A linkage disequilibrium map of the human major histocompatibility complex in singapore chinese conserved extended haplotypes and ancestral blocks 3

CHAPTER 3: RESULTS 49 Results 3.1 First Generation Linkage Disequilibrium and Haplotype Map of the Chromosome 6p and the Major Histocompatibility Complex To characterize the genetic variation and patterns of linkage disequilibrium (LD) of the human chromosome 6p and the MHC, separate genotyping projects were initiated and from these, distinct SNP maps with increasing marker densities were constructed SNP genotyping for all datasets were performed using the Illumina Golden Gate platform The descriptions of each SNP map are summarized in Table 3.1 The first map surveys the entire chromosome 6p arm at a density of approximately SNP every 100kb, the second focuses a higher density of SNPs (approximately SNP every 20kb) across a contiguous 7Mb stretch that contains the MHC These maps provide an overview of LD distribution across the MHC and allow comparisons to be made with the rest of the chromosome arm The third SNP map is focused solely within the MHC with a much greater density (1 SNP every 2.6kb) HLA haplotype and homozygosity data obtained from the first maps guided sample selection for the third map, allowing the detailed analysis of the common and conserved MHC haplotypes present in the Singapore Chinese population 50 Results Table 3.1: Summary of SNP Sets Used in this Study A first generation, low-density, SNP map was used to analyse the LD structure of the chromosome 6p and the MHC A high resolution SNP map was then constructed to analyse the LD of the MHC in greater detail The corresponding sections in the results chapter of this thesis is indicated for each SNP map 1st Generation Map 2nd Generation Map 1152 SNPs 2360 SNPs 198 Individuals 282 Individuals 1099 SNPs 2290 SNPs 192 Individuals 276 Individuals 909 SNPs 1877 SNPs Assayed Successfully Genotyped Informative SNPs Chr 6p Map (Section 3.1.2) MHC SNP Map (Section 3.1.3) High -Resolution MHC SNP Map (Section 3.2) Informative SNPs 615 345 1877 Chr Coordinates 175,572 58,750,370 30,133,482 37,000,199 28,970,148 33,882,048 SNP Density per 95.2kb per 19.9 kb per 2.6 kb SNP Sets 3.1.1 SNP Set of the First Generation Map A set of 1152 SNPs was selected and genotyped in 198 Singaporean Chinese individuals These individuals comprised of randomly selected, unrelated, healthy blood donors from whom appropriate informed consent were obtained The SNPs were chosen based on available genotype data deposited in dbSNP (build 121, Smigielski et al 2002) SNPs were initially selected to achieve a targeted density of SNP per 100kb across the entire Chromosome 6p arm, and a higher density of SNP per 10kb across the MHC and peri-MHC (physical coordinates 30.0Mb to 37.0Mb) 51 Results To reduce the chance of genotyping uninformative markers, priority was given to SNPs that has been shown to exist in high frequency in an East Asian population Of the 1152 SNP-genotyping attempted, 1099 were successful – this translates to a 95.4% marker success rate Of the 198 samples genotyped, samples failed to produce results that passed Illumina’s stringent quality checks, possibly due to inadequate DNA quality or quantity, thus attaining a sample success rate of 97.0% The overall genotype call rate and reproducibility was greater than 99.9% It has been noted that mis-genotyped and erroneously located SNPs may result in spurious linkage disequilibrium associations and false introduction of haplotype variants that not exist in nature (Gabriel et al 2002, Hosking et al 2004) The 1099 successfully genotyped SNPs were further passed through a series of quality control filters to remove possible genotyped errors First, the flanking sequences of the probes used in the assays were re-aligned to the human genome (NCBI build 36), ensuring that SNPs locations were as planned Eight loci could not be mapped to the chromosome 6p and were eliminated at this stage Next, to remove non-informative markers and identify possible genotyping errors, SNPs that had a minor allele frequency of less than 5% in the population, or did not satisfy the Hardy-Weinberg Equilibrium at a 0.1% significance level were removed 909 SNPs passed these sets of filters and were used to construct the SNP maps Allele frequencies for the SNPs assayed in these samples were compared to those reported for various populations in dbSNP, on which data it was relied on for the initial SNP selection As one would expect, the frequencies from this study had a 52 Results much higher correlation of determination (R2 = 0.86) with the aggregated East Asian population data as compared to that from other non-Asian populations (R2 = 0.35), thus providing a gauge to the reliability of the genotyping data (Figure 3.1) This also establishes that in the absence of any other information, the allele frequencies of SNPs reported in dbSNP would be sufficient in guiding informative marker selection in genotyping studies R2=0.86 Figure 3.1 Comparing Allele Frequencies Between Singapore Chinese and dbSNP Allele frequencies in the local Chinese population have a high correlation to frequencies reported in aggregated East Asian populations (panel A), in contrast with non-Asian populations (panel B) found in dbSNP R2=0.35 3.1.2 Chromosome 6p SNP LD Map By design, these 909 SNPs were not uniformly distributed across the Chromosome 6p; the MHC region and its perimeter were deliberately more densely surveyed In order to construct an unbiased linkage disequilibrium map across the chromosome 53 Results arm, a “picket-fence” approach was used to select genotyped SNPs from the denser 30.0Mb - 37.0Mb segment to achieve an approximate density of SNP per 100kb distribution consistent with the overall SNP density In all, 615 SNPs were used to construct a linkage disequilibrium map across the entire chromosome 6p, with the first marker starting at position 175,572bp and the last marker at position 58,750,370bp of the physical map The average SNP density is SNP per 95.2kb, with a median distance of 80.0kb between consecutive SNP pairs, ranging from 27kb to 485kb, with 414 pairs less than 100kb apart The average minor allele frequency of the data set is 28% with an average heterozygosity of 0.37 To evaluate the LD structure across the chromosome arm, the most commonly used measures of LD, r2 and D′, were calculated between all possible SNP pairs separated by less than 5Mb D′ and r2 are both based on the disequilibrium parameter D (Ott 1999), a difference between observed and expected frequencies of 2-locus haplotypes, although they differ in their interpretation; D′ is strictly an indicator of the absence of recombination in the history of the studied population samples, whereas a high r2 value has an additional requirement of correlation between allele frequencies ( Devlin and Risch 1995, Ardlie et al 2002) The distribution of LD between SNP pairs on this map is shown as a heatmap in Figure 3.2 and can be seen to vary greatly along the chromosome arm Stronger LD between consecutive marker pairs is seen towards the centre of the chromosome 6p especially in the region telomeric to the MHC The punctuate nature of LD can be seen with several islands of SNPs in high pairwise LD standing out in contrast with the relatively uniform level of equilibrium in the rest of the chromosome arm At an average marker density of SNP every 95kb, strong LD between consecutive SNPs is not expected (Gabriel et al 2002, International HapMap 54 Results Consortium 2005), and these islands of high LD are the exceptions rather than the rule Figure 3.2 Chromosome 6p SNP LD Map Gene density across the chromosome 6p (represented in blue) is plotted as sliding window gene counts per 100kb Locations of highlighted genes are represented green glyphs All gene annotations were taken from the Vertebrate Genome Annotation Database (Vega) (Wilming et al 2008) The set of SNPs used in the map are drawn as vertical grey lines Linkage-disequilibrium between pairs of SNPs is depicted using a heatmap produced by Haploview (Barret et al 2005), with darker shades of red representing pairs of SNPs with high D´ 55 Results Smoothing out the pairwise LD values by averaging them across 2Mb sliding windows, the distribution of LD across the chromosome arm was plotted as a function of physical distance in Figure 3.3 Figure 3.3 Distribution of LD Across the Chromosome 6p Averaged pairwise LD between SNPs within 2Mb windows was calculated and plotted against physical distance LD was calculated using both the D′ coefficient (blue shaded area) as well as r2 (red shaded area) The averaged pairwise LD value across the whole chromosome arm is indicated by the blue (D′) and yellow (r2) horizontal dotted lines The HapMap genetic map (release 22) in centiMorgans is also plotted in the green line Given that markers in LD not necessarily show strong allelic correlation (Ott 1999), averaged r2 values are much lower than D′ but the trend of these parameters track evenly across the chromosome arm With the high marker spacing and relative sparseness of this SNP map, high r2 values are not expected and the mean r2 value between pairs of markers less than 5Mb apart is only 0.03 (yellow dotted line) The average for D′ is 0.16 (blue dotted line) There is a noticeable elevation of linkage disequilibrium above the chromosomal-average at several locations, the most prominent of these being an 8Mb-long segment at the centre of the chromosome arm, with elevated LD seen in both D′ and r2 values This strong LD segment lies between 56 Results positions 25Mb and 33Mb with the peak being a 2Mb window centred at position 28.9Mb This peak between positions 27.9Mb and 29.9Mb of chromosome 6p contains 21 informative SNPs with pairwise D′ averaging 0.54 and pairwise r2 averaging 0.15 This segment of elevated LD is also underscored by fewer recombination hotspots and a lower recombination rate (0.46 cM/Mb as opposed to chromosome average of 1.27cM/Mb) in the genetic map reported recently by the International HapMap project (International HapMap Consortium 2005) The centromeric half of this high-LD segment contains the classical MHC loci (positions 30.0Mb – 33.4Mb), while the telomeric half is marked by the presence of the largest histone cluster in the human genome (there are over 40 loci coding for histone genes between 26 Mb to 28 Mb) as well as an 8-zinc finger cluster (between 27.5Mb to 28.7Mb) At the centre and peak of this high-LD segment is a large olfactory receptor cluster, with 13 olfactory receptor genes between 29.1Mb to 29.6 Mb The gene map showing the clusters in this region can be seen in Figure 3.4 57 Results Figure 3.4: Gene Clusters Telomeric to the MHC The list of genes in this region is obtained from the VEGA project (Wilming et al 2008) The large gene clusters in the region between 25Mb and 30Mb can be clearly seen in this figure The centre of the high-LD segment (28.9Mb) lies close to a large olfactory receptor cluster marked out in a red border 58 Results Table 3.3 Distribution of HLA Allele Frequencies HLA-A HLA-C HLA-DRB1 Allele Frequency Allele Frequency Allele Frequency Allele Frequency A*1101 A*2402 A*0207 A*3303 A*0201 A*0203 A*1102 A*0206 A*3101 A*2601 A*3001 A*0101 A*0301 A*2901 A*6801 A*2410 A*2420 30.21% (116) 13.28% (51) 12.76% (49) 10.42% (40) 8.59% (33) 8.07% (31) 4.43% (17) 3.13% (12) 2.08% (8) 1.56% (6) 1.56% (6) 0.78% (3) 0.78% (3) 0.78% (3) 0.52% (2) 0.26% (1) 0.26% (1) C*0102 C*0702 C*0304 C*0302 C*0801 C*0401 C*1202 C*0303 C*1402 C*1502 C*0602 C*1203 C*0403 C*1505 C*0701 C*0704 C*0103 22.14% (85) 20.83% (80) 13.28% (51) 8.85% (34) 8.59% (33) 4.95% (19) 3.65% (14) 3.39% (13) 3.39% (13) 3.13% (12) 2.34% (9) 1.56% (6) 1.04% (4) 0.78% (3) 0.52% (2) 0.52% (2) 0.26% (1) DRB1*0901 DRB1*1202 DRB1*1501 DRB1*0803 DRB1*1101 DRB1*1602 DRB1*0405 DRB1*0301 DRB1*1201 DRB1*0403 DRB1*0406 DRB1*0701 DRB1*1401 DRB1*0404 DRB1*0802 DRB1*1301 DRB1*1405 16.93% (65) 9.38% (36) 8.59% (33) 7.81% (30) 7.03% (29) 7.55% (27) 6.51% (25) 6.51% (25) 4.69% (18) 3.39% (13) 2.86% (11) 2.86% (11) 2.86% (11) 1.3% (5) 1.3% (5) 1.3% (5) 1.3% (5) DRB1*0809 DRB1*1302 DRB1*1001 DRB1*1439 DRB1*1502 DRB1*0101 DRB1*1312 DRB1*0307 DRB1*0327 DRB1*0801 DRB1*1104 DRB1*1106 DRB1*1303 DRB1*1403 DRB1*1404 DRB1*1504 1.04% (4) 1.04% (4) 0.78% (3) 0.78% (3) 0.78% (3) 0.52% (2) 0.52% (2) 0.26% (1) 0.26% (1) 0.26% (1) 0.26% (1) 0.26% (1) 0.26% (1) 0.26% (1) 0.26% (1) 0.26% (1) A*3201 A*3401 0.26% (1) 0.26% (1) C*0586 C*0802 C*1403 0.26% (1) 0.26% (1) 0.26% (1) HLA-B Allele Frequency Allele Frequency B*4001 B*4601 B*5801 B*1301 B*3802 B*1502 B*5502 B*5101 B*2704 B*5401 B*1501 B*3901 B*5102 B*4002 B*3501 B*4801 B*1302 B*0705 B*4006 B*4403 B*5601 B*0801 B*1511 15.63% (60) 15.1% (58) 8.85% (34) 6.77% (26) 5.47% (21) 5.21% (20) 4.95% (19) 3.91% (15) 3.39% (13) 2.86% (11) 2.6% (10) 2.6% (10) 2.08% (8) 2.08% (8) 1.82% (7) 1.82% (7) 1.56% (6) 1.3% (5) 1.04% (4) 0.78% (3) 0.78% (3) 0.52% (2) 0.52% (2) B*1518 B*1527 B*1801 B*3503 B*3505 B*5201 B*5501 B*5504 B*5604 B*5701 B*1401 B*1512 B*1521 B*1525 B*1558 B*1802 B*3701 B*3801 B*3909 B*5107 B*5512 B*5603 0.52% (2) 0.52% (2) 0.52% (2) 0.52% (2) 0.52% (2) 0.52% (2) 0.52% (2) 0.52% (2) 0.52% (2) 0.52% (2) 0.26% (1) 0.26% (1) 0.26% (1) 0.26% (1) 0.26% (1) 0.26% (1) 0.26% (1) 0.26% (1) 0.26% (1) 0.26% (1) 0.26% (1) 0.26% (1) Table 3.3 Distribution of HLA Allele Frequencies The complete list of alleles at the classical HLA loci (HLA-A, -B, -C and -DRB1) seen in 192 individuals from the Singaporean Chinese Population Absolute counts can be found in parentheses under the frequency column Common alleles (present at least 5% in the population) are listed in bold 65 Results The HLA haplotypes of each individual were reconstructed using the application PHASE, a Bayesian-based algorithm that has been shown to be accurate for biallelic and multi-allelic haplotype reconstruction (Stephens and Scheet 2005, Marchini et al 2006) The list of common 2- and 3-locus HLA haplotypes that are seen in more than chromosomes is shown in Table 3.4 Two long-range 3-locus haplotypes are seen to exist in remarkably high frequency in the population: the A*0207-B*4601DRB1*0901 and A*3303-B*5801-DRB1*0301 haplotypes are each present at a frequency of almost 5% in the population The HLA allele and haplotype frequencies presented here agree very well with a report published very recently with data from 536 Singaporean Chinese samples (Tang et al 2007) 3.1.3.3 Linkage Between HLA Alleles The HLA genes are the most polymorphic in the human genome; in this sample of 384 chromosomes from the Singaporean Chinese population, there are a total of 19 HLA-A, 20 HLA-C, 45 HLA-B and 33 HLA-DRB1 alleles seen However, due to allelic association between HLA alleles, the diversity of HLA haplotypes fall short of that as expected under linkage equilibrium, with some haplotypes over represented For example, given the individual frequencies of HLA-A*0207 and HLA-B*4601 (12.8% and 15.1% respectively, from Table 3.3), if the alleles are truly independent, only 2% of the samples are expected to carry the haplotype A*0207-B*4601 Yet, this haplotype is present in over 9% of the chromosomes (Table 3.4) In contrast, the frequency of the A*1101-B*4001 haplotype (5.47%) does not deviate from the expected, suggesting that allelic association may not be strong between all HLA allele pairs This allelic association is also reflected in the p-values of the difference between the expected and observed haplotype frequencies (Table 3.4) 66 Results Table 3.4 Common 2- and 3-locus HLA Haplotypes in Singaporean Chinese Population HLA haplotypes were reconstructed using the program PHASE and these are listed as observed frequencies Only haplotypes seen in more than individual chromosomes are listed in this table Expected frequencies are calculated from individual allele frequencies P-values for 2-locus haplotypes are calculated using 2X2 contingency tables and Fisher’s test, while pvalues for 3-locus haplotypes were calculated using the 1-sample z-test HLA-A, B Haplotype Count Obs Freq Exp Freq P-Value HLA-B, DRB1 Haplotype Count Obs Freq Exp Freq P-Value A*0207 B*4601 A*3303 B*5801 A*1101 B*4001 A*1101 B*1301 A*0203 B*3802 A*1101 B*1502 A*2402 B*4001 A*0201 B*4001 A*1101 B*4601 A*1101 B*5502 A*1102 B*2704 A*1101 B*5101 A*1101 B*1501 A*1101 B*5401 A*2402 B*4601 36 29 21 16 15 12 12 10 9 6 9.38% 7.55% 5.47% 4.17% 3.91% 3.13% 3.13% 2.60% 2.34% 2.34% 2.34% 2.08% 1.56% 1.56% 1.56% 1.93% 0.92% 4.72% 2.05% 0.44% 0.16% 2.08% 1.34% 4.56% 1.57% 0.15% 1.18% 0.79% 0.87% 2.01% 0.0001 0.0001 0.4410 0.0007 0.0001 0.0050 0.1003 0.0228 0.0080 0.1225 0.0001 0.0801 0.0727 0.0956 0.6740 B*4601 DRB1*0901 B*5801 DRB1*0301 B*1502 DRB1*1202 B*4001 DRB1*0901 B*4001 DRB1*1602 B*4601 DRB1*1101 B*4001 DRB1*1501 B*1301 DRB1*1501 B*3802 DRB1*1602 B*5401 DRB1*0405 B*4601 DRB1*0803 B*4001 DRB1*1101 B*1301 DRB1*1602 27 22 12 11 10 7 7 6 6 7.03% 5.73% 3.13% 2.86% 2.60% 1.82% 1.82% 1.82% 1.82% 1.56% 1.56% 1.56% 1.56% 2.56% 0.58% 0.49% 2.64% 1.18% 1.06% 1.10% 0.58% 0.41% 0.19% 1.18% 1.10% 0.51% 0.0001 0.0001 0.0001 0.7112 0.0074 0.1566 0.3250 0.0037 0.0004 0.0001 0.4276 0.4057 0.0087 HLA-C, B Haplotype Count Obs Freq Exp Freq P-Value HLA-A, B, DRB1 Haplotype Count Obs Freq Exp Freq P-Value C*0102 B*4601 C*0302 B*5801 C*0702 B*4001 C*0304 B*1301 C*0801 B*1502 C*0702 B*3802 C*0304 B*4001 C*1202 B*2704 C*0102 B*5401 C*0102 B*5502 C*0702 B*3901 C*1402 B*5101 C*0401 B*1501 C*0602 B*1302 C*0801 B*4801 54 34 32 24 20 19 17 12 11 10 10 10 6 14.06% 8.85% 8.33% 6.25% 5.21% 4.95% 4.43% 3.13% 2.86% 2.60% 2.60% 2.60% 1.82% 1.56% 1.56% 3.34% 0.78% 3.26% 0.90% 0.45% 1.14% 2.08% 0.12% 0.63% 1.10% 0.54% 0.13% 0.13% 0.04% 0.16% 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0006 0.0001 0.0001 0.0028 0.0001 0.0001 0.0001 0.0001 0.0001 A*0207 B*4601 DRB1*0901 A*3303 B*5801 DRB1*0301 A*1101 B*4001 DRB1*1501 A*1101 B*1502 DRB1*1202 A*0203 B*3802 DRB1*1602 19 18 7 4.95% 4.69% 1.82% 1.82% 1.56% 0.33% 0.06% 0.41% 0.15% 0.03% =0.7) across the segment are A*1101-B*1502 (HH=0.73), A*1102B*2704 (HH=0.58), A*0203-B*3802 (HH=0.74), A*0207-B*4601 (HH=0.84) and A*3303-B*5801 (HH=1.00) Within the HLA-B, -DRB1 haplotypes strong fixity is seen in the following haplotypes: B*1301-DRB1*1501 (HH=0.71), B*1502- DRB1*1202 (HH=1.00), B*3802-DRB1*1602 (HH=1.00), B*4601-DRB1*0901 (HH=0.92), B*5401-DRB1*0405 (HH=1.00) and B*5801-DRB1*0301 (HH=0.91) There are only three-locus HLA-A,-B,-DRB1 haplotypes that are represented more than times in the samples, and of these A*0203-B*3802-DRB1*1602 (HH=1.00), A*0207-B*4601-DRB1*0901 (HH=0.72) , A*1101-B*1502-DRB1*1202 (HH=1.00) and A*3303-B*5801-DRB1*0301 (HH=1.00) are highly conserved The genetic fixity of these haplotypes across unrelated chromosomes is remarkable For instance, 18 chromosomes carry the A*3303-B*5801-DRB1*0301 haplotype and they appear completely identical across the entire 2.6Mb segment In contrast A*1101-B*4001DRB1*1501 show very little identity across the segment, and with HH=0, indicates that none of the chromosomes in the samples which carry these alleles are identical The plots also show that HLA allele frequencies are not an indicator of homozygosity HLA alleles common in the local Chinese population such as A*0201, A*2402, B*1301, B*4001, DRB1*1101 and DRB1*1501 are not associated with haplotypes with high homozygosity Additionally, although the HLA-A*1101 allele (found in 73 Results over 30% of the chromosomes) is associated with the B*1502-DRB1*1202 haplotype, most A*1101 carrying haplotypes are not conserved Comparing the allelic association plots in Figure 3.4 with the homozygosity data, allele pairs that are strongly correlated (r2 > 0.5) all have high homozygosity These pairs are A*0203 - B*3802, A*0207 - B*4601, A*3305 - B*5801 and B*5801 DRB1*0301 However, HLA allele-pairs with low correlation as determined by r2 may still be associated with a conserved haplotype For example, B*3802 and DRB1*1602 have a low pairwise r2 (0.06), yet all haplotypes carrying these alleles are completely identical 74 Figure 3.8 SNP Homozygosity Plots for Common 2-Locus HLA-A HLA-B Haplotypes Homozygosity values for each successfully genotyped SNP between the HLA-A and -B loci are plotted for common HLA-A, -B haplotypes The number of chromosomes (out of a possible 384) carrying each HLA haplotype is shown indicated the plots (n = X) The overall haplotype homozygosity (HH) is also indicated Results 75 Figure 3.9 SNP Homozygosity Plots for Common 2-Locus HLA-B, HLA-DRB1 Haplotypes Homozygosity values for each successfully genotyped SNP between the HLA-B and -DRB1 loci are plotted for common HLA-B, -DRB1 haplotypes The number of chromosomes (out of a possible 384) carrying each HLA haplotype is shown indicated the plots (n = X) The overall haplotype homozygosity (HH) is also indicated Results 76 Figure 3.10 SNP Homozygosity Plots for Common 3-Locus HLA-A, HLA-B, HLA-DRB1 Haplotypes Homozygosity values for each successfully genotyped SNP between the HLA-A and -DRB1 loci are plotted for common HLA-A, -B, -DRB1 haplotypes The number of chromosomes (out of a possible 384) carrying each HLA haplotype is shown indicated the plots (n = X) The overall haplotype homozygosity (HH) is also indicated Results 77 Results The haplotype pattern seen in these SNP-HLA data is consistent with the hypothesis that the MHC is made up of segments (or blocks) of ancestral haplotypes These segments have shuffled through homologous recombination forming the large number of haplotypic variants seen in the population As a result of factors such as admixture, population bottlenecks, selection, recombination suppression and genetic drift, some of these haplotypes expanded in frequency within populations, leading to long stretches of genetic fixity that appears as conserved extended haplotypes The HLA-B,-DRB1 haplotypes carrying DRB1*1602 illustrate this well The ancestral haplotype carrying DRB1*1602 shuffled into other haplotypes carrying different HLA-B alleles, and as a result DRB1*1602 is seen with multiple HLA-B partners (B*1301, B*3802 and B*4001) with high frequency Multiple recombination events over time added to the diversity in DRB1*1602 haplotypes, leading to the low conservation within DRB1*1602-B*4001 or DRB1*1602-B*1301 haplotypes However, the DRB1*1602-B*3802 recombinant haplotype may have arose relatively recently and expanded in numbers rapidly, perhaps through selection or admixture, maintaining the genetic fixity in the haplotypes Conserved Extended Haplotypes that exist in the local Chinese population can be deduced from the preceding data From the 3-locus plots: A*0203-B*3802DRB1*1602, A*0207-B*4601-DRB1*0901, A*1101-B*1502-DRB1*1202 and A*3303-B*5801-DRB1*0301 are conserved haplotypes, stretching across and possibly beyond 2.6Mb Additionally from the 2-locus plots B*5401-DRB1*0405 and A*1102-B*2704 are conserved haplotypes stretching across more than 1Mb We cannot discount that other conserved haplotypes exist if other markers or gene loci, 78 Results such as the TNF and Complotype blocks (Yunis et al 2003, Alper et al 2006) are used to define haplotypes 79 ... 4 32 .155 32 .32 7 32 .414 32 .472 32 .541 32 .802 32 . 933 33 . 031 33 .185 33 .34 0 33 .37 6 33 .409 33 .467 33 .6 83 33. 718 33 . 739 33 .769 33 .848 33 .864 33 .952 34 .026 34 .065 34 .160 34 . 230 34 .36 3 34 .544 34 .659 34 .948... 34 .948 35 .117 35 .240 35 .277 35 .32 4 35 .486 35 .587 32 .166 32 .39 2 32 .4 43 32.485 32 .557 32 . 832 32 .941 33 .071 33 .194 33 .36 6 33 .38 8 33 .419 33 .546 33 .696 33 .728 33 .752 33 .788 33 .858 33 .880 33 .966 34 . 034 34 .068...Results 3. 1 First Generation Linkage Disequilibrium and Haplotype Map of the Chromosome 6p and the Major Histocompatibility Complex To characterize the genetic variation and patterns of linkage disequilibrium