DPB1 Research Articles

Association of valine and leucine at HLA-DRB1 position 11 with radiographic progression in rheumatoid arthritis, independent of the shared epitope alleles but not independent of anti-citrullinated protein antibodies.

For decades it has been known that the HLA-DRB1 shared epitope (SE) alleles are associated with an increased risk of development and progression of rheumatoid arthritis (RA). Recently, the following variations in the peptide-binding grooves of HLA molecules that predispose to RA development have been identified: Val and Leu at HLA-DRB1 position 11, Asp at HLA-B position 9, and Phe at HLA-DPB1 position 9. This study was undertaken to investigate whether these variants are also associated with radiographic progression in RA, independent of SE and anti-citrullinated protein antibody (ACPA) status. A total of 4,911 radiograph sets from 1,878 RA patients included in the Leiden Early Arthritis Clinic (The Netherlands), Umeå (Sweden), Hospital Clinico San Carlos-Rheumatoid Arthritis (Spain), and National Data Bank for Rheumatic Diseases (US) cohorts were studied. HLA was imputed using single-nucleotide polymorphism data from an Immunochip, and the amino acids listed above were tested in relation to radiographic progression per cohort using an additive model. Results from the 4 cohorts were combined in inverse-variance weighted meta-analyses using a fixed-effects model. Analyses were conditioned on SE and ACPA status. Val and Leu at HLA-DRB1 position 11 were associated with more radiographic progression (meta-analysis P = 5.11 × 10(-7)); this effect was independent of SE status (meta-analysis P = 0.022) but not independent of ACPA status. Phe at HLA-DPB1 position 9 was associated with more severe radiographic progression (meta-analysis P = 0.024), though not independent of SE status. Asp at HLA-B position 9 was not associated with radiographic progression. Val and Leu at HLA-DRB1 position 11 conferred a risk of a higher rate of radiographic progression independent of SE status but not independent of ACPA status. These findings support the relevance of these amino acids at position 11.

LBP04: APPLICATION OF SINGLE MOLECULE REAL-TIME (SMRT) SEQUENCING TECHNOLOGY FOR THE FIELD 4 LEVEL GENOTYPING OF CLASSICAL HLA LOCI

Aim Super high resolution-single molecule-sequence-based typing (SS-SBT) is an HLA DNA typing method for the field 4 level of allelic resolution to efficiently detect new and null alleles without phase ambiguity by combination of long ranged PCR amplification and next generation sequencing (NGS) technologies. We previously reported the development and application of the SS-SBT method such as long ranged PCR system from the promoter-enhancer region to 3’UTR for 11 classical HLA loci, A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1 and DPB1 using NGS platforms such as Ion PGM, GS Junior and MiSeq. However, HLA alleles could not be assigned at the field 4 level in some HLA loci such as HLA-DQA1, -DPA1 and -DPB1, because SNP and indel densities to separate both of the phases were much lower than in other HLA loci. In this respect, third generation sequencers such as single molecule, real-time (SMRT®) DNA sequencer PacBio RS II (2.5∼10 kb) are expected to solve this current genotyping problem to improve the SS-SBT method. Here we report an accuracy of genotyping results obtained from SMRT® technology. Method Ten genomic DNAs (TU1∼TU10) obtained from Japanese were provided for long ranged PCR amplification using eight HLA loci specific primer pairs (A, B, C, DRB1, DQA1, DQB1, DPA1 and DPB1). Consensus sequences were obtained via HlaTools/Long Amplicon analysis such as overlapping, clustering of filtered subreads, phasing and removing PCR artifacts. Results HLA sequences obtained from 10 pooled amplicon samples applied to SMRT® technology with HlaTools/Long Amplicon analysis were compared to published reference sequences for consensus alleles. A total of 153 amplicon sequences were compared, 117 with the field 4 level sequences and 36 with the field 3 level sequences. At the field 3 level 100% of sequences were identical to reference ones while ∼70% (80/117) of sequences were identical at the field 4 level. Error sequences were mostly localized to intronic single- and di-nucleotide homopolymer regions. Conclusion The SMRT® technology is useful for highly accurate DNA typing for all the classical HLA loci.

Establishment of a polymerase chain reaction sequencing based typing method for HLA-DPB1 exons 2 and 3 and investigation of their polymorphisms

To establish a polymerase chain reaction sequencing-based typing (PCR SBT) method for HLA-DPB1 exons 2 and 3, and to analyze their polymorphisms. Based on the sequences of HLA-DPB1 loci, locus-specific primers were designed and applied to amplify the target sequences encompassing the entire exons 2 and 3 of HLA-DPB1. The amplification products were digested by enzymes and directly sequenced in both directions. The genotype was assigned by Assign 3.5+ SBT software. Specific target fragment was obtained with the PCR amplification, and good quality electropherogram was derived by direct sequencing. Among 242 individuals from Zhejiang Han population, 18 HLA-DPB1 alleles were detected. Alleles with a frequency of > 0.05 have included DPB1*05:01:01/135:01 (0.4112), DPB1*02:01:02 (0.1901), DPB1*04:01:01 (0.1136) and DPB1*02:02 (0.0620). A novel HLA-DPB1*168:01 allele has also been identified. Nine polymorphism sites were founded in the exon 3 region, which included a new SNP site 517 A>T. The PCR-SBT method for exons 2 and 3 of HLA-DPB1 is reliable, which allowed detection of polymorphisms in exon 3 of the HLA-DPB1 gene.

HLA DPB1 rs9277535 polymorphism strongly predicts HBsAg clearance in interferon treated genotype D HBeAg-negative patients with chronic hepatitis B

Introduction and Aim: HLA DP polymorphisms have recently been associated to spontaneous hepatitis B virus (HBV) clearance in Asians patients. Whether these SNPs influence IFN-induced HBsAg response in difficult to cure HBeAg-negative genotype D chronic hepatitis B Caucasian patients, is currently unknown.

Genetic susceptibility to beryllium: a case–referent study of men and women of working age with sarcoidosis or other chronic lung disease

ObjectiveThe study was designed to investigate whether beryllium exposure was related to illness diagnosed as sarcoidosis. Chronic beryllium disease (CBD) and sarcoidosis are clinically and pathologically indistinguishable, with only the...

Polymorphisms in HLA-DPB1 are associated with differences in rubella virus-specific humoral immunity after vaccination.

Vaccination with live attenuated rubella virus induces a strong immune response in most individuals. However, small numbers of subjects never reach or maintain protective antibody levels, and there is a high degree of variability in immune response. We have previously described genetic polymorphisms in HLA and other candidate genes that are associated with interindividual differences in humoral immunity to rubella virus. To expand our previous work, we performed a genome-wide association study (GWAS) to discover single-nucleotide polymorphisms (SNPs) associated with rubella virus-specific neutralizing antibodies. We identified rs2064479 in the HLA-DPB1 genetic region as being significantly associated with humoral immune response variations after rubella vaccination (P = 8.62 × 10(-8)). All other significant SNPs in this GWAS were located near the HLA-DPB1 gene (P ≤ 1 × 10(-7)). These findings demonstrate that polymorphisms in HLA-DPB1 are strongly associated with interindividual differences in neutralizing antibody levels to rubella vaccination and represent a validation of our previous HLA work.

Clinical significance of HLA-DRB1 and HLA-DPB1 gene polymorphisms in cirrhosis patients

To investigate the genetic association between cirrhosis and polymorphisms in the genes encoding major histocompatibility complex, class II (HLA)-DR beta 1 (DRB1) and HLA-DP beta 1 (DPB1). A population of 168 parent/offspring trios, in which the proband had a diagnosis of hepatitis B virus infection with clinical signs of cirrhosis.The HLA-DRB1 and DPB 1 gene polymorphisms of rs24755213 and rs202176660 were detected by PCR and single nucleotide polymorphism (SNP) genotyping.Correlation analysis and haplotype relative risk analysis were carried out. A/G genotypes were detected in rs24755213 of HLA-DRB1 and C/T genotypes were detected in rs202176660 of DPB1.The rs24755213 allele was associated with cirrhosis (P=0.014), with the G allele identified as a protective factor (Z=-2.33) and the A allele identified as a hazard factor (Z=2.33).The rs202176660 allele was also associated with cirrhosis (P =0.026), with the T allele identified as a protective factor (Z=-2.06) and the C allele identified as a hazard factor (Z=2.06).The haplotypes of G/T and A/C in rs24755213 and rs202176660 respectively were associated with cirrhosis (P =0.037 and 0.002, Z=-2.12 and 2.09 respectively). In this group of Chinese patients with hepatitis B virus-related cirrhosis, polymorphisms in the HLA-DRB 1 and DPB1 genes were associated with cimhosis.

Abstract 4698: Gene expression profiling distinguishes between endometrial stromal tumors subtypes

Abstract Introduction Endometrial stromal tumors (EST) are the second most prevalent uterine sarcomas. EST are divided into three histosubtypes: benign endometrial stromal nodules (ESN), low-grade endometrial stromal sarcomas (ESS) and the most malignant undifferentiated stromal sarcomas (UES). Moreover, due to clinicopathological features intermediate between classical low-grade ESS and UES, some tumors have been described in the literature as “high-grade ESS”. The histological distinction between these three subtypes has important prognostic implications. The molecular biology of these tumors remains poorly understood because of their low incidence. Patients and Methods In the present study, we performed gene expression profiling of 22 EST using microarray, RNA-Seq and qRT-PCR techniques. Our cohort included 10 classical low-grade ESS (8 with ESS-associated gene fusions), 8 UES and 4 tumors which were classified as high-grade ESS based on the presence of YWHAE-FAM22A/B fusion and histological features. Results Using gene expression microrarrays, we have identified 81 entities differentially expressed in analyzed samples (fold change > 2, p < 0.005). Hierarchical clustering analysis revealed that high-grade ESS formed a separate sub-cluster with a distinct molecular profile as compared with the low-grade ESS and UES cases. Low-grade ESS cases formed 2 distinct sub-clusters, each containing 5 cases, one of which presented gene expression profile partially corresponding to high-grade ESS cases. GO analysis of genes strongly up-regulated in UES specimens showed over-representation of immune response (GO:0006955) and immune system process (GO:0002376) pathways (MARCO, MYO1F, FCN1, GPR183, HLA-DPB1, FCGR2B, NCF1, CCL18, CD28, PTAFR, CCL13, STK4, CD14, NCF4 and CTLA4 genes) (p < 0.05). UES cases were also characterized by significant down-regulation of WT1 tumor suppressor gene as compared to the low- and high-grade ESS. Moreover, both high-grade ESS and UES cases were characterized by significantly lower expression level of PEG3 gene, which has been reported as a tumor suppressor gene. All low-grade ESS cases presented significant over-expression of estrogen receptor ESR1 gene. Microarray results were validated by RNA-Seq and qRT-PCR experiments. Conclusions This study provides gene expression profiles that distinguish between EST subtypes and sheds light on the biology of these tumors. Our results point to immune system involvement in the pathogenesis of UES and support the notion that the current EST classification should discriminate high-grade ESS as a distinct subtype. Citation Format: Joanna Przybyl, Magdalena Kowalewska, Anna Quattrone, Barbara Dewaele, Vanessa Vanspauwen, Julio Finalet-Ferreiro, Michal Swierniak, Elwira Bakula-Zalewska, Janusz A. Siedlecki, Mariusz Bidzinski, Jan Cools, Maria Debiec-Rychter. Gene expression profiling distinguishes between endometrial stromal tumors subtypes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4698. doi:10.1158/1538-7445.AM2014-4698

OR02: DEVELOPMENT OF ADVANCED NGS BASED HLA DNA TYPING METHOD: SS-SBT

Super high resolution-single molecule-sequence-based typing (SS-SBT) is an HLA DNA typing method to the field 4 level of allelic resolution (8-digit typing) by combination of long ranged PCR amplification and next generation sequencing (NGS) technologies. We previously reported the development and application of the SS-SBT method such as long ranged PCR system from promoter-enhancer to 3’UTR for the 11 classical HLA loci, A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1 and DPB1, setting of NGS parameters, and automated NGS data processing system, Sequence Alignment Based Assigning Software (SeaBass). Here we report the development of the advanced SS-SBT method. (1) Since allelic imbalance was observed in some particular alleles, the PCR primer sequences were modified for some HLA loci. (2) We newly designed middle ranged PCR primer sets for amplification from intron 1 to exon 4 in DRB1, DQB1 and DPB1 (3.8 kb to 5.3 kb). (3) We improved the program (SeaBass) to avoid allele mis-assignment. (1) By application of the SS-SBT using over 300 DNA samples that were observed with phase ambiguities when defined by conventional HLA DNA typing methods (SBT, PCR-SSO etc.), all of them, except for DQA1, DPA1 and DPB1, were typed to single HLA alleles to the field 4 level with minimum allelic imbalance. (2) The new PCR system including primers previously designed A, B and C (4.6 kb to 5.5 kb) is specialized to typing for polymorphic exons (the field 3 level or 6-digit typing), contributing to a convenient replacement model from conventional typing methods to NGS based typing method. (3) In comparison of three different automated NGS data processing systems such as Omixon Target 1.6.0 and NGSengine 1.2, SeaBass could call all of HLA alleles assigned by conventional methods with 100% accuracy. The advanced SS-SBT method is a powerful tool that provides precise allele data for efficient detection of new and null alleles without phase ambiguity.

Low expression of HLA-DRA, HLA-DPA1, and HLA-DPB1 is associated with poor prognosis in pediatric adrenocortical tumors (ACT).

Low expression of HLA class II antigens has been associated with more aggressive disease in several human malignancies including adult adrenocortical tumors (ACT), but their clinical relevance in pediatric ACT needs to be investigated. This study analyzed the expression profile of three class II histocompatibility genes (HLA-DRA, HLA-DPA1, and HLA-DPB1) in 58 consecutive pediatric ACT (13 adenomas and 45 carcinomas) by quantitative real time PCR and their association with clinical and biological features. HLA-DPA1 protein level was determined by immunohistochemistry. A significant association (P < 0.01) was observed between lower expression levels of the three genes analyzed and poor prognostic factors such as age ≥ 4 years, tumor size ≥ 200 cm(3), tumor weight ≥ 100 g, and metastatic disease; the presence of an unfavorable event and death. Underexpression of the HLA-DRA, HLA-DPA1, and HLA-DPB1 genes were associated with lower 5-year event-free survival (EFS) (P = 0.017, P < 0.001, and P = 0.017, respectively). Cox multivariate analysis showed that HLA-DPA1 was an independent prognostic factor (P = 0.029) when analyzed in association with stage IV, age and tumor size. Significantly lower EFS was also observed in patients with negative/weak immunostaining for HLA-DPA1 (P = 0.002). Similar results were observed when only patients classified as having carcinomas were analyzed. Our results suggest that lower expression of HLA-DRA, HLA-DPA1, and HLA-DPB1 genes may contribute to more aggressive disease in pediatric ACT. HLA-DPA1 immunostaining may represent potential aggressiveness marker in this tumor.

A Study on Polymorphisms and Common and Well-Documented Alleles of HLA-DPA1 and-DPB1 in Southern Chinese Han Population

Objective To explore the polymorphisms of human leukocyte antigen (HLA)-DPA1 and-DPB1,and determine the common and well-documented (CWD) alleles in southern Chinese Han population.Methods From February 2010 to September 2011,a total of 1 730 cases of peripheral blood samples from unrelated healthy voluntary blood donors of Chinese Han population in Shenzhen blood center,were collected in this study by compater random number table.All these samples were subjected to HLA-DPA1 and-DPB1 sequencing-based typing at exon 2 in both directions.In-house group specific primers were designed and utilized to identify the ambiguous allele combinations.Products of sequencing reactions subjected to sequence cleaning by EtOH/ethylene diamine tetraacetic acid (EDTA) method.The purified products were run electrophoresis on the ABI 3730 DNA sequencer,then obtained sequences were imported into the Assign 3.5 software,and the HLA-DPA1 and-DPB1 allelic genotype were assigned.Results In 1 730 unrelated healthy southern Chinese Han individuals,a total of 7 HLA-DPA1 alleles were identified,including 5 common alleles of which the frequencies were more than 0.1％.Frequencies of each common allele were 54.65％ of DPA1 * 02 ∶ 02,31.24％ of * 01 ∶ 03,10.26％ of * 02 ∶ 01,3.64％ of * 04 ∶ 01,and 0.12％ of * 01 ∶ 04.Two rare novel alleles were observed in less than 3 times.Thirty three HLA-DPB1 alleles including 20 common alleles with a frequency more than 0.1％,and 2 well-documented alleles with a frequency less than 0.1％ but were detected more than 3 times and 11 rare alleles of which two were novel alleles were determined.Conclusions Technique for simultaneous HLA-DPA1 and-DPB1 sequencebased typing at exon 2 and assay for determining ambiguous allele combinations using the in-house group specific primers have been established in this study.The information of allele frequencies and CWD alleles for HLA-DPA1 and-DPB1 in southern Chinese Han population were obtained. Key words: Human leukocyte antigens; Sequencing-based typing; Group specific primer; Common and well-documented alleles

Genetic variants in human leukocyte antigen-DP influence both hepatitis C virus persistence and hepatitis C virus F protein generation in the Chinese Han population.

Chronic hepatitis C is a serious liver disease that often results in cirrhosis or hepatocellular carcinoma. The aim of this study was to assess the association of human leukocyte antigen-DP (HLA-DP) variants with risk of chronic hepatitis C virus (HCV) or anti-F antibody generation. We selected two single nucleotide polymorphisms (SNPs) in a region including HLA-DPA1 (rs3077) and HLA-DPB1 (rs9277534) and genotyped SNPs in 702 cases and 342 healthy controls from the Chinese population using TaqMan SNP genotyping assay. Moreover, the exon 2 of the HLA-DPA1 and HLA-DPB1 genes were amplified and determined by sequencing-based typing (SBT). The results showed that rs3077 significantly increased the risk of chronic HCV infection in additive models and dominant models (odds ratio (OR) = 1.32 and 1.53). The rs3077 also contributed to decrease the risk of anti-F antibody generation in additive models and dominant models (OR = 0.46 and 0.56). Subsequent analyses revealed the risk haplotypes (DPA1*0103-DPB1*0501 and DPA1*0103-DPB1*0201) and protective haplotypes (DPA1*0202-DPB1*0501 and DPA1*0202-DPB1*0202) to chronic HCV infection. Moreover, we also found that the haplotype of DPA1*0103-DPB1*0201 and DPA1*0202-DPB1*0202 were associated with the anti-F antibody generation. Our findings show that genetic variants in HLA-DP gene are associated with chronic HCV infection and anti-F antibody generation.

A study of HLA-DPA1 and DPB1 matching status for unrelated donor-recipient pairs matched at allele level for HLA-A, -B, -C, -DRB1 and -DQB1 loci

To analyze the status of HLA-DPA1 and DPB1 matching for unrelated donor-recipient pairs matched at high-resolution allele level for HLA-A, B, C, DRB1 and DQB1 loci. A total of 76 unrelated donor-recipient pairs matching at allele level for HLA-A, B, C, DRB1 and DQB1 loci were subjected to HLA-DPA1 and DPB1 sequence-based typing (SBT). HLA-DPA1and DPB1 matching status at high-resolution allelic level was also analyzed. The allelic identity ratio for single HLA-DPA1 and DPB1 were 17.1% and 9.2%, respectively. HLA-DPA1 and DPB1 allelic identity ratio were both very low. The majority of unrelated donor-recipient pairs (73.7%) had an incompatibility at 1 HLA-DPA1 allele, 9.2% of pairs had an incompatibility at 2 DPA1 alleles. As for the high-polymorphic HLA-DPB1 gene, 57.9% of studied donor-recipient pairs had an incompatibility at 1 HLA-DPB1 allele, almost 1/3 (32.9%) of them were completely incompatible. When HLA-DPA1 and DPB1 genes were analyzed together, the donor-recipient pairs matched at 2/4 was the most common (51.4%), 4/4 allelic complete matched pairs accounted for 5.6%, and 0/4 matched pairs accounted for 8.3%. Our results indicated that the ratio of HLA-DPA1 and DPB1 complete match in the unrelated donor-recipient pairs matching at allelic level for HLA-A, B, C, DRB1 and DQB1 loci were very low. The effect of HLA-DPA1 and DPB1 matching status on clinical unrelated stem cell transplantation still needs to be elucidated.

21-OR

Aim The clonal property of next generation sequencing systems allows for the quantitative analysis of mixed samples by simply counting sequences corresponding to the component alleles. Previously, we developed a method using 454 amplicon sequencing and Conexio software for high resolution and high throughput genotyping of the HLA class I and class II loci. This system was used to analyze the blood of a SCID child and estimate the proportion of maternal cells by counting HLA-C allelic sequence reads corresponding to the non-transmitted maternal allele. Here we report the development of a system that allows us to quantify HLA allelic mixtures in plasma. Methods DNA from plasma or contrived mixtures of cell lines was amplified using primers that targeted a short region (∼150 bp to amplify small DNA fragments in plasma) of HLA DPB1 or DQB1 exon 2. Amplicons were further amplified by emulsion PCR and sequenced on a 454 GS FLX or GS Junior. Sequences were examined using modified Conexio Assign ATF 454 software. This software allowed for rapid digital analysis of each DPB1 or DQB1 allele. We also analyzed mixtures using the Illumina MiSeq. Results Using the modified Conexio software, minority HLA alleles in a mixture were readily identified and separated from background “noise” i.e., sequences generated by PCR or sequencing errors. In mixtures of two heterozygous cell lines, the minority HLA alleles could be detected at 0.5% with 1 ng (∼140 diploid genomes) DNA input. In 3/3 plasma samples from kidney transplant recipients undergoing acute or chronic rejection, donor HLA alleles were detected at an average of 0.6% of the total of donor plus recipient alleles. Conclusions We have developed a system that allows the sensitive and precise analysis of mixtures using either 454 or Illumina MiSeq sequencing technology. This method could be useful in numerous clinical applications; we have demonstrated the detection of donor HLA alleles in the plasma of kidney transplant recipients undergoing rejection. Holcomb: Roche: Employee. Rastrou: Roche: Employee. Hoglund: Roche: Employee. Goodridge: Conexio Genomics: Employee. Erlich: Roche: Employee.

Association of granulomatosis with polyangiitis (Wegener's) with HLA-DPB1*04 and SEMA6A gene variants: evidence from genome-wide analysis.

To identify genetic determinants of granulomatosis with polyangiitis (Wegener's) (GPA). We carried out a genome-wide association study (GWAS) of 492 GPA cases and 1,506 healthy controls (white subjects of European descent), followed by replication analysis of the most strongly associated signals in an independent cohort of 528 GPA cases and 1,228 controls. Genome-wide significant associations were identified in 32 single-nucleotide polymorphic (SNP) markers across the HLA region, the majority of which were located in the HLA-DPB1 and HLA-DPA1 genes encoding the class II major histocompatibility complex (MHC) DPβ chain 1 and DPα chain 1 proteins, respectively. Peak association signals in these 2 genes, emanating from SNPs rs9277554 (for DPβ chain 1) and rs9277341 (DPα chain 1) were strongly replicated in an independent cohort (in the combined analysis of the initial cohort and the replication cohort, P = 1.92 × 10(-50) and 2.18 × 10(-39) , respectively). Imputation of classic HLA alleles and conditional analyses revealed that the SNP association signal was fully accounted for by the classic HLA-DPB1*04 allele. An independent single SNP, rs26595, near SEMA6A (the gene for semaphorin 6A) on chromosome 5, was also associated with GPA, reaching genome-wide significance in a combined analysis of the GWAS and replication cohorts (P = 2.09 × 10(-8) ). We identified the SEMA6A and HLA-DP loci as significant contributors to risk for GPA, with the HLA-DPB1*04 allele almost completely accounting for the MHC association. These two associations confirm the critical role of immunogenetic factors in the development of GPA.

A Study on the Correlation of HLA-DPA1 and-DPB1 Polymorphisms With Nasopharyngeal Carcinoma in the Southern Chinese Han Population

Objective To study the correlation of HLA-DPA1 and-DPB1 polymorphisms with nasopharyngeal carcinoma (NPC) in the southern Chinese Han population.Methods From February to July 2012,a total of 74 patients who diagnosed as NPC by pathological examination in Guangxi Wuzhou Red Cross Hospital and Shenzhen Armed Police Hospital were included in this study as study group.And during the same period,a total of 160 healthy donors who were selected by computer randomly in Shenzhen blood center were included in this study as control group (The study protocol was approved by the Ethical Review Board of Investigation in Human Being of our hospital).Both the study group and control group were from southern Chinese Han.All the samples were subjected to sequencing-based typing (PCR-SBT) at exon 2 of HLA-DPA1 and-DPB1 genes in both directions.The allelic frequencies of HLA-DPA1 and-DPB1 loci for the NPC patients were compared with control group by x2 test,the P value and relative risk (RR) value were calculated according to the Wolf method.Results A total of 4 HLA-DPA1 alleles (DPA1 * 01:03,02:01,02:02 and 04:01) were identified in both study group and control group.As for the highly polymorphic HLA-DPB1 locus,14 HLA-DPB1 alleles in the NPC patient group and 18 DPB1 alleles in the healthy controls were identified.The allele frequencies for both HLA-DPA1 and-DPB1 loci showed no significant differences between study group and control group (x2＜3.84,P＞0.05).Conclusions The present study implied that the polymorphisms of HLA-DPA1 and-DPB1 showed no independent association with NPC in the southern Chinese Han individuals. Key words: human leukocyte antigens; HLA-DP; allelic polymorphisms; sequencing based typing; nasopharyngeal carcinoma

Genome-wide association study of aspirin-exacerbated respiratory disease in a Korean population

Aspirin-exacerbated respiratory disease (AERD) is a nonallergic clinical syndrome characterized by a severe decline in forced expiratory volume in one second (FEV1) following the ingestion of non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin. The effects of genetic variants have not fully explained all of the observed individual differences to an aspirin challenge despite previous attempts to identify AERD-related genes. In the present study, we performed genome-wide association study (GWAS) and targeted association study in Korean asthmatics to identify new genetic factors associated with AERD. A total of 685 asthmatic patients without AERD and 117 subjects with AERD were used for the GWAS of the first stage, and 996 asthmatics without AERD and 142 subjects with AERD were used for a follow-up study. A total of 702 SNPs were genotyped using the GoldenGate assay with the VeraCode microbead. GWAS revealed the top-ranked variants in 3' regions of the HLA-DPB1 gene. To investigate the detailed genetic effects of an associated region with the risk of AERD, a follow-up targeted association study with the 702 single nucleotide polymorphisms (SNPs) of 14 genes was performed on 802 Korean subjects. In a case-control analysis, HLA-DPB1 rs1042151 (Met105Val) shows the most significant association with the susceptibility of AERD (p = 5.11 × 10(-7); OR = 2.40). Moreover, rs1042151 also shows a gene dose for the percent decline of FEV1 after an aspirin challenge (p = 2.82 × 10(-7)). Our findings show that the HLA-DPB1 gene polymorphism may be the most susceptible genetic factor for the risk of AERD in Korean asthmatics and confirm the importance of HLA-DPB1 in the genetic etiology of AERD.

Genetics of graft-versus-host disease: The major histocompatibility complex

Graft-versus-host disease (GVHD) is a potentially life-threatening complication of allogeneic hematopoietic cell transplantation. Many genes are presumed to be involved in GVHD, but the best characterized genetic system is that of the human major histocompatibility complex (MHC) located on chromosome 6. Among the hundreds of genes located within the MHC region, the best known and characterized are the classical HLA genes, HLA-A, C, B, DRB1, DQB1, and DPB1. They play a fundamental role in T cell immune responses, and HLA-A, C, and B also function as ligands for the natural killer cell immunoglobulin-like receptors involved in innate immunity. This review highlights the state-of-the art in the field of histocompatibility and immunogenetics of the MHC with respect to genetic risk factors for GVHD.

HLA-DP genetic variation, proxies for early life immune modulation and childhood acute lymphoblastic leukemia risk

AbstractThe human leukocyte antigen (HLA) genes are candidate genetic susceptibility loci for childhood acute lymphoblastic leukemia (ALL). We examined the effect of HLA-DP genetic variation on risk and evaluated its potential interaction with 4 proxies for early immune modulation, including measures of infectious exposures in infancy (presence of older siblings, daycare attendance, ear infections) and breastfeeding. A total of 585 ALL cases and 848 controls were genotyped at the HLA-DPA1 and DPB1 loci. Because of potential heterogeneity in effect by race/ethnicity, we included only non-Hispanic white (47%) and Hispanic (53%) children and considered these 2 groups separately in the analysis. Logistic regression analyses showed an increased risk of ALL associated with HLA-DPB1*01:01 (odds ratio [OR] = 1.43, 95% CI, 1.01-2.04) with no heterogeneity by Hispanic ethnicity (P = .969). Analyses of DPB1 supertypes showed a marked childhood ALL association with DP1, particularly for high-hyperdiploid ALL (OR = 1.83; 95% CI, 1.20-2.78). Evidence of interaction was found between DP1 and older sibling (P = .036), and between DP1 and breastfeeding (P = .094), with both showing statistically significant DP1 associations within the lower exposure categories only. These findings support an immune mechanism in the etiology of childhood ALL involving the HLA-DPB1 gene in the context of an insufficiently modulated immune system.

156-P

Aim Anorexia nervosa (AN) is a complex disorder with many genetic factors implicated in disease onset and pathology. In some cases the presence of a particular allele appears to modulate the immune response to certain exogenous antigens and protects the host from illness, whereas in other cases the host may mount an immune response against self-antigens. Our aim is to study the distribution of DPB1 ∗ alelles in the in Anorexia Nervosa. Methods We have been studied 18 restrictive AN, ANR: 10 compulsive AN, ANC, and a control group (n = 128) of the Castilla y Leon (Spain) population. DNA was extracted by resin balls with metallic core (kit DNA Direct II from Dynal), obtaining 1-4 mcg of DNA per sample. We have employed for our study SSP Dynal kits for low resolution for the locus DPB ∗ , with minor experimental changes. We have reduced the amount of DNA per tube to 2-4 ng and the concentration of the Taq Polymerase to 0,1 U/tube, instead of 100 ng of DNA and 0,4 U/tube of Taq Polymerase recommended by the kit instructions. The conditions of the PCR have been: 1) 94 °C, 2 min, 1 cycle. 2) 94 °C 10 s, 65 °C 60 s 10 cycles. 3) 94 °C 10 s, 61 °C 50 s, 72 °C 30 s 30 cycles. 4) 72 °C 3 min. The PCR products were revealed by agarose electrophoresis. Results The allele frequencies in our study in the control group were: HLA DPB1 ∗ 0101, 3,6%; DPB1 ∗ 0201, 13,6%; DPB1 ∗ 0202, 3,2%; DPB1 ∗ 0301, 10,6%; DPB1 ∗ 0401, 34,4 %; DPB1 ∗ 0402, 7,6%; DPB1 ∗ 0501, 1,8%; DPB1 ∗ 0601, 2,4%; DPB1 ∗ 0901, 2,2%; DPB1 ∗ 1001, 2,4%; DPB1 ∗ 1101, 7,6%; DPB1 ∗ 1301, 0,6%; DPB1 ∗ 1401, 1,2%; DPB1 ∗ 1501, 1,2%; DPB1 ∗ 1701, 2,8%. Conclusions No significative differences with our control and AN groups for these alleles were found.