Genotype List String: a grammar for describing HLA and KIR genotyping results in a text string.

Knowledge of an individual's human leukocyte antigen (HLA) genotype is essential for modern medical genetics, and is crucial for hematopoietic stem cell and solid-organ transplantation. However, the high levels of polymorphism known for the HLA genes make it difficult to generate an HLA genotype that unambiguously identifies the alleles that are present at a given HLA locus in an individual. For the last 20 years, the histocompatibility and immunogenetics community has recorded this HLA genotyping ambiguity using allele codes developed by the National Marrow Donor Program (NMDP). While these allele codes may have been effective for recording an HLA genotyping result when initially developed, their use today results in increased ambiguity in an HLA genotype, and they are no longer suitable in the era of rapid allele discovery and ultra-high allele polymorphism. Here, we present a text string format capable of fully representing HLA genotyping results. This Genotype List (GL) String format is an extension of a proposed standard for reporting killer-cell immunoglobulin-like receptor (KIR) genotype data that can be applied to any genetic data that use a standard nomenclature for identifying variants. The GL String format uses a hierarchical set of operators to describe the relationships between alleles, lists of possible alleles, phased alleles, genotypes, lists of possible genotypes, and multilocus unphased genotypes, without losing typing information or increasing typing ambiguity. When used in concert with appropriate tools to create, exchange, and parse these strings, we anticipate that GL Strings will replace NMDP allele codes for reporting HLA genotypes.

Common and well-documented HLA alleles: 2012 update to the CWD catalogue.

We have updated the catalogue of common and well-documented (CWD) human leukocyte antigen (HLA) alleles to reflect current understanding of the prevalence of specific allele sequences. The original CWD catalogue designated 721 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, and -DPB1 loci in IMGT (IMmunoGeneTics)/HLA Database release 2.15.0 as being CWD. The updated CWD catalogue designates 1122 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 loci as being CWD, and represents 14.3% of the HLA alleles in IMGT/HLA Database release 3.9.0. In particular, we identified 415 of these alleles as being 'common' (having known frequencies) and 707 as being 'well-documented' on the basis of ~140,000 sequence-based typing observations and available HLA haplotype data. Using these allele prevalence data, we have also assigned CWD status to specific G and P designations. We identified 147/151 G groups and 290/415 P groups as being CWD. The CWD catalogue will be updated on a regular basis moving forward, and will incorporate changes to the IMGT/HLA Database as well as empirical data from the histocompatibility and immunogenetics community. This version 2.0.0 of the CWD catalogue is available online at cwd.immunogenomics.org, and will be integrated into the Allele Frequencies Net Database, the IMGT/HLA Database and National Marrow Donor Program's bioinformatics web pages.

16(th) IHIW: population global distribution of killer immunoglobulin-like receptor (KIR) and ligands.

In the last fifteen years, published reports have described KIR gene-content frequency distributions in more than 120 populations worldwide. However, there have been limited studies examining these data in aggregate to detect overall patterns of variation at regional and global levels. Here, we present a summary of the collection of KIR gene-content data for 105 worldwide populations collected as part of the 15th and 16th International Histocompatibility and Immunogenetics Workshops, and preliminary results for data analysis.

Multi-locus HLA class I and II allele and haplotype associations with follicular lymphoma.

Follicular lymphoma (FL) is an indolent, sometimes, fatal disease characterized by recurrence at progressively shorter intervals and is frequently refractive to therapy. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the human leukocyte antigen (HLA) region on chromosome 6p21.32-33 that are statistically significantly associated with FL risk. Low to medium resolution typing of single or multiple HLA genes has provided an incomplete picture of the total genetic risk imparted by this highly variable region. To gain further insight into the role of HLA alleles in lymphomagenesis and to investigate the independence of validated SNPs and HLA alleles with FL risk, high-resolution HLA typing was conducted using next-generation sequencing in 222 non-Hispanic White FL cases and 220 matched controls from a larger San Francisco Bay Area population-based case-control study of lymphoma. A novel protective association was found between the DPB1*03:01 allele and FL risk [odds ratio (OR) = 0.39, 95% confidence interval (CI) = 0.21-0.68]. Extended haplotypes DRB1*01:01-DQA1*01:01-DQB1*05:01 (OR = 2.01, 95% CI = 1.22-3.38) and DRB1*15-DQA1*01-DQB1*06 (OR = 0.55, 95% CI = 0.36-0.82) also influenced FL risk. Moreover, DRB1*15-DQA1*01-DQB1*06 was highly correlated with an established FL risk locus, rs2647012. These results provide further insight into the critical roles of HLA alleles and SNPs in FL pathogenesis that involve multi-locus effects across the HLA region.

A multi-site study using high-resolution HLA genotyping by next generation sequencing.

The high degree of polymorphism at human leukocyte antigen (HLA) class I and class II loci makes high-resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results because of incomplete genomic coverage and inability to set phase for HLA allele determination. The 454 Life Sciences Genome Sequencer (GS FLX) next generation sequencing system coupled with conexio atf software can provide very high-resolution HLA genotyping. High-throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double-blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and CONEXIO ATF software allows reliable identification of HLA genotypes at high resolution.

Report from the killer immunoglobulin-like receptor (KIR) anthropology component of the 15th International Histocompatibility Workshop: worldwide variation in the KIR loci and further evidence for the co-evolution of KIR and HLA.

The killer immunoglobulin-like receptor (KIR) anthropology component of the 15th International Histocompatibility Workshop (IHIWS) sought to explore worldwide population variation in the KIR loci, and to examine the relationship between KIR genes and their human leukocyte antigen (HLA) ligands. Fifteen laboratories submitted KIR genotype and HLA ligand data in 27 populations from six broad ethnic groups. Data were analyzed for correlations between the frequencies of KIR and their known HLA ligands. In addition, allelic typing was performed for KIR2DL2 and 3DL1 in a subset of populations. Strong and significant correlations were observed between KIR2DL2, 2DL3 genotype frequencies and the frequency of their ligand, HLA-C1. In contrast, only weak associations were seen for 3DL1, 3DS1 and the HLA-Bw4 ligand. Although some aspects of the correlations observed here differ from those reported in other populations, these data provide additional evidence of linked evolutionary histories for some KIR and HLA loci. Investigation of allele-level variation for the B haplotype locus KIR 2DL2 showed that two alleles, *001 and *003, predominate in all populations in this study. Much more allelic variation was observed for the A haplotype locus 3DL1, with several alleles observed at moderate frequencies and extensive variation observed between populations.

High-resolution, high-throughput HLA genotyping by next-generation sequencing.

The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome. Hematopoietic stem cell transplantation requires allele-level HLA typing at multiple loci to select the best matched unrelated donors for recipient patients. In current methods for HLA typing, both alleles of a heterozygote are amplified and typed or sequenced simultaneously, often making it difficult to unambiguously determine the sequence of the two alleles. Next-generation sequencing methods clonally propagate in parallel millions of single DNA molecules, which are then also sequenced in parallel. Recently, the read lengths obtainable by one such next-generation sequencing method (454 Life Sciences, Inc.) have increased to >250 nucleotides. These clonal read lengths make possible setting the phase of the linked polymorphisms within an exon and thus the unambiguous determination of the sequence of each HLA allele. Here we demonstrate this capacity as well as show that the throughput of the system is sufficiently high to enable a complete, 7-locus HLA class I and II typing for 24 or 48 individual DNAs in a single GS FLX sequencing run. Highly multiplexed amplicon sequencing is facilitated by the use of sample-specific internal sequence tags (multiplex identification tags or MIDs) in the primers that allow pooling of samples yet maintain the ability to assign sequences to specific individuals. We have incorporated an HLA typing software application developed by Conexio Genomics (Freemantle, Australia) that assigns HLA genotypes for these 7 loci (HLA-A, -B, -C, DRB1, DQA1, DQB1, DPB1), as well as for DRB3, DRB4, and DRB5 from 454 sequence data. The potential of this HLA sequencing system to analyze chimeric mixtures is demonstrated here by the detection of a rare HLA-B allele in a mixture of two homozygous cell lines (1/100), as well as by the detection of the rare nontransmitted maternal allele present in the blood of a severe combined immunodeficiency disease syndrome (SCIDS) patient.

The HLA-B/-C haplotype block contains major determinants for host control of HIV.

A genome-wide association study of people with incident human immunodeficiency virus (HIV) infection selected from nine different cohorts identified allelic polymorphisms, which associated with either viral set point (HCP5 and 5' HLA-C) or with HIV disease progression (RNF39 and ZNRD1). To determine the influence of these polymorphisms on host control of HIV, we carried out a population-based association study. The analysis revealed complete linkage disequilibrium between HCP5 and HLA-B*5701/HLA-Cw*06, a modest effect of 5' HLA-C on viral set point in the absence of HLA-B*5701, and no influence of the RNF39 /ZNRD1 extended haplotype on HIV disease progression. No correlation was found between the infection status and any of these genetic variants (P>0.1, Fisher's exact test). These findings suggest a pattern of strong linkage disequilibrium consistent with an HLA-B/-C haplotype block, making identification of a causal variant difficult, and underscore the importance of validating polymorphisms in putative determinants for host control by association analysis of independent populations.

HLA class I (A, B, C) and class II (DRB1, DQA1, DQB1, DPB1) alleles and haplotypes in the Han from southern China.

In this study, polymerase chain reaction-sequence-specific oligonucleotide prode (SSOP) typing results for the human leukocyte antigen (HLA) class I (A, B, and C) and class II (DRB1, DQA1, DQB1, and DPB1) loci in 264 individuals of the Han ethnic group from the Canton region of southern China are presented. The data are examined at the allele, genotype, and haplotype level. Common alleles at each of the loci are in keeping with those observed in similar populations, while the high-resolution typing methods used give additional details about allele frequency distributions not shown in previous studies. Twenty distinct alleles are seen at HLA-A in this population. The locus is dominated by the A*1101 allele, which is found here at a frequency of 0.266. The next three most common alleles, A*2402, A*3303, and A*0203, are each seen at frequencies of greater than 10%, and together, these four alleles account for roughly two-thirds of the total for HLA-A in this population. Fifty alleles are observed for HLA-B, 21 of which are singleton copies. The most common HLA-B alleles are B*4001 (f= 0.144), B*4601 (f= 0.119), B*5801 (f= 0.089), B*1301 (f= 0.068), B*1502 (f= 0.073), and B*3802 (f= 0.070). At the HLA-C locus, there are a total of 20 alleles. Four alleles (Cw*0702, Cw*0102, Cw*0801, and Cw*0304) are found at frequencies of greater than 10%, and together, these alleles comprise over 60% of the total. Overall, the class II loci are somewhat less diverse than class I. Twenty-eight distinct alleles are seen at DRB1, and the most common three, DRB1*0901, *1202, and *1501, are each seen at frequencies of greater than 10%. The DR4 lineage also shows extensive expansion in this population, with seven subtypes, representing one quarter of the diversity at this locus. Eight alleles are observed at DQA1; DQA1*0301 and 0102 are the most common alleles, with frequencies over 20%. The DQB1 locus is dominated by four alleles of the 03 lineage, which make up nearly half of the total. The two most common DQB1 alleles in this population are DQB1*0301 (f= 0.242) and DQB1*0303 (f= 0.15). Eighteen alleles are observed at DPB1; DPB1*0501 is the most common allele, with a frequency of 37%. The class I allele frequency distributions, expressed in terms of Watterson's (homozygosity) F-statistic, are all within expectations under neutrality, while there is evidence for balancing selection at DRB1, DQA1, and DQB1. Departures from Hardy-Weinberg expectations are observed for HLA-C and DRB1 in this population. Strong individual haplotypic associations are seen for all pairs of loci, and many of these occur at frequencies greater than 5%. In the class I region, several examples of HLA-B and -C loci in complete or near complete linkage disequilibrium (LD) are present, and the two most common, B*4601-Cw*0102 and B*5801-Cw*0302 account for more than 20% of the B-C haplotypes. Similarly, at class II, nearly all of the most common DR-DQ haplotypes are in nearly complete LD. The most common DRB1-DQB1 haplotypes are DRB1*0901-DQB1*0303 (f= 0.144) and DRB1*1202-DQB1*0301 (f= 0.131). The most common four locus class I and class II combined haplotypes are A*3303-B*5801-DRB1*0301-DPB1*0401 (f= 0.028) and A*0207-B*4601-DRB1*0901-DPB1*0501 (f= 0.026). The presentation of complete DNA typing for the class I loci and haplotype analysis in a large sample such as this can provide insights into the population history of the region and give useful data for HLA matching in transplantation and disease association studies in the Chinese population.

A novel multilocus genotyping assay to identify genetic predictors of stroke in sickle cell anaemia.

We describe a novel multilocus genotyping assay permitting simultaneous identification of 60 candidate markers for stroke in sickle cell anaemia (SCA). Based on cerebral magnetic resonance imaging (MRI), 69 patients were divided into stroke and control groups. The variant allele, CBS 278thr, showed protection from stroke, whereas the apoE3 allele showed a trend towards association with increased stroke risk. Several other variant alleles [TNFalpha (-308)A, CETP (-628)A, apoCIII (-641)A] showed a trend towards significant associations with stroke risk. These preliminary results on a small group of patients suggest that a multilocus genotyping assay may be valuable in identifying genes that increase the risk of stroke in SCA.

Genetic variability and linkage disequilibrium within the HLA-DP region: analysis of 15 different populations.

In order to understand the forces governing the evolution of the genetic diversity in the HLA-DP molecule, polymerase chain reaction (PCR)-based methods were used to characterize genetic variation at the DPA1 and DPB1 loci encoding this heterodimer on 2,807 chromosomes from 15 different populations including individuals of African, Asian, Amerindian, Indian and European origin. These ethnically diverse samples represent a variety of population substructures and include small, isolated populations as well as larger, presumably admixed populations. Ten DPA1 and 39 DPB1 alleles were identified and observed on 87 distinct DP haplotypes, 34 of which were found to be in significant positive linkage disequilibrium in at least one population. Some haplotypes were found in all ethnic groups while others were confined to a single ethnic group or population. Strong positive global linkage disequilibrium (Wn) between DPA1 and DPB1 was present in all 15 populations. The African populations displayed the lowest values of Wn whereas the Amerindian populations displayed near absolute disequilibrium. Analysis of the distribution of haplotypes using the normalized deviate of the Ewens-Watterson homozygosity statistic, F, suggests that DP haplotypes encoding the functional heterodimer are subject to much lower degrees of balancing selection than other loci within the HLA region. Finally, neighbor joining tree analyses demonstrate the power of haplotype diversity for inferring the relationships between the different populations.

HLA class II antigens positively and negatively associated with hepatosplenic schistosomiasis in a Chinese population.

To identify possible associations between host genetic factors and the onset of liver fibrosis following Schistosoma japonicum infection, the major histocompatibility class II alleles of 84 individuals living on an island (Jishan) endemic for schistosomiasis japonica in the Poyang Lake Region of Southern China were determined. Forty patients exhibiting advanced schistosomiasis, characterised by extensive liver fibrosis, and 44 age and sex-matched control subjects were assessed for the class II haplotypes HLA-DRB1 and HLA-DQB1. Two HLA-DRB1 alleles, HLA-DRB1*0901 (P=0.012) and *1302 (P=0.039), and two HLA-DQB1 alleles, HLA-DQB1*0303 (P=0.012) and *0609 (P=0.037), were found to be significantly associated with susceptibility to fibrosis. These associated DRB1 and DQB1 alleles are in very strong linkage disequilibrium, with DRB1*0901-DQB1*0303 and DRB1*1302-DQB1*0609 found as common haplotypes in this population. In contrast, the alleles HLA-DRB1*1501 (P=0.025) and HLA-DQB1*0601 (P=0.022) were found to be associated with resistance to hepatosplenic disease. Moreover, the alleles DQB1*0303 and DRB1*0901 did not increase susceptibility in the presence of DQB1*0601, indicating that DQB1*0601 is dominant over DQB1*0303 and DRB1*0901. The study has thus identified both positive and negative associations between HLA class II alleles and the risk of individuals developing moderate to severe liver fibrosis following schistosome infection.

Non-conservative substitutions distinguish previously uncharacterized HLA-A molecules.

The extent of class I HLA polymorphism is not yet realized, and to provide a glimpse of the HLA-A polymorphism which remains undetected, we have analyzed approximately 3,700 National Marrow Donor Program (NMDP) Donor/Recipient Pair Retrospective Study Samples with HLA-A DNA sequence-based typing (SBT). Seventeen new HLA-A alleles were detected, with a total of 19 nucleotide substitutions distinguishing these new alleles from their closest HLA-A relatives. Nearly all of the new alleles differ by single nucleotide substitutions; a majority of these substitutions can be explained by gene conversion events but 6 alleles likely originated by point mutation. Fifteen of the 19 nucleotide substitutions translate into amino acid differences in the molecule. Structurally, the inferred amino acid alterations were non-conservative in terms of chemical property, and most substitutions were positioned in 1 or more of the specificity pockets which determine peptide binding. Although these new alleles were identified in a primarily Caucasian sample population, 9 of the 17 new HLA-A alleles were found in samples of non-Caucasoid origin. A new allele detection rate of 1 in approximately 200 individuals in our data set would, therefore, be higher in a non-Caucasoid sample population. In summary, the single nucleotide substitutions that distinguish undetected HLA-A alleles translate into functionally distinct HLA-A molecules. Further studies of the role of HLA-A in transplantation, in disease association, and in evolution must therefore accommodate the discovery of new alleles differing by single nucleotides.

Detection of microchimerism by PCR is a function of amplification strategy.

BACKGROUND: Suitable detection methods are needed to support larger studies of microchimerism and the allogeneic exposures that may be etiologically related to it. STUDY DESIGN AND METHODS: A twotier PCR strategy for microchimerism detection was developed on the basis of the observation that assay sensitivity for the detection of microchimerism depends on the specificity with which primer pairs recognize sequences unique to the minor population. First, specimens are tested to determine the host HLA class II genotype by using a locus-specific PCR strategy with low sensitivity for microchimerism. Then, a sequence-specific PCR analysis having high sensitivity for detection of microchimerism is applied to detect and quantitate the minor population. Locus-specific, group-specific, and sequence-specific amplification strategies for the detection of distinct minor WBC populations prepared ex vivo were compared. In addition, 39 clinical samples from patients with known transfusion-associated microchimerism and 20 umbilical cord blood (CB) specimens containing maternal WBCs were studied. RESULTS: Locus-specific amplification detected 17 (94%) of 18 cases in which microchimerism was present at 10 percent, but only 1 of 51 cases with microchimerism < or = 1 percent. Group-specific amplification detected all 63 cases with minor populations present at > or = 0.10 percent, but only 16 of 21 cases at the 0.01 percent level. Sequence-specific amplification detected 100 percent of cases down to the 0.01 percent level. When applied to clinical samples, locus-specific amplification reliably identified the major population but proved insensitive to low-level minor populations. CONCLUSIONS: For the detection of microchimerism, assay sensitivity is a function of amplification strategy. These results suggest a simple approach to population screening for microchimerism: the background population of WBCs is typed by a locus-specific method, while minor population(s) can then be sought by using one or several sequence-specific amplifications.

Sibling donor cord blood banking for children with sickle cell disease.

Although hematopoietic stem cell transplantation has curative potential for selected patients with sickle cell disease (SCD), most patients who are eligible for transplantation do not have a suitable donor. Cord blood (CB) from a sibling could provide an alternative stem cell source that, while not as well established as marrow, may offer certain advantages for selected families. These potential advantages include low risk to the infant donor, the possibility that mismatched CB units from sibling donors may be acceptable for transplantation, prompt availability of a stored CB unit for transplant, and decreased risk of clinically significant graft-versus-host disease. When families with SCD (or other transplant-treatable condition) conceive a sibling, no comprehensive research resource exists to assist the family in collecting the new infant's CB. With support from the National Heart Lung and Blood Institute, we are developing a noncommercial research-based CB Banking Program specifically for medically indicated sibling donations. In preliminary experience, we have collected CB from 52 SCD families across 19 states. Of these, 2 CB units have thus far been used for transplantation and 9 others are HLA-identical. We conclude that a CB bank focusing on sibling-donations may be feasible, but further study is required to determine whether such a bank can collect CB units of sufficient quantity and quality to support controlled trials of sibling CB transplantation. Families with a specific medical need, such as those already caring for a child with SCD, should consider collecting sibling CB as part of comprehensive care if the opportunity becomes available.

Evolution of Pacific/Asian populations inferred from HLA class II allele frequency distributions.

The allele frequency distributions for the HLA class II loci, DRB1, DQB1 and DPB1, in eight Pacific/Asian populations: Hawaiian, Samoan, Malay, Papua New Guinea (PNG) Highlands, and two Indonesian and PNG Lowland groups, were determined using high-resolution polymerase chain reaction/sequence-specific oligonucleotide probe (PCR/SSOP) typing methods. The allele frequency distributions for the HLA-DRB1 locus were determined for a third Indonesian population as well as for an additional Filipino population. DRB1 alleles in the DR2 serogroup (or allelic lineage) are very common in this region; in some populations, more than 50% of the alleles belong to this serogroup. The DRB1*1502 allele is frequent in nine of the ten populations studied, reaching a frequency of 0.48 in one Indonesian population and among Filipinos. Extensive DR-DQ haplotype diversity was detected in these populations. Seven different DR2-DQB1 haplotypes were observed in the Indonesian and PNG Lowland populations, eight in the PNG Highlands and ten in Malays and Filipinos. The DRB1*0410 allele, commonly observed in Australia, is observed in the PNG Highlands at a low frequency (f=0.03) and is absent in the other populations. Two additional DRB1 alleles commonly observed in Australia, DRB1*0405 and *1407, are also observed in the PNG Highlands at high frequencies (f=0.132 and 0.126), while they are rare in the PNG Lowlands (f=0.039 and 0.013). These alleles are generally rare or absent in the other populations. The DPB1*0501 allele, common in Chinese and Japanese populations, is most frequent in the Samoan, Hawaiian, Indonesian, and Malay populations, and the *0401 allele is the most frequent DPB1 allele in the PNG Lowlands. Both of these alleles have the same very high frequency (f=0.34) in the PNG Highlands. Analyses of homozygosity (the Ewens-Watterson F statistic) in these and other populations indicate that, while most allele frequency distributions are consistent with balancing selection, values of F for the Indonesian and Javan populations may reflect positive directional selection. Phylogenetic trees constructed using the allele frequencies at the DRB1 locus of the populations reported here, as well as those for additional Pacific, Asian, and Australian populations, indicate that the PNG Highland population is more closely related to Australian populations than to PNG Lowland populations, while the PNG Lowlands are more closely related to other Melanesian populations.

Evidence for HLA-related susceptibility for stroke in children with sickle cell disease.

Cerebral infarction occurs in one quarter of all children with sickle cell anemia (SCA). There is an increased risk of stroke in siblings with SCA, suggesting genetic factors may influence risk of stroke. The authors investigated whether HLA type was associated with risk of stroke in children with SCA. Fifty-three patients with SCA underwent complete HLA typing at both HLA class I (HLA-A, B) and HLA class II (HLA-DR, DQ, DP) loci. Of the 53 patients, 22 had magnetic resonance imagining (MRI)-documented evidence of cerebral infarction, and the remaining 31 patients had negative MRI scans. Comparison of the results of HLA typing between the SCA patients with a positive and those with a negative MRI documented that the 2 groups differed with respect to the class I HLA-B (P =.012), and the class II HLA-DRB1 (P =.0008) and DQB1 (P =.029). Susceptibility associations at the HLA-DRB1 locus included both DR3 alleles, where DRB1*0301 and *0302 were both associated with an increased risk of stroke. Protective associations were found in the DR2 group, where DRB1*1501 was protective for stroke. DQB1*0201, which is in linkage disequilibrium with DRB1*0301, was also associated with stroke. Similarly, DQB1*0602, in linkage disequilibrium with DRB1*1501, was protective. Specific HLA alleles may influence the risk of stroke in children with SCA. HLA typing may prove useful in identifying SCA patients at higher risk for stroke.

HLA class II haplotype associations with inflammatory bowel disease in Jewish (Ashkenazi) and non-Jewish caucasian populations.

Ulcerative colitis (UC) and Crohn's disease (CD) are the clinical entities comprising idiopathic inflammatory bowel disease (IBD). Previous studies on the association of IBD and human leukocyte antigen (HLA) class II genes suggested a role for HLA in this disease. Here we present HLA class II (DRB1, DQB1, DQA1, DPB1) allele and haplotype distributions determined using the polymerase chain reaction and sequence-specific oligonucleotide probe methods. A total of 578 UC and CD Caucasian patients and controls from Jewish (Ashkenazi) and non-Jewish populations was examined. Our previously reported association of DR1-DQ5 with CD was attributable to DRB1*0103. A dramatic association with IBD and the highly unusual DRB1*0103-DQA1*0501-DQB1*0301 haplotype (OR = 6.6, p = 0.036) was found. The more common DR1 haplotype, DRB1*0103-DQA1*0101-DQB1*0501, was also associated with IBD (OR = 3.1, p = 0.014), a result suggesting that interaction between DR and DQ may determine the extent of disease risk. Our previously reported association of DR2 with UC was attributable to DRB1*1502 (OR = 2.6, p = 0.006). At the DPB1 locus, a significant association of DPB1*0401 with CD was observed for the combined populations (OR = 1.85, p = 0.007). These observations indicate that some class II alleles and haplotypes confer susceptibility to both UC and CD, implying common immunogenetic mechanisms of pathogenesis, while others confer risk to only one of these diseases, and illustrate the value of DNA HLA typing in disease susceptibility analyses.

Evolution of HLA class II molecules: Allelic and amino acid site variability across populations.

Analysis of the highly polymorphic beta1 domains of the HLA class II molecules encoded by the DRB1, DQB1, and DPB1 loci reveals contrasting levels of diversity at the allele and amino acid site levels. Statistics of allele frequency distributions, based on Watterson's homozygosity statistic F, reveal distinct evolutionary patterns for these loci in ethnically diverse samples (26 populations for DQB1 and DRB1 and 14 for DPB1). When examined over all populations, the DQB1 locus allelic variation exhibits striking balanced polymorphism (P < 10(-4)), DRB1 shows some evidence of balancing selection (P < 0.06), and while there is overall very little evidence for selection of DPB1 allele frequencies, there is a trend in the direction of balancing selection (P < 0.08). In contrast, at the amino acid level all three loci show strong evidence of balancing selection at some sites. Averaged over polymorphic amino acid sites, DQB1 and DPB1 show similar deviation from neutrality expectations, and both exhibit more balanced polymorphic amino acid sites than DRB1. Across ethnic groups, polymorphisms at many codons show evidence for balancing selection, yet data consistent with directional selection were observed at other codons. Both antigen-binding pocket- and non-pocket-forming amino acid sites show overall deviation from neutrality for all three loci. Only in the case of DRB1 was there a significant difference between pocket- and non-pocket-forming amino acid sites. Our findings indicate that balancing selection at the MHC occurs at the level of polymorphic amino acid residues, and that in many cases this selection is consistent across populations.

Hardy-Weinberg testing for HLA class II (DRB1, DQA1, DQB1, and DPB1) loci in 26 human ethnic groups.

Testing the fit of population data to Hardy-Weinberg proportions is crucial in the validation of many current approaches in population genetic studies. In this paper, we tested fit to Hardy-Weinberg proportions using exact approaches for both the overall and individual heterozygote genotype data of four HLA Class II loci: DRB1, DQA1, DQB1, and DPB1, from 26 human populations. Eighty of 99 overall tests fit the Hardy-Weinberg expectation (73% for DRB1, 89% for DQA1, 81% for DQB1 and 81% for DPB1). Deviations from Hardy-Weinberg proportions were both locus and group specific. Although we could not rule out other mechanisms at work, the individual test results indicated that the departure was possibly partly due to recent admixture. Evidence for selection and other sources of deviation are also discussed.