Rapid DNA typing for HLA-C using sequence-specific primers (PCR-SSP): Identification of serological and non-serologically defined HLA-C alleles including several new alleles

Abstract: Detection of HLA-C antigens by complement mediated cytotoxicity using human alloantisera is often difficult. Between 20 to 40% of individuals in every race have undectectable HLA-C locus antigens and 9 out of the 29 sequenced HLA-C alleles so far published encode serologically undetected antigens. In addition, HLA-C molecules are expressed at the cell surface at about 10% of the levels of HLA-A and HLA-B. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable and rapid method for typing HLA-DR, HLA-DQA and HLA-DQB genes. PCR-SSP takes two hours to perform and is therefore suitable for the genotyping of cadaveric donors. We have designed a set of primers which will positively identify the HLA-C alleles corresponding to the serologically defined series HLA-Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7 and Cw8. The serologically undetectable alleles have also been detected in groups according to sequence homology. In addition, three new unsequenced variants have been identified. DNA samples from 56 International Histocompatibility Workshop reference cell lines and 103 control individuals have been typed by the HLA-C PCR-SSP technique. 4/56 cell line types and 11/103 normal control individuals types were discrepant with the reported serological types. All combinations of serologically detectable and most of the serologically blank HLA-C antigens can be readily identified. DNA typing for HLA-Cw by PCR-SSP can take as little as 130 minutes from start to finish, including DNA preparation.     

HLA-B51 and haplotypic diversity of B-Cw associations: implications for matching in unrelated hematopoietic stem cell transplantation

In unrelated hematopoietic stem cell transplantation (HSCT), human leukocyte antigen (HLA)-C locus incompatibilities occur frequently and are associated with increased risk of posttransplant complications. Because HLA-B51 is associated with a high rate of Cw disparities, we performed a comprehensive four-digit typing analysis of 140 ABCDRB1 B51 genotypes proven by pedigree analysis and 311 unrelated donors selected for 75 B51-positive patients. In addition, 145 A1/Ax-B8/B51-DR3/DRx donors were HLA typed at a high-resolution level and tested for three microsatellite (Msat) polymorphisms located in the HLA class I and III regions. Based on these data sets, 182 different ABCDR haplotypes with 14 different B-Cw associations were detected. Rates of Cw mismatches were shown to be highly correlated with the ABDRB1 haplotypes. We have computed 21 B51 haplotypes that disclose a high probability of HLA-C allele matching and 30 haplotypes with a low (<25%) probability. The HLA-C allele frequency profiles were quite different in these two groups, with a more heterogeneous distribution in the low matching probability group. HLA-Cw*1502 was inversely correlated with the likelihood to identify a Cw-mismatched donor: it was present in 61% of the high vs 18% of the low probability group ( P  < 0.0001). The analysis of three Msats in the class I and III regions showed a higher allelic diversity in B51-positive haplotypes compared with the conserved A1-B8-DR3 haplotype. HLA-B51 haplotypes therefore exhibit a high diversity at the level of B-Cw associations and of non-HLA polymorphisms in the class I and III regions. Such heterogeneity negatively impacts on overall matching in HSCT.     

Donor HLA-C Genotype Has a Profound Impact on the Clinical Outcome Following Liver Transplantation

Late allograft dysfunction is a significant problem following liver transplantation and its pathogenesis is uncertain. HLA-C is the major inhibitory ligand for killer immunoglobulin-like receptors (KIRs) that regulate the cytotoxic activity of natural killer (NK) cells. HLA-C alleles can be allocated into two groups, termed HLA-C1 and HLA-C2, based on their KIR specificity. HLA-C2 interactions are more inhibiting to NK cell activation. We studied the clinical importance of HLA-C genotype in a large liver transplant cohort and found that possession of at least one HLA-C2 allele by the donor allograft was associated with less histological evidence of chronic rejection and graft cirrhosis, a 16.2% reduction in graft loss (p = 0.003) (hazard ratio: 2.7, 95% CI 1.4–5.3) and a 13.6% improvement in patient survival (p = 0.01) (hazard ratio: 1.9, 95% CI 1.1–3.3) at 10 years. Transplantation of an HLA-C2 homozygous allograft led to a particularly striking 26.5% reduction in graft loss (p < 0.001) (hazard ratio: 7.2, 95% CI 2.2–23.0) at 10 years when compared to HLA-C1 homozygous allografts. Donor HLA-C genotype is therefore a major determinant of clinical outcome after liver transplantation and reveals the importance of NK cells in chronic rejection and graft cirrhosis. Modulation of HLA-C and KIR interactions represents an important novel approach to promote long-term graft and patient survival.     

SPR1 gene near HLA-C is unlikely to be a psoriasis susceptibility gene

Although genetics analyses have identified the HLA-Cw6 allele to be the major risk allele for psoriasis vulgaris (PV) in many racial groups, it has been proposed that other putative genes near the HLA-C locus are involved in PV susceptibility and that the association of Cw6 is a result of linkage disequilibrium. The SPR1 gene, a predicted gene located 128 kb telomeric to the HLA-C locus, is considered to be one potential candidate gene of PV. Until now, no association study of the SPR1 gene has been conducted on psoriasis patients. We investigated the SPR1 gene for disease association by direct sequencing of the SPR1 gene in 116 Chinese patients with PV and 116 normal subjects. Genotyping for HLA-Cw6 was also carried out using polymerase chain reaction/restriction fragment length polymorphism. Significant increase of the HLA-Cw6 allele was found in psoriasis patients (32.8% vs. 13.8%, P = 0.001). We found that the SPR1 gene is a highly polymorphic gene containing 13 single nucleotide polymorphisms (SNPs), two of which have not been previously reported, and four SNPs cause amino acid change. No significantly different allelic distribution of 13 SPR1 SNPs could be found between the patients with PV and controls after correction for multiple testing. If the frequencies of SPR1 SNPs were compared between the early onset psoriatics and control subjects, early onset patients were more likely to have G allele at position 988 (60% vs. 35.3%, P = 0.001). However, the significance disappeared upon stratification for the Cw6 status. Haplotype-based association analysis showed two susceptibility haplotypes (types 8 and 19) in early onset psoriasis patients. Nonetheless, the significance also disappeared after stratification of the Cw6 status. Our results suggest that HLA-Cw6 remains the major risk allele in Chinese psoriatics, and that the SPR1 gene might not play an important role in the causation of PV.     

STG does not associate with psoriasis in the Swedish population

Psoriasis is a chronic inflammatory skin disease that is known to have a strong genetic predisposition. Several psoriasis-susceptibility loci have been previously found through genomic scans. Of these, psoriasis-susceptibility region 1 (PSORS1) on chromosome 6p21 remains the most consistently identified region across populations with the highest association with disease. STG is a gene that was previously isolated from rhesus monkey taste buds, and its ortholog in humans was found to be part of the cluster of genes in PSORS1, which is telomeric to HLA-C. Upon characterization of STG, we identified several sequence variants and investigated their association with psoriasis in cases and controls from the Swedish population. None of these STG single-nucleotide polymorphisms were found to be significantly associated with psoriasis. However, HLA-Cw*0602 status was strongly associated with disease. STG expression was investigated in human tissues and found not to be restricted to taste buds, with signals also being detected in skin and tonsils.     

HLA-Cw基因常规检测区外第1、5、6、7外显子核苷酸序列测定的研究及临床应用

目的 研究中国人群HLA-Cw基因第1、5、6、7外显子的分子遗传多态性,探讨增加第1、5 6 7外显子核苷酸序列测定在临床组织配型工作中的重要性及意义.方法 应用PCR-SBT法,对324份样本的HLA-Cw基因第2、3、4外显子作常规测序分型.对检出的模棱两可结果,设计HLACw第1、5 6 7外显子序列测序引物并优化测序反应条件,增加第1、5、6、7外显子核苷酸序列分析.结果 对HLA-Cw基冈第2、3、4外显子常规检测,一次性获得等位基因前4位数分型结果 的样本占23.8%(77/324);出现模棱两可结果的样本数占76.2%(247/324),检出的模棱两可等位基因组合有73种;增加HLA-Cw基因的第1、5、6、7外显子多态性检测,可解决Cw* 030201/030202、030301/0320N、Cw* 040101/0409N/0430、Cw* 070201/0750、Cw* 0403/0409N/0430和Cw* 080101/0822等10种常见的模棱两可等位基因组合.结论 在临床HLA-Cw基因配型中增加第1、5、6、7外显子多态性检测,有助于解决测序分型中的模棱两可的结果 和提高HLA-Cw基因分型精确度,对临床组织配型工作具有重要意义.     

母体KIR与胎儿HLA-C基因的组合形式对子痫前期发病的影响

【目的】探索子宫蜕膜自然杀伤细胞（uNk细胞）免疫球蛋白受体基因（KIR基因）人类白细胞抗原C类分子基因（HLA—C基因）的结合对妊娠期高血压疾病发病的影响。【方法]30例早发型重度子痫前期孕妇及其胎儿作为观察组,20例正常健康孕妇及其胎儿作为对照组,孕妇抽取静脉血进行KIR基因分型,胎儿在出生时抽取脐静脉血进行HLA—C基因分型。【结果】两组HLA—C2的百分率相比差异无显著性（x2=0．67,P〉0．05）。子痫前期组母体KIR基因AA型的有14例,对照组母体AA型有3例,子痫前期组有更多孕妇的KIR基因为AA型,差异有显著性（x2=5．31,P〈0．05）。在子痫前期组,胎儿为HLA-C2型基因共16例,有11例母体KIR基因型为AA型,胎儿为HL艮c1共14例,有3例母体KIR基因型为AA型,在对照组,胎儿为HLA—C2基因型共13例,有3例母体KIR基因型为AA型,比较发现,予痫前期组中,胎儿为HLA—C2基因型时,有更多的母体KIR基因型为AA型,差异有显著性（确切概率P值=0．0109）。子痫前期组与对照组比较,子痫前期组有更多的母体KIR为AA基因型与胎儿HLA—C为C2基因型的结合形式（确切概率P=0．0161）。【结论】母体KIR基因为AA型,所生胎儿HLA—C基因为C2型时,子痫前期发病的风险增加。     

一个罕见HLA-C等位基因间重组家系的遗传背景研究

目的 研究HLA-C等位基因座位上的交换重组,探讨HLA新等位基因产生的分子遗传背景.方法 采集拟进行造血干细胞移植的1例慢性粒细胞白血病患者、患者父母以及2名哥哥的静脉血,采用AlleleSEQR HLA 测序分型试剂进行HLA-A、C、B、DRB1和DQB1共5个位点的高分辨基因分型;再采用Protrans S4 HLA单倍体测序技术分离2个HLA-C等位基因,以确定序列异常的1个C等位基因;进一步采用分子克隆和测序方法进行患者及父母HLA-C等位基因的全长单倍体序列分析.按遗传规律分析该家系的HLA 5个位点的连锁单倍型,同时将HLA-C等位基因全长序列经IMGT/HLA Database中的"BLAST"程序进行序列的对比,以确定C等位基因重组发生的精确位置.结果 经HLA测序及遗传家系分析,该家系中父亲的2条HLA连锁单倍型分别为a:A*0207-C*010201-B*550201-DRB1*090102-DQB1*030302与b:A*240201-C*120202-B*5201-DRB1*1502-DQB1*0601;母亲的分别为:c:A*300101-C*060201-B*130201-DRB1*0405-DQB1*0401与d:A*110101-C*070201-B*4001-DRB1*080302-DQB1*0601.患者的2名哥哥均分别正常遗传了父母的2条单倍体a、d及b、c.患者的2条单倍体分别为母源单倍体c及父源重组单倍体a/b,其中A*240201来自父亲的1条染色单体b,而B*550201-DRB1*090102-DQB1*030302则来自父亲的另1条染色单体a.对比患者携带的C未知新等位基因与父亲的1对C*010201 、C*120202等位基因的全长单倍体序列,推断父亲的2条染色单体在减数分裂时,由于HLA-C等位基因的第273位至330位碱基区域之间发生了交换,重组后形成新的C等位基因及单倍型并遗传给了患者.该C新等位基因的全长序列已递交GenBank,并被世界卫生组织HLA因子命名委员会正式命名为C*0121.结论 本研究发现了1个罕见的中国汉族人群HLA-C基因座位上的基因重组家系,阐明了HLA-C*0121新等位基因及单倍型的产生来源,为更深入研究基因重组及HLA多态性形成机制提供了理论依据.

Abstract：

Objective To study the inter-allelic recombination event occurring in the HLA-C locus in a family of Chinese Han nationality, and to evaluate the molecular genetic background of the new HLA allele.Methods Peripheral blood samples were collected from a Chinese leukemia woman patient, as well as her healthy parents and two brothers.HLA-A, C, B, DRB1 and DQB1 alleles were typed by high-resolution PCR-sequence-based typing (SBT) method using Atria Genetic AlleleSEQR HLA SBT kits.The Protrans S4 HLA-C single allele-specific sequencing strategy was used to separate the two HLA-C alleles and to determine novelty of the allele.The full length sequences of HLA-C alleles of the patient and her parents were further analyzed using cloning and haplotype sequencing method. The HLA five loci linked haplotypes and the recombination site were analyzed by family study, meanwhile the full length sequences of the five HLA-C alleles were compared with the IMGT/HLA database by the program "BLAST".Results The two haplotypes of the father and mother were a:A*0207-C*010201-B*550201-DRB1*090102-DRQ1*030302 and b:A*240201-C*120202-B*5201-DRB1*1502-DRQ1*0601, c:A*300101-C*060201-B*130201-DRB1*0405-DRQ1*0401 and d:A*110101-C*070201-B*4001-DRB1*080302-DRQ1*0601,respectively.The two brothers inherited their parent′s haplotypes a, d and b, c respectively.The two haplotypes of the patient were the maternal c and paternal recombinant a/b haplotype.The recombinant a/b haplotype A*240201-C*new-B*550201-DRB1*090102-DRQ1*030302, A*240201 came from the paternal haplotype b,while B*550201-DRB1*090102-DRQ1*030302 came from the other paternal haplotype a.When comparing the full length sequences of the HLA-C new allele with the father′s allele C*010201 and C*120202, it could deduce that the recombinant a/b haplotype derived from a recombination event occurring between the paternal chromosome 6 during meiosis.The crossover site was between genomic nt273 and nt330 of HLA-C alleles, which created a HLA-C new allele and the fifth haplotype of the family, and inherited it to the patient.The full length sequences of the new allele had been submitted to Genbank, and officially named C*0121 by WHO nomenclature committee.Conclusion This study demonstrates a rare inter-allelic recombination event occurring in the HLA-C locus within a Chinese Han family and illustrates the process of novel allele and haplotype, and provides direct theory for further studying the mechanisms of gene recombination and HLA polymorphism.     

一个无效新等位基因C*01∶37N的鉴定及其序列分析

目的 鉴定中国汉族人群中新发现的一个HLA-C无效等位基因,并对国际上业已公布的HLA-C无效等位基因的突变情况进行分析.方法 采用分子克隆和单倍体测序的方法,鉴定1例HLA-C基因测序分型结果异常样本的分子生物学基础.结果 检出了一个C*01新变异等位基因,其序列与C*01∶02∶01最相近,但存在编码区nt 363 G＞A点突变,位于第三外显子的第97密码子由TGG直接变成终止密码子TGA,导致一个无效等位基因,其序列提交国际GenBank(序列号:GU592508)和IMGT/HLADatabase(HWS10010188).结论 该无效等位基因已被世界卫生组织(WHO)HLA因子命名委员会正式命名为C*01∶37N.

Abstract：

Objective To identify a novel HLA-C null allele in a Chinese Han individual and characterize the nucleotides mutations of HLA-C null alleles reported currently. Methods The molecular basis of a sample with inconclusive sequencing result was clarified by traditional cloning and haplotype sequencing. Results A novel HLA-C * 01 variant allele was identified. Its sequence was very similar to allele C * 01∶02∶01. There was a single nucleotide mutation at the coding sequence nt 363 G＞ A (codon 97TGG＞TGA) in exon 3. The genomic sequence of this novel allele was submitted to GenBank with the accession number GU592508 and the IMGT/HLA Database (HWS10010188). Conclusions The novel variant null allele has been officially named C * 01∶37N by the WHO Nomenclature Committee for factors of the HLA System.     

HLA-C and guttate psoriasis

BACKGROUND: Psoriasis is a heterogeneous disease in its clinical expression. Both genetic and environmental factors are thought to contribute to the pathogenesis of the inflammatory and hyperproliferative components of the typical skin lesions. Predisposing genetic influences include associations with human leucocyte antigens (HLA) of which that with HLA-Cw6 is the strongest. Guttate psoriasis is a specific clinical manifestation of psoriasis frequently associated with group A beta-haemolytic streptococcal throat infection. OBJECTIVES: We set out to determine whether further clinical subdivision of psoriasis is associated with tighter correlation with HLA-C alleles. PATIENTS/METHODS: We determined the HLA-C locus genotype of 29 caucasian patients with guttate psoriasis presenting consecutively with guttate psoriasis associated with a history of a sore throat and/or an antistreptolysin O titre > 200 IU mL-1. Polymerase chain reaction typing using sequence-specific primers was used to detect all known HLA-C alleles. These data were compared with a control population of 604 random caucasian cadaver donors. RESULTS: All patients (100%) with guttate psoriasis carried the Cw*0602 allele compared with 20% of the control population (odds ratio = infinity; 95% confidence limits 25.00-infinity; Pcorrected < 0.0000002). CONCLUSIONS: This result is consistent with HLA-Cw*0602 playing a part directly in the pathogenesis of guttate psoriasis.