 and fi2 are the
frequencies of the i-th allele in two populations. The
magnitude of c is the principal determinant of the
efficiency for admixture mapping (Chakraborty and Weiss 1988;
Chakraborty et al. 1991). We first calculated the c value
for each SNP and next the c values for a block by
considering block haplotypes as alleles.
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WEB SITE REFERENCES
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http://www.ncbi.nlm.nih.gov/Omim; Online Mendelian Inheritance in
Man (OMIM).
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Acknowledgements
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We thank Hongyu Zhao for helpful comments. We thank Donghui Kan for
his assistance in programming. This work was supported by grants from
the National Heart, Lung and Blood Institute (UOI–HL54485; HL54466;
HL65702).
The publication costs of this article were
defrayed in part by payment of page charges. This article must
therefore be hereby marked "advertisement" in accordance with 18
USC section 1734 solely to indicate this fact.
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Footnotes
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4 These authors contributed equally to this work. 
5 Corresponding author.
E-MAIL xzhu1{at}lumc.edu; FAX (708) 327-9009.
Article and publication are at
http://www.genome.org/cgi/doi/10.1101/gr.302003. Article published online before print in January 2003.
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REFERENCES
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Received March 22, 2002;
accepted in revised format October 22, 2002.
13:173-181 © by 2003 Cold Spring Harbor Laboratory Press ISSN 1088-9051/03 $5.00
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