猪SLA-DR二级结构预测及同源模建

应用EXPASY服务器（http：／／www．expasy．oh／tools）上的SOPMA法对SLA—DR的α链和口链进行二级结构的预测,并与人的HLA—DR相应的α链和β链的氨基酸和二级结构成分比较,在此基础上同源模建SLA—DRα链、β链及复合体SLA—DR的三级结构。结果显示,SLA—DR的α链各二级结构成分α螺旋、β折叠、转角和无规则卷曲的数目分别为36、61、4和69,β链中分别为42、56、16和74。α链和β链各二级结构成分与复合体SLA—DR具有高度的符合率,分别达到98．7％（α螺旋）、99．1％（β折叠）、83．3％（转角）和97．2％（无规则卷曲）。各功能区分析,SLA—DR的α1和β1区为结合抗原的高变化区。同源模建和三级结构分析表明SLA—DR的α链和口链具有独立的三级结构,并可以组成复合体SLA—DR。     

实验用SPF大白猪和长白猪SLAⅡ类基因多态性研究

目的研究实验用SPF大白猪和长白猪SLA II类基因的多态性。方法分别采集15头SPF大白猪和22头长白猪抗凝血,分离外周血淋巴细胞,提取总RNA,反转录后RT-PCR扩增DQB1、DRB1和DQA基因并进行测序,分析获得的SLA II类等位基因序列多态性。结果 3个基因共获得25个等位基因,包括8个DQB1,10个DRB1和7个DQA,全部获得ISAG SLA命名委员会的官方命名,其中3个等位基因首次提交完整序列,命名为DQB1*02:12(KU754590)、DQB1*02:03(KU754591)和DRB1*06:07(KU754601),3个DQA等位基因为新发现等位基因。SPF大白猪和长白猪DQB1等位基因与外源性抗原结合的15个氨基酸中,共有5个氨基酸具有高度保守性;DRB1等位基因的16个外源性抗原识别位点中,仅1个位点高度保守;DQA等位基因19个抗原结合位点中,有11个高度保守。SLA II类基因氨基酸序列分子进化树表明,3个基因分别聚为两大类,与国外Yucatan小型猪具有较近的亲缘关系,而与其他猪种未表现明显遗传距离相隔。结论成功鉴定了大白猪和长白猪的25个SLA II类等位基因,发现其具有较为丰富的多态性,所获得SLA II类等位基因在其他品种猪也广泛分布,具有多样性,这一研究结果对大白猪和长白猪发展为经典实验动物模型具有重要作用。     

白鱀豚MHC基因类DQB1座位第二外元的序列变异分析

测定了 4 5个克隆的白豚 (Lipotesvexillifer)MHCⅡ类基因DQB座位第二外元 172bp的核苷酸序列 ,共获得 15种序列 ,发现了 2 2个变异位点。核苷酸的非同义替换明显多于同义替换 ,并造成了 15个氨基酸的改变。氨基酸的替换趋于集中在假定的与抗原的选择性识别相关的位点附近。白豚DQB基因的核苷酸和氨基酸序列与文献报道的白鲸 (Delphinapterusleucas)和一角鲸 (Monodonmonoceros)DQB1序列具有较高的同源性。氨基酸序列不具备人及其它一些灵长类动物DQB2基因所共有的基序 (Motif) ,而与牛DQB1基因的基序相近 ,说明本研究得到的白豚MHC序列应属于类DQB1基因。同一个体出现了多种序列的情况 ,提示白豚的DQB基因可能存在着座位重复。白豚的类DQB1座位的序列中存在多种基序的不同组合 ,推测是由于基因转换造成的.     

中国大陆野猪SLA DRB基因exon2的序列变异分析

本文基于135头野猪样本中检测到的15种SLA DRB基因exon 2序列的遗传变异分析,探讨DRB基因在不同地理区域中自然选择的变化特征.研究发现DRB基因exon 2抗原结合位点的非同义替换率为同义替换率的2.95倍,表明该位点受到平衡选择的强烈作用.在系统发生分析中,在基于核苷酸序列构建的NJ树中,野猪DRB位点的15种等位基因全部聚为一枝,没有发现跨种多态现象;而在基于氨基酸序列构建的系统发生树中,野猪SLA-DRB*nnu6和人DRB基因exon 2片段聚在一起,这种氨基酸残基的高度相似可能是由于自然选择压力下产生的趋同.野猪DRB基因exon 2的序列多态性分析表明,东北野猪基因多态性低于四川野猪和华南野猪,这可能与不同气候条件和地理环境下的寄生虫等因素对SLA等位基因变异的影响存在差异有关.     

白(既鱼)豚MHC基因类DQB1座位第二外元的序列变异分析

测定了45个克隆的白(既鱼)豚(Lipotes vexillifer)MHC Ⅱ类基因DQB座位第二外元172 bp的核苷酸序列,共获得15种序列,发现了22个变异位点.核苷酸的非同义替换明显多于同义替换,并造成了15个氨基酸的改变.氨基酸的替换趋于集中在假定的与抗原的选择性识别相关的位点附近.白(既鱼)豚DQB基因的核苷酸和氨基酸序列与文献报道的白鲸(Delphinapterus leucas)和一角鲸(Monodon monoceros)DQB1序列具有较高的同源性.氨基酸序列不具备人及其它一些灵长类动物DQB2基因所共有的基序(Motif),而与牛DQB1基因的基序相近,说明本研究得到的白(既鱼)豚MHC序列应属于类DQB1基因.同一个体出现了多种序列的情况,提示白(既鱼)豚的DQB基因可能存在着座位重复.白(既鱼)豚的类DQB1座位的序列中存在多种基序的不同组合,推测是由于基因转换造成的[动物学报 49(4):501～507,2003].     

猪白细胞Ⅱ类抗原基因多态性研究进展

本文对SLAⅡ类基因的染色体图谱定位、分子结构、基因分型及其多态性的研究进展作了介绍。重点介绍了SLAⅡ类基因的RFLP基因分型及其多态性研究。猪的主要组织相容性复合物Ⅱ类抗原基因座位存在不同程度的等位基因多态性。RFLP技术是一种简便而快捷的SLAⅡ类等位基因分型方法。SLAⅡ类基因的多态性和抗原肽结合位点密切相关,这些位点能结合处理过的抗原,然后递呈给T细胞。另外,在编码β1链的SLA-DRB1基因和SLA-DQB基因中有4个富含GC序列区。 Abstract:This article gave a detailed introduction about regional map,molecular structure,genotyping and polymorphism of SLA class Ⅱ genes.The pig major histocompatibility complex (MHC) class Ⅱ antigens have been known to exhibit a different degree of allelic polymorphism.The locus-specific oligonucleotide primers and RFLP analysis provide a simple and rapid method for genotyping expressed SLA class Ⅱ from genomic DNA.SLA class Ⅱ polymorphism was related to the antigenic peptide binding sites.Detailed analysis of sequences showed that there were 4 GC-rich sequences in exon 2 of SLA-DQB and SLA-DRB1 genes.     

黄麂Mhc-DRB 基因多态性及其维持机制

利用牛DRB3 特异性引物(LA31 和LA32),通过聚合酶链式反应(PCR)、单链构象多态性(SSCP)以及克隆测序技术,从12 只黄麂个体中共获得20 个DRB 第二外显子等位基因,其中6 个个体具有3 ～ 4 个等位基因,提示利用该引物从黄麂中至少可以扩增出2 个DRB 位点。所有序列均无插入、缺失和终止密码子。基于序列比对(与牛DRB3 和鹿科DRB 基因同源性非常高),以及所检测到的氨基酸变异位点主要位于抗原结合区,推测本文所获得的黄麂序列为表达的、且具有重要功能的DRB 位点。抗原结合区氨基酸位点的非同义替换(d_N )显著大于同义替换(d_S )(P < 0.01),说明历史上黄麂DRB 基因经历过正选择作用。CODMEL 程序中的模型M7 和M8 似然比检测(Likelihood ratio test,LRT)结果同样支持上述推论。进一步利用经验贝叶斯法准确地检测出6 个受正选择作用的氨基酸位点(位点11、37、61、67、71、86),其中的5 个位点位于PBR 区。因此,正选择作用可能是维持黄麂DRB 基因多态性的主要机制之一。基于DRB 外显子2 序列利用邻接法(NJ)
构建了部分偶蹄动物系统发生关系,在NJ 树上,黄麂DRB 基因与其它鹿科动物DRB 基因呈镶嵌式分布,提示跨物种进化是维持黄麂DRB 基因多态性的另一重要机制。此外,黄麂两个等位基因(Mure-DRB1 和Mure-DRB11)和马鹿的两个等位基因(Ceel-DRB34 和Ceel-DRB46)与牛科的等位基因构成一个独立的进化枝,说明黄麂和马鹿的某些DRB 基因具有非常古老的谱系。     

中华菊头蝠MHCⅡ-DRB 基因遗传多态性及进化

采样自云南同一种群的中华菊头蝠共16 个个体,用于DRB 基因的分子进化和多态性研究。利用翼膜组织提取DNA 基因组,并PCR 克隆测序分析。获得了相差3 bp 的两种不同长度序列类型,A 序列类型263 bp,在研究群体中有15 个等位基因;B 序列类型260 bp,在研究群体中有8 个等位基因。在分析的74 个氨基酸变异位点上检测到12 个正向选择位点。在9 个个体中检测到分布频率最高的等位基因,也有多个等位基因只存在一个个体中。单个个体中最多存在6 个等位基因。遗传多态性分析表明中华菊头蝠DRB 基因具有较高的多态性。中华菊头蝠DRB 基因可能至少存在3 个重复座位。利用已发表的翼手目DRB 第二外显子序列构建的系统进化树表明中华菊头蝠MHCⅡ-DRB 基因处于独立进化支。     

民猪的SM-DRB基因的PCR-RFLP多态性分析

采用克隆测序和PCR—RFLP相结合的方法,对民猪的SLA—DRB基因的整个编码区进行了扫描和多态性分析．结果表明该基因的第2外显子是整个基因的高变区,突变率达到5．6％,其中80％为有效突变,但没有发现新的等位基因;PCR-RFLP结果表明外显子1用内切酶Alu Ⅰ酶切可获得3种基因型;外显子2用内切酶TaqⅠ酶切可获得3种基因型：外显子3用内切酶BcnⅠ酶切可获得3种基因型：外显子4用内切酶MboⅠ酶切可获得2种基因型：外显子5用内切酶HinlⅠ酶切可获得3种基因型．Hardy-Weinberg平衡分析表明民猪在Alu Ⅰ和HinlⅠ处于平衡状态（P〉0．05）．在TaqⅠ、BcnⅠ和MboⅠ处于不平衡状态．     

猪MHC-DQB、DRB近端调控区序列及其多态性

根据人MHC—DRB和MHC—DQB基因组序列和猪MHC-DRB和MHC-DQB基因外显子1设计引物,应用PCR扩增及克隆测序技术,首次得到了猪MHC-DRB和MHC-DQB基因的5上游近端调控区(URR)序列。分析发现所得序列中存在与MHCⅡ类基因表达调控有关的高度保守的W、X、Y、CCAAT及类TATA调控元件,调控元件的空间组织顺序也与其他物种相应序列的相同。利用SSCP技术在313头猪中共发现12个DRB-URR复等位基因和14个DQB-URR复等位基因,序列比对结果表明在这些复等位基因中存在丰富的多态位点,为进一步深入研究猪MHCⅡ类基因近端调控区的多态性及抗病育种研究奠定了基础。     

Molecular characterization of swine leucocyte antigen class II genes in outbred pig populations

The highly polymorphic swine leucocyte antigen (SLA) genes are among the most important determinants of swine immune responses to disease and vaccines. Accurate and effective SLA genotyping methods are required to understand how SLA gene polymorphisms affect immunity, especially in outbred pigs with diverse genetic backgrounds. In this study, we present a simple and rapid molecular‐based typing system for characterizing SLA class II alleles of the DRB1, DQB1 and DQA loci. This system utilizes a set of 47 sequence‐specific PCR primers developed to differentiate alleles by groups that share similar sequence motifs. We applied this typing method to investigate the SLA class II diversity in four populations of outbred pigs (n = 206) and characterized a total of 19 SLA class II haplotypes, six of which were shared by at least three of the sampled pig populations. We found that Lr‐0.1 (DRB1*01XX–DQB1*01XX–DQA*01XX) was the most prevalent haplotype with a combined frequency of 16.0%, followed by Lr‐0.2 (DRB1*02XX–DQB1*02XX–DQA*02XX) with 14.6% and Lr‐0.15b (DRB1*04XX–DQB1*0202–DQA*02XX) with 14.1%. Over 70% of the pigs (n = 147) had at least one copy of one of these three haplotypes. The PCR‐based typing system described in this study demonstrates a reliable and unambiguous detection method for SLA class II alleles. It will be a valuable tool for studying the influence of SLA diversity on various immunological, pathological and physiological traits in outbred pigs.     

Molecular characterization of swine leukocyte antigen gene diversity in purebred Pietrain pigs

The porcine major histocompatibility complex (MHC) harbors the highly polymorphic swine leukocyte antigen (SLA) class I and II gene clusters encoding glycoproteins that present antigenic peptides to T cells in the adaptive immune response. In Austria, the majority of commercial pigs are F ₂ descendants of F ₁ Large White/Landrace hybrids paired with Pietrain boars. Therefore, the repertoire of SLA alleles and haplotypes present in Pietrain pigs has an important influence on that of their descendants. In this study, we characterized the SLA class I ( SLA‐1 , SLA‐2 , SLA‐3 ) and class II ( SLA‐DRB1 , SLA‐DQB1 , SLA‐DQA ) genes of 27 purebred Pietrain pigs using a combination of the high‐resolution sequence‐based typing (SBT) method and a low‐resolution (Lr) PCR‐based method using allele‐group, sequence‐specific primers (PCR‐SSP). A total of 15 class I and 13 class II haplotypes were identified in the studied cohort. The most common SLA class I haplotype Lr‐43.0 ( SLA‐1 *11XX– SLA‐3 *04XX– SLA‐2 *04XX) was identified in 11 animals with a frequency of 20%. For SLA class II, the most prevalent haplotype, Lr‐0.14 [ SLA‐DRB1 *0901– SLA‐DQB1 *0801– SLA‐DQA *03XX], was found in 14 animals with a frequency of 26%. Two class II haplotypes, tentatively designated as Lr‐Pie‐0.1 [ SLA‐DRB1 *01XX/be01/ha04– SLA‐DQB1 *05XX– SLA ‐ DQA*blank] and Lr‐Pie‐0.2 [ SLA‐DRB1 *06XX– SLA‐DQB1 *03XX– SLA‐DQA *03XX], appeared to be novel and have never been reported so far in other pig populations. We showed that SLA genotyping using PCR‐SSP‐based assays represents a rapid and cost‐effective way to study SLA diversity in outbred commercial pigs and may facilitate the development of more effective vaccines or identification of disease‐resistant pigs in the context of SLA antigens to improve overall swine health.     

Characterization of swine leukocyte antigen alleles and haplotypes on a novel miniature pig line,Microminipig

Microminipigs are extremely small‐sized, novel miniature pigs that were recently developed for medical research. The inbred Microminipigs with defined swine leukocyte antigen (SLA) haplotypes are expected to be useful for allo‐ and xenotransplantation studies and also for association analyses between SLA haplotypes and immunological traits. To establish SLA‐defined Microminipig lines, we characterized the polymorphic SLA alleles for three class I (SLA‐1, SLA‐2 and SLA‐3) and two class II (SLA‐DRB1 and SLA‐DQB1) genes of 14 parental Microminipigs using a high‐resolution nucleotide sequence‐based typing method. Eleven class I and II haplotypes, including three recombinant haplotypes, were found in the offspring of the parental Microminipigs. Two class I and class II haplotypes, Hp‐31.0 (SLA‐1*1502–SLA‐3*070102–SLA‐2*1601) and Hp‐0.37 (SLA‐DRB1*0701–SLA‐DQB1*0502), are novel and have not so far been reported in other pig breeds. Crossover regions were defined by the analysis of 22 microsatellite markers within the SLA class III region of three recombinant haplotypes. The SLA allele and haplotype information of Microminipigs in this study will be useful to establish SLA homozygous lines including three recombinants for transplantation and immunological studies.     

HLA class II associations with rheumatic heart disease among clinically homogeneous patients in children in Latvia

Genetic control of immune reactions has a major role in the development of rheumatic heart disease (RHD) and differs between patients with rheumatic fever (RF). Some authors think the risk of acquiring RHD is associated with the HLA class II DR and DQ loci, but other views exist, due to the various HLA-typing methods and ways of grouping cases. Our goal was to determine the relations between HLA class II alleles and risk of or protection from RF in patients with relatively homogeneous clinical manifestations. A total of 70 RF patients under the age of 18 years were surveyed in Latvia. HLA genotyping of DRB1*01 to DRB1*18 and DQB1*0201-202, *0301-305, *0401-402, *0501-504, and *0601-608 was performed using polymerase chain reaction sequence-specific primers. Data for a control group of 100 healthy individuals typed for HLA by the same method were available from the databank of the Immunology Institute of Latvia. Of the RF patients, 47 had RHD and 8 had Sydenham's chorea. We concluded that HLA class II DRB1*07-DQB1*0401-2 and DRB1*07-DQB1*0302 could be the risk alleles and HLA class II DRB1*06 and DQB1*0602-8, the protective ones. Patients with mitral valve regurgitation more often had DRB1*07 and DQB1*0401-2, and patients with multivalvular lesions more often had DRB1*07 and DQB1*0302. In Sydenham's chorea patients, the DQB1*0401-2 allele was more frequent. Genotyping control showed a high risk of RF and RHD in patients with DRB1*01-DQB1*0301-DRB1*07-DQB1*0302 and DRB1*15-DQB1*0302-DRB1*07-DQB1*0303.     

湖南沙子岭猪SLA-DR基因克隆及生物信息学分析

为了克隆湖南沙子岭猪SLA-DRA和SLA-DRB基因, 分析SLA基因特性和抗原多态性, 评价湖南沙子岭猪在异种移植中的应用前景奠定基础。应用RT-PCR分别扩增湖南沙子岭猪SLA-DRA和SLA-DRB基因, 鉴定后克隆到PUCm-T载体并进行测序, 用NCBI中的BLAST和ExPASY中相关软件进行生物信息学分析。得到的湖南沙子岭猪SLA-DRA 和SLA-DRB 基因特异性基因片段, 大小分别为1 177 bp和909 bp。生物信息学分析发现, 所扩增的SLA-DRA 和SLA-DRB 基因片段均包含完整的开放阅读框, 分别编码252和266个氨基酸残基。将湖南沙子岭猪SLA-DRA和SLA-DRB基因在GenBank 登录, 登录号分别为EF143987和EF143988。同源性分析发现, 湖南沙子岭猪SLA-DRA 和SLA-DRB 与人类相应的DRA、DRB相比, 核苷酸序列同源性分别为83% 和83 %, 编码氨基酸同源性分别为83% 和79%。与GenBank 登录的其他猪种相比, SLA-DRA基因同源性最高可达100 %, 而SLA-DRB基因具有多态性。     

Gene duplications and sequence polymorphism of bovine class II DQB genes

The genetic diversity of bovine class II DQB genes was investigated by polymerase chain reaction amplification and DNA sequencing. The first domain exon was amplified from genomic DNA samples representing 14 class II haplotypes, defined by restriction fragment length polymorphism (RFLP) analysis. The presence of a polymorphism in the copy number of DQB genes was confirmed since two DQB sequences were isolated from certain haplotypes. Four subtypes of bovine DQB genes were found. DQB1 is the major type and was found in almost all haplotypes. DQB2 is very similar to DQB1 but was found only in the duplicated haplotypes DQ9 to 12. DQB3 and DQB4 are two quite divergent genes only present in certain duplicated haplotypes. The bovine DQB complexity thus resembles that in the human DRB region. Bovine DQB genes were found to be highly polymorphic as ten DQB1 alleles and four DQB2 alleles were identified. The observed sequence polymorphism correlated well with previously defined DQB RFLPs. Bovine and human DQB alleles show striking similarities at the amino acid level. In contrast, the frequency of silent substitutions is much higher in comparisons of DQB alleles between species than within species ruling out the possibility that any of the contemporary DQB alleles have been maintained since the divergence of humans and cattle. The frequency of silent substitutions between DQB alleles was markedly lower in cattle than in humans, in agreement with a previous comparison of human and bovine DRB alleles.     

Regression mapping of association between the human leukocyte antigen region and Graves disease

The human leukocyte antigen class II genes DRB1, DQB1, and DQA1 are associated with Graves disease (GD), but, because of strong linkage disequilibrium within this region, the primary etiological variant(s) remains unknown. In the present study, 871 patients with GD and 621 control subjects were genotyped at the DRB1, DQB1, and DQA1 loci. All three loci were associated with GD (P=1.45 x 10(-12), P=3.20 x 10(-5), and P=9.26 x 10(-12), respectively). Stepwise logistic-regression analysis showed that the association could be explained by either DRB1 or DQA1 but not by DQB1. To extend previous results, the amino acid sequence of the exon 2-encoded peptide-binding domain of DRB1 was predicted for each subject, and, by use of logistic regression, each position was analyzed for association with GD. Of 102 amino acids, 70 were uninformative; of the remaining 32 amino acids, 13 were associated with GD (P values ranged from 2.20 x 10(-4) to 1.2 x 10(-12)). The strongest association was at position beta 74. This analysis is consistent with the possibility that position beta 74 of exon 2 of the DRB1 molecule may have a specific and central role in autoantigen presentation by DRB1 to T lymphocytes. However, we cannot yet exclude a primary role for DQA1 or for other polymorphisms that affect DRB1 function or expression.     

Evolution of MHC class II SLA‐DRB1 locus in the Croatian wild boar (Sus scrofa) implies duplication and weak signals of positive selection

The wild boar is an ancestor of the domestic pig and an important game species with the widest geographical range of all ungulates. Although a large amount of data are available on major histocompatibility complex (MHC) variability in domestic pigs, only a few studies have been performed on wild boars. Due to their crucial role in appropriate immune responses and extreme polymorphism, MHC genes represent some of the best candidates for studying the processes of adaptive evolution. Here, we present the results on the variability and evolution of the entire MHC class II SLA‐DRB1 locus exon 2 in 133 wild boars from Croatia. Using direct sequencing and cloning methods, we identified 20 SLA‐DRB1 alleles, including eight new variants, with notable divergence. In some individuals, we documented functional locus duplication, and SLA‐DRB1*04:10 was identified as the allele involved in the duplication. The expression of a duplicated locus was confirmed by cloning and sequencing cDNA‐derived amplicons. Based on individual genotypes, we were able to assume that alleles SLA‐DRB1*04:10 and SLA‐DRB1*06:07 are linked as an allelic combination that co‐evolves as a two‐locus haplotype. Our investigation of evolutionary processes at the SLA‐DRB1 locus confirmed the role of intralocus recombination in generating allelic variability, whereas tests of positive selection based on the dN/dS (non‐synonymous/synonymous substitution rate ratio) test revealed atypically weak and ambiguous signals.     

MHC class II genes in European wolves: a comparison with dogs

The genome of the grey wolf, one of the most widely distributed land mammal species, has been subjected to both stochastic factors, including biogeographical subdivision and population fragmentation, and strong selection during the domestication of the dog. To explore the effects of drift and selection on the partitioning of MHC variation in the diversification of species, we present nine DQA, 10 DQB, and 17 DRB1 sequences of the second exon for European wolves and compare them with sequences of North American wolves and dogs. The relatively large number of class II alleles present in both European and North American wolves attests to their large historical population sizes, yet there are few alleles shared between these regions at DQB and DRB1. Similarly, the dog has an extensive array of class II MHC alleles, a consequence of a genetically diverse origin, but allelic overlap with wolves only at DQA. Although we might expect a progression from shared alleles to shared allelic lineages during differentiation, the partitioning of diversity between wolves and dogs at DQB and DRB1 differs from that at DQA. Furthermore, an extensive region of nucleotide sequence shared between DRB1 and DQB alleles and a shared motif suggests intergenic recombination may have contributed to MHC diversity in the Canidae.     

RFLP analysis of SLA haplotypes in Swiss large white and American Hampshire pigs using SLA class I and class II probes

Summary

Restriction fragment length polymorphism (RFLP) analyses of swine leukocyte antigen (SLA) class I and class II genes from Swiss Large White and American Hampshire families were performed using porcine DNA probes. Class I and class II RFLPs associated with the serologically‐defined haplotypes SLA H1, H8, H16 and H24 and with serotypes SLA 15, 16; SLA 14; and SLA 6, SB 19, were identified. Seven allelic class I RFLP patterns were observed. For genes in the SLA class II region, six allelic RFLP patterns of DQA and DQB; five allelic RFLP patterns of DRA; and seven allelic RFLP patterns of DRB were observed. The serologically‐defined H8 haplotype was subtyped based on differences in class II RFLPs.