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Published in final edited form as: Immunogenetics. 2015 Jul 22;67(9):479–485. doi: 10.1007/s00251-015-0857-y

Very long haplotype tracts characterized at high resolution from HLA homozygous cell lines

Paul J Norman 1, Steve Norberg 2, Neda Nemat-Gorgani 1, Thomas Royce 2, Jill A Hollenbach 3, Melissa Shults Won 2, Lisbeth A Guethlein 1, Kevin L Gunderson 2, Mostafa Ronaghi 2, Peter Parham 1,*
PMCID: PMC4540692  NIHMSID: NIHMS710085  PMID: 26198775

Abstract

The HLA region of chromosome 6 contains the most polymorphic genes in humans. Spanning ~5Mbp the densely packed region encompasses approximately 175 expressed genes including the highly polymorphic HLA class I and II loci. Most of the other genes and functional elements are also polymorphic, and many of them are directly implicated in immune function or immune-related disease. For these reasons this complex genomic region is subject to intense scrutiny by researchers with the common goal of aiding further understanding and diagnoses of multiple immune-related diseases and syndromes. To aid assay development and characterization of the classical loci, a panel of cell lines partially or fully homozygous for HLA class I and II was assembled over time by the International Histocompatibility Working Group (IHWG). Containing a minimum of 88 unique HLA haplotypes, we show this panel represents a significant proportion of European HLA allelic and haplotype diversity (60–95%). Using a high-density whole genome array that includes 13,331 HLA region SNPs, we analyzed 99 IHWG cells to map the coordinates of the homozygous tracts at a fine scale. The mean homozygous tract length within chromosome 6 from these individuals is 21Mbp. Within HLA the mean haplotype length is 4.3Mbp, and 65% of the cell lines were shown to be homozygous throughout the entire region. In addition, four cell lines are homozygous throughout the complex KIR region of chromosome 19 (~250kbp). The data we describe will provide a valuable resource for characterizing haplotypes, designing and refining imputation algorithms and developing assay controls.

Keywords: HLA class I, HLA class II, HLA region genes, Homozygous, HLA imputation, KIR

Introduction

The HLA region of chromosome 6 harbours the most polymorphic human genes (Horton et al. 2004; Tennessen et al. 2012; Robinson et al. 2015), has vital roles in immune defence and reproduction, and exerts a dominant impact on immune-mediated diseases and syndromes (Parham et al. 2013; Trowsdale et al. 2013). The most direct application for clinical HLA genotyping is in transplantation, where millions of individuals are genotyped worldwide to enable tissue matching of donors with patients (Terasaki 1969; Petersdorf 2013). HLA genotyping is also critical for avoiding specific drug sensitivity (Illing et al. 2013) and assists accurate diagnoses or analyses of many autoimmune diseases (de Bakker et al. 2012). To aid both the characterization of HLA variation and development of techniques to detect it, a panel of immortalized HLA homozygous cell lines was established almost 30 years ago by the International Histocompatibility Working Group (IHWG) (Yang et al. 1987). The cell panel and its genetic characterization continue to be updated and remain valuable resources for discovering and mapping HLA region variation (Marsh et al. 1996; Horton et al. 2004; Dorak et al. 2006; Mickelson E et al. 2006; Hosomichi et al. 2013; Larsen et al. 2014). As many of the cell lines were generated from consanguineous individuals, they also present an opportunity to study HLA haplotypes that are identical by descent (IBD). Thus, to reduce complexity during long-range haplotype assembly and ultimately provide unambiguously-phased reference sequences for the entire HLA region we genotyped these cell lines to define the precise coordinates of their homozygous tracts. Using a high-density whole genome SNP array, we analyzed 13,331 HLA region SNPs from 99 HLA homozygous cells. The mean IBD haplotype length was 4.3Mbp, and 65% were homozygous throughout the entire 5.0Mbp HLA region of chromosome 6. In addition, four of the cell lines were homozygous throughout the KIR region of chromosome 19.

Materials and Methods

Target region coordinates

The classical HLA region encompasses 4.7Mbp of chromosome 6p21, and includes all of the HLA Class I and II genes (Horton et al. 2004). The region is extended herein to 5.0Mbp to account for non-overlapping segments of the two haplotypes that have been fully sequenced, COX and PGF (Stewart et al. 2004). Thus we define the HLA region as spanning chr6:28477897-33500000 bp, and flanked by SNP numbers rs2108925-rs449242. The closest genes within these markers are GPX5 (28493788-28502728) and ZBTB9 (33422355-33377699). The ‘extended HLA region’ (Horton et al. 2004) begins approximately at SNP rs9358871 (chr6:25726675): encompassing HIST1H2AA (25726290-25726790) as the gene at the 5’ extreme. The KIR region of chromosome 19 is flanked by SNP numbers rs1325158-rs17771967 (chr19:55226402-55380214 bp). All coordinates are shown according to Human Genome build 37 (hg19).

DNA samples and conventional HLA genotypes

DNA was extracted from 99 EBV-transformed B cell lines. These were chosen from knowledge of their HLA Class I and II genotypes (http://www.ihwg.org/), because they have the lowest possible heterozygosity within the 5.0Mbp HLA complex (Supplementary Fig. 1). Low passage cells were grown from archive material available at Stanford, or purchased from the IHWG repository. All the cells are homozygous for HLA-B, 97% are homozygous for HLA-A, -C or -DRB1, 95% for -DQB1 and 78% for -DPB1. The panel consisted of 87 cells from the 10th IHWC (Yang et al. 1987) and 12 cells from the 12th and 13th IHWC (Marsh et al. 1996; Mickelson E et al. 2006), and included all eight cells targeted by the MHC sequencing consortium (Horton et al. 2004). At least fifty of the cells derive from consanguineous donors (Supplementary Fig. 1), so their haplotype pairs are likely identical by descent (IBD). All of these cells have been genotyped for HLA class I and II by multiple investigators and clinical transplant centres worldwide (Mickelson E et al. 2006; Hosomichi et al. 2013). DNA samples were genotyped for confirmation to ‘4-digit’ resolution of HLA-A, -B and -C using LABType SSO reagents (One Lambda, Canoga Park, CA) and a Luminex 100 reader (Luminex, Austin TX). In addition, the cells were genotyped for presence of the HLA-Y pseudogene, using primers and conditions described previously (Gleimer et al. 2011). HLA-Y marks the presence of a ~70kb structural variant detected on some HLA haplotypes (Watanabe et al. 1997). We also used data obtained from 90 Europeans, 44 Chinese Han, 45 Japanese and 90 Yorubans studied in the HapMap phase II project (Myers et al. 2005) and who were genotyped using the same whole-genome 1.1M SNP array as the IHWG panel. From this set, we were able to assemble a control sample set of 86 individuals, sex and ethnically matched with 86 of the HLA-homozygous cell line donors.

Whole genome SNP analysis

Whole genome SNP genotyping (Steemers et al. 2006) was performed using the Infinium HumanOmni1-Quad BeadChip array (Illumina, San Diego CA) according to the manufacturer’s instructions, and analysed using PLINK 1.07 (Purcell et al. 2007). Whole-genome SNP data from one sample (WAR) was obtained as part of another study (Moreno-Estrada et al. 2013). There were 123,995 genome-wide markers specific for copy number variants excluded from further analysis as were all (from 14,490) HLA-region SNPs with >10% failure rate. Of the SNPs that passed filtering, 402 occurred within HLA class I or II genes and only 32 of these were in exons. This reflects the high sequence divergence and non-binary nature of HLA allelic polymorphism. Following the filtering process 13,331 SNPs in the HLA region were analyzed. Any further SNPs with missing data were excluded per individual so that a mean of 13,269 SNPs were used to estimate the homozygous tract lengths.

The size and location of each homozygous tract that is greater than 1Mbp was estimated using PLINK (Purcell et al. 2007), set to tolerate two heterozygous SNPs per 500kbp window. The coordinates of the homozygous tracts overlapping the HLA region were adjusted manually to indicate the estimated start point, which likely occurs between the last known heterozygous SNP and the first homozygous SNP within the segment indicated by PLINK. To search for homozygous tracts in the KIR region, the analysis was repeated using 200kbp as the minimum homozygous tract size. The coordinates of HapMap II recombination hotspots were obtained from Myers et al. 2005 (Myers et al. 2005) and mapped to hg19 using the UCSC genome browser liftOver tool (Karolchik et al. 2014). The mean resolution of these hotspots within the HLA region is +/− 5kb. Principal components analysis (PCA) plots were generated from genome-wide SNP data in PLINK binary files using the SNPRelate package in R. Only autosomal, non-monomorphic SNPs were considered for analysis, and the first two principal components plotted.

Results and Discussion

To characterize full-length HLA haplotypes at high-resolution and with unambiguous phase, 99 individuals were selected from a panel of HLA homozygous cell lines (Yang et al. 1987). Of 1,013,490 genome-wide SNPs that were genotyped successfully, 13,331 fall within the 5.0Mbp HLA region (median: 1 SNP per 152bp) and a further 3,947 correspond to the additional 3.1Mbp known as the ‘extended HLA’ region (median: 1 SNP per 390bp). Comparison of the entire genome data with that from major world populations using PCA confirmed that the majority (80%) of the cell lines were derived from European-origin donors (Fig. 1). Also included in our panel are two Africans, five Amerindians, seven East Asians, one Hispanic and one South Asian individual (Fig. 1). The genome-wide SNP data from the IHWG cell panel are available in human-readable and PLINK binary format at ImmPort under SDY295: EXP13576 (https://immport.niaid.nih.gov/).

Figure 1. The IHWG panel comprises mostly European individuals.

Figure 1

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Shown is a PCA plot using ~1 million genome wide SNP markers from four HapMap populations (Eur, CHB, JPT, Yoruba) and the IHWG panel. There were no Amerindian populations in HapMap II, thus the five Amerindian IHWG samples form a separate cluster (top centre). AMA1 is North African, MANIKA is South Asian (Indian) and MGAR is Hispanic. Also labelled are the two cells, COX and PGF completed by the MHC sequencing consortium (Stewart et al. 2004). PGF is the hg19 reference sequence for the HLA region. The genome-wide data set is available at ImmPort under SDY295: EXP13576 (https://immport.niaid.nih.gov/).

Present in the HLA-homozygous cell line panel are 24 HLA-A, 34 HLA-B, 17 HLA-C, 31 HLA-DRB1, 16 HLA-DQB1 and 15 HLA-DPB1 alleles (Fig. 2 and Supplementary Figure 1). HLA-Y was detected in 12 of the cell lines, and linked to A*02:05, 30:01, 31:01, 33:01 or 68:02, as observed previously (Watanabe et al. 1997; Williams et al. 1999; Coquillard et al. 2004). A minimum of 75 distinct A-B-C and 88 A-B-C-DRB1 haplotypes are present in the IHWG sample panel (Fig. 2 and Supplementary Figure 1). Two are common European haplotypes. Each of the two common haplotypes is present in five homozygous cell lines and these include the COX (A*01:01, B*08:01, C*07:01, DRB1*03:01) and PGF (A*03:01, B*07:02, C*07:02, DRB1*15:01) haplotypes that were determined to completion by the MHC sequencing project (Stewart et al. 2004). We estimate the allele distribution of the cell lines represents 90–95% of the European and 60–80% of the world population by allele frequency (from data available at dbMHC and allelefrequencies.net; Fig. 2). We also estimate the sample panel represents 20–50% of the HLA haplotype diversity of Europeans. The high-density SNP genotyping showed that 88.3% SNP markers within the HLA region vary in this sample set, which is similar to 86% in a sex and ethnically matched control set of samples that were not selected for HLA genotype. Together, these findings show that the sample set represents a significant proportion of European HLA diversity and provides a smaller but reasonable representation of worldwide HLA allelic diversity, despite having been selected for HLA homozygosity.

Figure 2. The IHWG panel represents a significant proportion of European HLA diversity.

Figure 2

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The distribution of HLA alleles and haplotypes present in the IHWG panel was compared with their frequencies available at dbMHC and allelefrequencies.net (Gonzalez-Galarza et al. 2015; Sayers 2015) to estimate the proportion of individuals in Europe and worldwide who possess them.

We used the high-density SNP data to measure the coordinates of the homozygous tracts that intersect the HLA region. These are shown in Supplementary Figure 2. The mean size of homozygous tract within chromosome 6 of the IHWG cell lines is 21Mbp and the largest is 135.0Mbp (79% of chromosome 6: cell CB6B). Sixty-three of the cells are homozygous through the entire 5.0Mbp HLA region and 53 of these are also homozygous through the ‘extended HLA’ (Fig. 3). Whilst 49–62% of the IHWG panel is derived from consanguineous donors (Supplementary Figure 1), 66–80% of the cells that are homozygous through the entire HLA region are consanguineous (Supplementary Figure 2). Thus the set of longest haplotypes that we describe is likely to be enriched for ones that are identical by descent (IBD). The IHWG cell lines are homozygous for an average 4.3Mbp (87%) of the 5.0Mbp region and the minimum HLA homozygous tract length is 1.0Mbp (cell JY). Four of the homozygous tracts are not continuous through the region, each being broken by a single heterozygous segment of ~200–600kb (BER, EJ32B, HOKKAIDO and SA1: Supplementary Figure 2). One sample (HOM2) had unexpectedly high heterozygosity in the intervals between the classical HLA genes that had been genotyped with conventional methods (Fig. 4). As the SNP diversity within these segments was similar to that of the heterozygous samples, this shows that the two HLA haplotypes of HOM2 are not IBD, despite the cell line being listed as consanguineous (Supplementary Figure 1). We show similarly that the PGF cell line is heterozygous through the first ~500kb of the targeted region, as previously suspected (Stewart et al. 2004). Four of the cell lines (AWELLS, LKT3, MT14B and SPL) are fully homozygous through the KIR region of chromosome 19q13.4, and PF04015 and T7526 have >50% homozygosity of KIR (not shown).

Figure 3. IWHG cell homozygous tracts that intersect the HLA region.

Figure 3

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Shown are homozygous segments determined from high density whole genome SNP analysis. Blue lines denote the interval between the last observed heterozygous SNP and the first homozygous SNP at each end of the homozygous tracts. Red dashed line indicates the 5Mb HLA region and the grey dotted line indicates the ‘extended’ HLA region. The precise genomic coordinates of the homozygous tracts for each of the cell lines are given in Supplementary Figure 2.

Figure 4. HLA haplotypes of HOM2 are not identical by descent.

Figure 4

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SNP diversity was calculated in sliding windows of 100bp through the HLA region from four cell lines; EN1NOT (heterozygous), COX (homozygous), PGF (partially homozygous; one haplotype is huRef build 19), and HOM2. HOM2 is homozygous for HLA-A, -B, -C, -DP, -DQ and -DR by conventional genotyping methods (Fig. 1). The locations of the classical HLA genes are shown in the lower panel.

The Hapmap II project mapped 29 recombination hotspots to the HLA region (Myers et al. 2005). We determined that 52% of the homozygous tract breakpoints in the HLA region of the IHWG cells occur within one of these recombination hotspots (Fig. 5). This finding is consistent with a genome-wide average of 50–60% recombination events occurring within hotspots (Myers et al. 2005; Coop et al. 2008). Within the HLA region the most frequent haplotype breakpoints of the IHWG cell lines overlap with the previously identified (Jeffreys et al. 2001; Cullen et al. 2002; Stenzel et al. 2004) hotspots, GABBR1 (29571319-29600162), TAP2 (32790065-32806010), DMB (32902747-32908584) and DOA (32974614-32977313) (Fig. 5). A further understanding of the characteristics and commonalities of these hotspots is likely to be revealed through complete sequencing of the surrounding regions.

Figure 5. Recombination hotspots and HLA haplotype breakpoints.

Figure 5

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Top; shows the number of heterozygous haplotypes in the IHWG panel (N=99) at each point through the HLA region. Inverted triangles show the location of the 29 recombination hotspots predicted to occur within the HLA region (Myers et al. 2005). The triangles are filled in red for hotspots overlapping with breakpoints in the IHWG panel, and the size corresponds to the frequency of use in the panel. Lower; shows the number of cells that become heterozygous at each point (53% of breakpoints overlap with the predicted hotspots).

Whole-genome SNP arrays can provide a low resolution scan of genomic structural variation. By virtue of the extended homozygosity in the IHWG panel we were able to identify nine large (>35kb) structural variants that associate with specific HLA alleles in this panel (Fig. 6). Surprisingly, the presence of HLA-Y is associated with two distinctive patterns of structural variation. Both of these occur near the predicted site of the ~70kb insertion that harbours this gene (telomeric of HLA-A and -G), but one pattern associates with HLA-A*31/33 alleles and the other with A*30:01/68:02 alleles (Fig. 6). This observation suggests presence of two divergent types of HLA-Y segment insertion in the HLA region. Other major structural variants we detected were associated with A*03 and DRB1*15/16 (Fig. 6). Although the method employed here measures structural variation at very low resolution, this knowledge will be beneficial towards full haplotype characterization.

Figure 6. Structural variants detected in HLA homozygous regions.

Figure 6

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Shown are the approximate coordinates of structural variants that track with specific HLA alleles (indicated as the Y axis). Green are likely insertions compared to the hg19 reference haplotype as determined by unexpected heterozygosity in homozygous segments. Red is a likely deletion as determined by a tract of failed SNP genotypes. Presence of distinct patterns that were identical amongst individuals sharing HLA alleles at a given locus showed these structural variants associate with the specific alleles.

The data presented here will be a valuable resource for several reasons. Extreme polymorphism, structural diversity and high repeat content render haplotype assembly from heterozygous individuals prohibitively difficult with most current sequencing technologies. Thus, the principle goal of mapping the homozygous tracts is to reduce complexity during long-range haplotype assembly and ultimately to provide unambiguously-phased reference sequences for the entire HLA region. Another goal is to examine true linkage between potential disease-causing markers, which can be achieved through analysis of these haplotypes that are identical by descent. Further, because the HLA class I and II genotypes of the IHWG cell lines have been thoroughly characterized, unambiguous knowledge of marker phase can be used to strengthen or test HLA genotype imputation algorithms. Finally, although selected for HLA homozygosity, the mean tract length genome-wide in the panel is 20% greater than the matched control set (data not shown), and so the HLA-homozygous cells will be informative for more general genetic studies. For these reasons we have made all of the data freely available on ImmPort (SDY295: EXP13576). In summary, the IHWC cell line panel represents a significant proportion of European HLA allelic and haplotype diversity. The data we describe will provide a valuable resource for characterizing haplotypes, refining imputation algorithms and developing assay controls.

Supplementary Material

251_2015_857_MOESM1_ESM. Supplementary Figure 1. The HLA homozygous cell line panel.

a. Columns from left to right: the cell line common names (bold indicates eight cells targeted previously by the MHC sequencing consortium (Horton et al. 2004), IHWG ID numbers, ethnicity, consanguineous (yes/no/unknown) status, HLA-Y presence/absence (Y indicates presence) and classical HLA locus genotype (N/A indicates unknown genotype, red indicates heterozygous at that locus). Data was obtained from the IHWG and IPD websites. Ninety-nine of the cells have IHWG designations and we included one homozygous (WAR) and one heterozygous (EN1NOT) cell line that were not part of the IHWG.

251_2015_857_MOESM2_ESM. Supplementary Figure 2 . HLA region homozygous tract coordinates in the IHWC panel.

The homozygous tract coordinates were estimated using PLINK (Purcell et al. 2007) and refined manually to show the interval between the last observed heterozygous SNP and the first homozygous SNP in each direction. At the left (Red text) indicates the donor is known to be consanguineous, (Blue text) indicates status unknown. The length of the longest homozygous tract in each cell is shown and (Green text) indicates this extends through the entire HLA region. Over 60% of the cell lines are homozygous through the classical 5.0Mbp HLA region. Shown at the right, four of the cells also have heterozygous tracts that interrupt their homozygous segment. The genome-wide data set is available at ImmPort under SDY295: EXP13576 (https://immport.niaid.nih.gov/).

Acknowledgments

We extend thanks to the members of the John Hansen laboratory at the Fred Hutchinson Cancer Research Center for supplying some of the (IHWG) cell line/DNA samples. This study was supported by U.S. National Institutes of Health grant UO1 AI090905.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

251_2015_857_MOESM1_ESM. Supplementary Figure 1. The HLA homozygous cell line panel.

a. Columns from left to right: the cell line common names (bold indicates eight cells targeted previously by the MHC sequencing consortium (Horton et al. 2004), IHWG ID numbers, ethnicity, consanguineous (yes/no/unknown) status, HLA-Y presence/absence (Y indicates presence) and classical HLA locus genotype (N/A indicates unknown genotype, red indicates heterozygous at that locus). Data was obtained from the IHWG and IPD websites. Ninety-nine of the cells have IHWG designations and we included one homozygous (WAR) and one heterozygous (EN1NOT) cell line that were not part of the IHWG.

NIHMS710085-supplement-251_2015_857_MOESM1_ESM.xlsx (26.8KB, xlsx)
251_2015_857_MOESM2_ESM. Supplementary Figure 2 . HLA region homozygous tract coordinates in the IHWC panel.

The homozygous tract coordinates were estimated using PLINK (Purcell et al. 2007) and refined manually to show the interval between the last observed heterozygous SNP and the first homozygous SNP in each direction. At the left (Red text) indicates the donor is known to be consanguineous, (Blue text) indicates status unknown. The length of the longest homozygous tract in each cell is shown and (Green text) indicates this extends through the entire HLA region. Over 60% of the cell lines are homozygous through the classical 5.0Mbp HLA region. Shown at the right, four of the cells also have heterozygous tracts that interrupt their homozygous segment. The genome-wide data set is available at ImmPort under SDY295: EXP13576 (https://immport.niaid.nih.gov/).

NIHMS710085-supplement-251_2015_857_MOESM2_ESM.pdf (401.3KB, pdf)

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