Molecular genetic studies of HLA-DRB1 alleles in patients with unexplained recurrent abortion in the Japanese population

BACKGROUND: Recently, evidence that HLA antigens are markers for recurrent spontaneous abortion has gained increased attention. Although the association between HLA class II antigens and patients with unexplained recurrent abortion was elucidated by a large population study in a Caucasian population, such analyses have been conducted in only a small Japanese population. The aim of the present study was to determine whether HLA-DR antigens are associated with patient populations with unexplained recurrent abortion in the Japanese population. METHODS: HLA-DRB1 genotypes were determined using a PCR-restriction fragment length polymorphism (PCR-RFLP) method in 93 patients with unexplained recurrent abortion (79 primary recurrent aborters and 14 secondary recurrent aborters) and in 115 normal fertile women. The rate of possession of each HLA-DRB1 genotype was compared among the three populations. RESULTS: The rate of possession of the HLA-DRB1*1502 in patients with secondary recurrent abortion was significantly higher (P < 0.01 after correction for multiple comparisons) compared with the control, fertile women. The rate of possession of HLA-DRB1*1502 was also higher in patients with primary recurrent abortions than in controls, but the difference was not statistically significant after correction. CONCLUSIONS: These findings suggest that HLA-DRB1*1502 might be a risk allele for unexplained recurrent abortion in the Japanese population.     

Human Leukocyte Antigen DQ α Sharing Is Not Increased in Couples With Recurrent Miscarriage

PROBLEM : The results regarding human leukocyte antigen (HLA) DQ a allele sharing in recurrent miscarriage couples are conflicting. The purpose of this study was to determine the frequency of HLA DQ α allele sharing in our unexplained recurrent spontaneous abortion (RSA) patients using modern DNA analytical techniques. METHODS : DNA was extracted from whole blood samples of 1) 51 couples with at least three miscarriages, and 2) 43 fertile couples (with at least seven children and no known history of recurrent miscarriage). The polymerase chain reaction (PCR) was used to amplify the second exon of the HLA DQ α locus on chromosome 6. Genotypes were identified by allele specific hybridization with 12 sequence-specific oligonucleotide probes. RESULTS : 47% of recurrent miscarriage couples and 35% of fertile couples shared no alleles. 47% of recurrent miscarriage couples compared to 58% of fertile couples shared one allele, and 6% of recurrent miscarriage couples and 7% of fertile couples shared two alleles. CONCLUSIONS : Reproductive partners with unexplained recurrent pregnancy loss have no increased frequency of HLA DQ α allele sharing. It is unlikely that HLA DQ α genotyping will be helpful in the management of patients with RSA.     

PCR-SBT法分析内蒙古地区鄂温克族人群HLA-DPB1等位基因型别

Objective： To study the HLA-DPB1 allele potymorphism in Ewenki from Inner Mongolian. Methods： HLA-DPB1 allele polymorphism in normal Ewenki were detemined by PCR with sequnncing-based-typing（SBT）. Results： 20 HLA-DPB1 alleles were observed and compared with other ethnic groups, the allele frequency of HLA-DPB1＊ 02012 （24.4%） and DPB1＊ 0402 （22.6%）, DPB1 ＊ 0401（20.2%）, DPB1＊ 0501（ 10.7 %） are highest, while others are lower. Conclusion： The distributions of HLA-DPB1 alleles frequencies in nounal Ewenki from Inner Mongolia has a unique style. It is most important to further study anthropology and related to illness in Ewenki nationality.     

HLA-DPB1 polymorphism in Blang and Puyi ethnic groups of southwest China inferred from sequence-based typing

In the present study, DNA typing for HLA-DPB1 was performed using polymerase chain reaction (PCR)-sequence-based typing method in two isolated Chinese populations: the Blangs (n = 94) in Shuangjiang County and the Puyis (n = 76) in Luoping County from Yunnan province of Southwest China. These two populations exhibited certain similarity in their allelic distributions of the HLA-DPB1 gene. A total of 11 and 12 alleles at the DPB1 locus were found in the Blang and Puyi groups, respectively. In the Blang group, the most frequent alleles were DPB1*0501 (51.0%) and DPB1*1301 (17.0%). DPB1*030101 was also common with a frequency of 6.4%. In the Puyi group, the most frequent allele was also DPB1*0501 with a frequency of 47.5%, followed by DPB1*1301 (21.1%). Two alleles DPB1*2101 and DPB1*0202 followed, with frequencies ranging between 5% and 8%. The alleles DPB1*4101, DPB1*3301, DPB1*6801 and DPB1*8401 were found for the first time in Chinese populations. A dendrogram constructed by neighbor-joining method showed that the Blang and Puyi ethnic minorities, which had the closest relationship belonged to the southern Chinese.     

浙江汉族人群HLA-DPA1和-DPB1基因多态性分析

目的 检测浙江地区汉族人群HLA-DPA1和HLA-DPB1等位基因及单倍型频率.方法 应用PCR-直接测序分型法对100名健康、无血缘关系的浙江汉族人外周血标本进行HLA-DPA1和HLADPB1等位基因分析.结果 在100份标本中检出8个HLA-DPA1等位基因和19个HLA-DPB1等位基因.HLA-DPA1等位基因频率较高的依次为DPA1*020202(47.0%)、DPA1*010301(38.5%)和DPA1*020101(10.5%).HLA-DPB1等位基因中,频率较高的依次为DPB1*0501(39.5%)、DPB1*020102(13.5%)和DPB1*040101(13.0%).连锁分析发现共有44个HLA-DPA1-DPB1单倍型,单倍型频率最高为DPA1*020202-DPB1*0501(29.5%).结论 浙江地区汉族人群HLA-DPA1和DPB1基因座等位基因具有丰富的多态性,2个基因座呈现连锁不平衡.     

浙江汉族人群HLA-DPA1和-DPB1基因多态性分析

目的 检测浙江地区汉族人群HLA-DPA1和HLA-DPB1等位基因及单倍型频率.方法 应用PCR-直接测序分型法对100名健康、无血缘关系的浙江汉族人外周血标本进行HLA-DPA1和HLADPB1等位基因分析.结果 在100份标本中检出8个HLA-DPA1等位基因和19个HLA-DPB1等位基因.HLA-DPA1等位基因频率较高的依次为DPA1*020202(47.0%)、DPA1*010301(38.5%)和DPA1*020101(10.5%).HLA-DPB1等位基因中,频率较高的依次为DPB1*0501(39.5%)、DPB1*020102(13.5%)和DPB1*040101(13.0%).连锁分析发现共有44个HLA-DPA1-DPB1单倍型,单倍型频率最高为DPA1*020202-DPB1*0501(29.5%).结论 浙江地区汉族人群HLA-DPA1和DPB1基因座等位基因具有丰富的多态性,2个基因座呈现连锁不平衡.     

A study of HLA-DPB1 phenotypes reveals DPB1*6301 in a rural population from Cameroon

Several recently reported HLA-DPB1 alleles have only been identified in a single family or individuals and are of unknown distribution world-wide. Many new DPB1 alleles appear to arise as a result of gene conversion-like events, which may localize variant DPB1 alleles to the population in which they were first identified. Using two SSOP-based typing methods in parallel, we have identified HLA-DPB1*6301 in an individual from rural Cameroon which has previously only been reported in a family of Mexican-American origin. The presence of DPB1*6301 was confirmed by sequence-based typing of exon 2.     

浙江汉族人群HLA-DPA1和-DPB1基因多态性分析

目的 检测浙江地区汉族人群HLA-DPA1和HLA-DPB1等位基因及单倍型频率.方法 应用PCR-直接测序分型法对100名健康、无血缘关系的浙江汉族人外周血标本进行HLA-DPA1和HLADPB1等位基因分析.结果 在100份标本中检出8个HLA-DPA1等位基因和19个HLA-DPB1等位基因.HLA-DPA1等位基因频率较高的依次为DPA1*020202(47.0%)、DPA1*010301(38.5%)和DPA1*020101(10.5%).HLA-DPB1等位基因中,频率较高的依次为DPB1*0501(39.5%)、DPB1*020102(13.5%)和DPB1*040101(13.0%).连锁分析发现共有44个HLA-DPA1-DPB1单倍型,单倍型频率最高为DPA1*020202-DPB1*0501(29.5%).结论 浙江地区汉族人群HLA-DPA1和DPB1基因座等位基因具有丰富的多态性,2个基因座呈现连锁不平衡.     

HLA-DPB1 allele mismatches between unrelated HLA-A,B,C,DR (generic) DQA1-identical unrelated individuals with unreactive MLC

We have used a PCR-RFLP method with one generic amplification of HLA-DPB1 second exon and 6 endonucleases to differentiate the 19 HLA-DPB1 alleles and 171 heterozygous combinations. The set of primers used in our studies produced fragment sizes different from those published before (1). The HLA-DPB1 alleles in Caucasians showed a higher frequency of DPB1*0401 and DPB1*0402, when compared to a small group of Colombians who showed a higher frequency of DPB1*0402 and DPB1*0201. We found three HLA-DPB1 alleles associated with two HLA haplotypes that result from non-random association of alleles: DPB1*0401 with HLA-A26, B38, DR4, DQA1*0301 and DPB1*0101 and DPB1*0401 with HLA-A1, B8, DR3, DQA1*0501. We also report that 70% of combinations between HLA (generic A,B,C,DR) and DQA1-identical MLC-unreactive cell mixtures showed HLA-DPB1 mismatches, suggesting that HLA-DPB1 differences are not important in MLC reactivity.     

用核酸序列测定1型糖尿病HLA-DPB1和DQB1第2外显子基因

目的 研究山东省汉族1型糖尿病与HLA－DPB1和HLA－DQB1等位基因的相关性。方法 采用基于核酸序列测定的基因分型技术对52例1型糖尿病患者及38例正常对照进行了DPB1和DQB1基因分析。结果 DPB1＊2201（P＜0．01）和DQB1＊0201（P＜0．01）、＊0303（P＜0．05）及＊0604（P＜0．05）等位基因频率在糖尿病患者组显著高于对照组,而PB1＊0402（P＜0．01）和DQB1＊0301（P＜0．01）等位基因在糖尿病患者组显著低于对照组。结论 DPB1＊2201和DQB1＊0201、＊0303及＊0604等位基因可能是山东省汉族1型糖尿病的易感性等位基因,而DPB1＊0402和DQB1＊0301等位基因可能是山东省汉族1型糖尿病的保护性等位基因。     

Gene polymorphism of HLA-DPB1 and DPA1 loci in caucasoid population: Frequencies and DPB1-DPA1 associations

The genetic polymorphism of the HLA-DPB1 and DPA1 loci was studied in 60 unrelated caucasoid individuals by PCR-RFLP. The polymorphic second exon of DPB1, the third exon of DPA1, and the trans-membrane DPA1 exon were specifically amplified in vitro by polymerase chain reaction (PCR). Amplified DNAs were digested with selected enzymes. Twenty patterns were obtained with DPB1 defining 20 DPB1 alleles. Thirty-nine homozygous cell lines were used as HLA-DP reference cells. The results obtained with these cell lines were compared to those obtained by PLT, RFLP, and SSO. Although three subdivisions of the allele DPA1^*01 were reported, DPA1^*0103 was the only represented one in the caucasoid population. In the studied population, it was the most frequent DPA1 allele (76.6%), whereas DPA1^*0201 frequency is 23.3%. DPB1^*0401 and DPB1^*0402 are the most frequent among the DPB1 alleles (40.0% and 13.3%, respectively). This may lead to a lower HLA-DPB1 diversity among caucasoids. Certain HLA-DPB1 alleles associate exclusively with one DPA1 allele (DPB1^*0401, 0402, and 0301 with DPA1^*01 and DPB1^*0101, 0501, and 1701 with DPA1^*0201) whereas the others can associate with both DPA1 alleles. This by itself can create another kind of polymorphism, indicating the importance of HLA-DPA1 typing. Thus, PCR-RFLP seems to be one of the best DNA typing methods: it represents direct, accurate, fast, and nonradioactive typing for both HLA-DPA1 and HLA-DPB1 alleles.     

Interleukin 1 receptor antagonist gene polymorphisms are not risk factors for recurrent pregnancy loss: evaluation of couples

PROBLEM: The interleukin-1 system has been implicated in pregnancy outcome. Fetal carriage of interleukin-1 receptor antagonist (IL-1Ra) specific alleles has been associated with adverse pregnancy outcomes including spontaneous abortion and pre-term labor. This study was undertaken to compare the frequency of IL-1RN 2 alleles among both male and female partners of couples experiencing recurrent pregnancy loss with that of fertile control couples. METHOD OF STUDY: Buccal swabs were obtained from 42 couples experiencing recurrent pregnancy loss and from 20 fertile control couples. DNA was extracted from the buccal swabs and analyzed for the presence of IL-1RN variable number tandem repeat. RESULTS: No significant differences were found when the frequency of IL-1RN 2 polymorphisms were compared between fertile control couples and couples experiencing recurrent pregnancy loss. Similar results were also obtained when comparing women or men respectively from each group. CONCLUSION: IL-1RN 2 allele is not a risk factor for recurrent pregnancy loss.     

BTNL2 allele associations with chronic beryllium disease in HLA-DPB1*Glu69-negative individuals

Butyrophilin-like 2 (BTNL2) polymorphisms have been associated with sarcoidosis. We hypothesized that BTNL2 variants might confer a human leukocyte antigen (HLA)-independent risk for chronic beryllium disease (CBD), a granulomatous lung disease with similar clinical, radiological, and pathological features to sarcoidosis. Genomic DNA was obtained from CBD (n= 88), beryllium sensitized (BeS, n= 86), and beryllium exposed nondiseased control subjects (Be-exp, n= 173). Six BTNL2 polymorphisms, HLA-DPB1, DRB1, and DQB1 alleles were determined by sequence-specific primer-PCR. All BTNL2 polymorphisms were in Hardy-Weinberg equilibrium. No significant differences were found between BTNL2 polymorphisms or haplotypes and CBD, BeS, or Be-exp. In HLA-DPB1*Glu69-negative subjects (n= 10 CBD, n= 13 BeS, n= 102 Be-exp), DRB1*13 and BTNL2 rs3117099TT homozygosity were increased in CBD (70% and 40%, respectively) vs Be-exp (16%, P= 0.001 and 2.9%, P= 0.001, respectively). The BTNL2 rs3117099T-HLA-DRB1*13 combination was significantly increased in CBD (50%) compared with Be-exp (6.9%, P= 0.001). In conclusion, both DRB1*13 and rs3117099TT homozygosity are associated with CBD in *Glu69-negative subjects, while DPB1*Glu69 is associated with CBD and BeS compared with Be-exp. As a result of the small sample size and strong linkage disequilibrium between DRB1*13-DQB1*0603/4/9 and the BTNL2 rs3117099T allele, it is difficult to assess the primary association in DPB1*Glu69-negative CBD cases.     

Genetic susceptibility to childhood common acute lymphoblastic leukaemia is associated with polymorphic peptide-binding pocket profiles in HLA-DPB1*0201

In a previous study, we obtained preliminary evidence in a small series of patients (n = 63) suggesting that susceptibility to childhood common acute lymphoblastic leukaemia (c-ALL) was associated with an allele at the HLA-DPB1 locus, DPB1*0201. We have now tested this hypothesis by comparing the frequency of children with leukaemia (n = 982) who typed for specific DPB1 alleles and two groups of non-leukaemic children, one consisting of children with solid tumours, excluding lymphomas (n = 409), the other consisting of normal infants (n = 864). We found that significantly more children with c-ALL and T-ALL, but not pro-B ALL or acute non-ALL typed for DPB1*0201 as compared with children with solid tumours [odds ratio (OR), 95% confidence interval (CI) for c-ALL: 1.76, 1.20-2.56; T-ALL: 1.93, 1.01-3.80] and normal infants (OR, 95% CI for c-ALL: 1.83, 1.34-2.48; T-ALL: 2.00, 1.10-3.82). In childhood c-ALL, significantly more children than those with solid tumours or normal infants typed for DPB1 alleles coding specific polymorphic amino acids lining the antigen-binding site of the DPbeta1*0201 allotypic protein, suggesting that susceptibility to childhood c-ALL may be influenced by DPbeta ABS amino acid polymorphisms shared by DPbeta1*0201 and other DPbeta1 allotypes. These results point to a mechanism of c-ALL susceptibility that involves the presentation of specific antigenic peptides, possibly derived from infectious agents, by DPbeta1*0201-related allotypic proteins, leading to the activation of helper T cells mediating proliferative stress on preleukaemic cells.     

HLA-DPB1*0401 is associated with dominant protection against type 1 diabetes in the general Saudi population and in subjects with a high-risk DR/DQ haplotype.

The human leukocyte antigen (HLA) class II DQB1*0201/0202-DRB1*04 genotype has been identified as predisposing to type 1 diabetes [insulin-dependent diabetes mellitus (IDDM)] in the Saudi Arabian population (P = 0.0002; odds ratio = 0.67; 95% confidence interval = 0.009-0.381). In this study, we searched for a factor at the DPB1 locus by analysing DPB1 polymorphism using sequence-based typing in 86 Saudi IDDM patients and control subjects, all carrying the HLA-DRB1*04/DQB1*02 haplotype or the known susceptibility allele DQB1*0201/0202. Significant protection was conferred by DPB1*0401, which was observed in 17 of 50 control subjects (55%) and 2 of 36 IDDM patients (5%) with the DQB1*0201/0202 allele (P = 0.0012; odds ratio = 8.75; confidence interval = 1.72-59.70). Our data showing a high frequency of the DPB1*0401 allele even in the presence of the predisposing DQB1*02 allele in healthy subjects may indicate a protective effect of this combination of HLA alleles against type 1 diabetes. This finding supports the hypothesis that protective HLA class II genes can override the risk conferred by HLA-DQ susceptibility alleles. Further studies using larger cohorts of control subjects and patients should be undertaken to confirm this observation.     

FREQUENCY OF HLA-DPB1 ALLELES IN A SPANISH POPULATION: THEIR CONTRIBUTION TO RHEUMATOID ARTHRITIS SUSCEPTIBILITY

HLA-DPB1 allele frequencies in 181 unrelated control individuals and 70 rheumatoid factor-positive RA patients from Seville (Spain) were determined using oligonucleotide typing methods. All frequencies shown concern the percentage of individuals positive for a certain allele. HLA-DPB1*0401 was the most common DPB1 allele in the healthy individuals, possessed by 65.7% of them. In addition to HLA-DPB1 *0401, only the following alleles were found in normal subjects at frequencies greater than 10%: DPB1*0101 (15.5%), DPB1*0201 (12.2%), DPB1*0301 (16.6), and DPB1*0402 (29.3%). When HLA-DPB1 allelic frequencies were compared between seropositive RA patients and controls, a negative association for DPB1*0301 and DPB1*0401 was found in RA patients, although it failed to reach statistical significance after correction for the number of comparisons made. The other DPB1 alleles exhibited almost identical frequencies in both groups. However, when only DR4⁺ patients and controls were considered, the decrease in the frequency of the DPB1*0301 and DPB1*0401 alleles lacked statistical significance. On the other hand, when DR4^- RA patients and controls were compared, the frequency of DPB1*0301 was found decreased significantly again, even more than in the whole group of patients.     

Association of HLA-G alleles and 3' UTR 14 bp haplotypes with recurrent miscarriage in Brazilian couples

Human leukocyte antigen (HLA)-G expression is restricted, expressed on trophoblast, with a major role in fetus acceptance. In addition to the 46 HLA-G alleles, the presence or absence of a 14 bp polymorphism located in the 3' UTR contributes to gene polymorphism that may influence both HLA-G mRNA stability and HLA-G isoform's splicing and consequently could play an immunomodulatory function in pregnancy. To elucidate the role of HLA-G polymorphism in pregnancy, HLA-G allele frequencies and the 14 bp polymorphisms were analyzed and compared in 60 couples with recurrent miscarriage (RM) and 68 fertile control couples. Two haplotypes showed a significant elevated frequency in patients (HLA-G*01:01:08/+14, p(c) < 0.0001 and HLA-G*01:04:01/-14, p(c) < 0.0001). The haplotype HLA-G*01:01:A/+14 exhibited a significant protective effect against RM in women (p(c) = 0.0238). Remarkably, significant differences in linkage disequilibrium were observed between patient and control groups. Two alleles showed a positive association with the +14 bp segment in RM patients and a strong negative association with fertile controls (HLA-G*01:01:08 = patients D' = 0.295-0.371; controls D' = -0.715 to -1.000; HLAG* 01:05N = patients D' = 0.728-1.000; controls D' = -1.000). HLA-G*01:04:01 showed a negative association with the 14 bp segment in patients and a positive association in controls (patients D' = -0.249 to - 0.674; controlss D' = 0.688-1.000). Our results suggest that haplotypic combinations of HLA-G alleles and the 14 bp segment may be associated with RM.     

慢性鼻窦炎伴鼻息肉与HLA-DPB1、DQB1基因相关性研究

目的:探讨慢性鼻窦炎伴鼻息肉患者在HLA-DPB1、DQB1两个基因位点的易感因素及保护性因素,初步探讨HLA-DPB1、DQB1两个基因与慢性鼻窦炎伴鼻息肉的相关性,寻找易感基因类型.方法:通过对HLA-DPB1、DQB1两个基因位点进行PCR扩增,用ABI 3700自动测序仪对扩增产物进行测序,从而对46例广东籍慢...     

Molecular genetic studies on the compatibility of HLA class II alleles in patients with unexplained recurrent miscarriage in the Japanese population

It is hypothesized that patients having unexplained recurrent miscarriage lack an appropriate immune reaction against their partner's antigens, which means possible compatibility of HLA antigens between the patient couples. The conclusion, however, has not yet been achieved, so the purpose was to determine whether significant compatibility of HLA class II exists between the couples. The HLA-DRB1 and -DQB1 genotypes were determined using PCR-RFLP method in 91 patient couples and in 72 normal couples. The number of patient couples with zero-allele mismatch was not significantly different compared with that of control couples regarding HLA-DRB1 genotype and phenotype, as well as regarding HLA-DQB1 genotype and phenotype. While the number of patient couples with zero- and one-allele mismatch was significantly higher compared with that in control as to HLA-DR and -DQ phenotype (P=0.029 by Chi-square test). In conclusion, it is suggested that the compatibility of HLA class II antigens between couples might be involved in the genesis of unexplained recurrent miscarriage.     

The HLA-G genotype is potentially associated with idiopathic recurrent spontaneous abortion

The causes for recurrent spontaneous abortion (RSA) remain unknown in a large proportion of the cases. Human leukocyte antigen (HLA)-G and HLA-E are expressed on invasive trophoblast cells, and are supposed to confer to materno-fetal tolerance. A total of 14 different nucleotide sequences have been described for HLA-G, including one dysfunctional null allele (HLA-G*0105N), while five different sequences have been described for HLA-E. In this study, 78 RSA couples and 52 fertile controls were typed for HLA-G and HLA-E by direct sequencing or single strand conformational polymorphism (SSCP) respectively. The overall analysis showed no significant difference in allele frequencies for either HLA-G or HLA-E between the two groups. However, HLA-G allele frequencies in women who had suffered from five or more RSA differed significantly from fertile controls (P: = 0.001), and from women who had undergone three or four RSA (P: = 0.027). Detailed analysis demonstrated a significant increase in the proportion of the HLA-G alleles *01013 and *0105N in the whole group of RSA women compared with fertile controls (P: = 0.007). When studying the prognostic value of HLA genotyping for pregnancy outcome (n = 41), 31 patients (76%) gave birth to a living child without performing immunotherapy. Seven out of 10 (70%) couples suffering from a further RSA carried the HLA-G*01013 or *0105N allele, compared with 10 out 31 (32%) couples giving birth (P: = 0.06). This study suggests that the HLA-G genotype may be a contributing factor in RSA.