TAP polymorphism is not associated with ankylosing spondylitis and complications with acute anterior uveitis in HLA-B27-positive Japanese

Abstract: HLA-B27 is associated with the etiology of ankylosing spondylitis (AS) and acute anterior uveitis (AAU). Transporter associated with antigen processing (TAP) 1 and TAP2 polymorphism influences the range of peptide presented by HLA class I molecules. In this report, contribution of TAP polymorphism to the susceptibility to AS and AAU was studied in HLA-B27-positive Japanese individuals. Patients were classified into three groups: 16 AS patients, 14 AAU patients and 22 patients with both AS and AAU. Twelve HLA-B27-positive healthy individuals were included as a control. TAP polymorphism was detected by PCR-RFLP methods. Significant differences in frequencies of TAP1 alleles were not found between patient groups. None of the TAP2 frequencies showed increased or decreased frequencies compared with HLA-B27-positive healthy controls. In comparison with a random Japanese control, TAP2D allele frequency was significantly increased in the AAU group, but failed to reach a significant level in a group consisting of the AAU-only patients and the patients with both AS and AAU. All of the patient groups were noted to have a significantly increased prevalence of the TAP2H allele as compared to random controls; however, the higher frequency of this allele was detected in HLA-B27 healthy controls as well. These observations suggest a linkage disequilibrium between TAP2D, TAP2H and HLA-B27 in Japanese.     

中国汉人HLADQAl基因对系统性红斑狼疮的遗传易感性研究

利用ＨＬＡ基因的ＰＣＲ－ＲＦＬＰ核苷酸分型技术，以等位基因特异性的限制性内切酶（ＡｐａｌⅠ、ＢｓａｊⅠ、ＨｐｈⅠ、ＦｏｋⅠ、ＭｂｏⅡ、ＭｎｌⅡ）消化ＤＱＡｌ座位特异的ＰＣＲ扩增产物，研究了上海及其附近地区中国汉人ＨＬＡ－ＤＱＡｌ基因与系统红斑狼疮的遗传关联。发现系统红斑狼疮ＤＱＡｌ＊０１０２（３６．５％，ＲＲ＝２．２５，Ｐ＜０．０５，ＥＦ＝０．２０）及＊０４０１（１５．４％，ＲＲ＝１２．４２，Ｐ＜０．００５，ＥＦ＝０．１４）显著增加，而ＤＱＡｌ＊０５０１（１１．５％，ＲＲ＝０．２１，Ｐ＜０．００５，ＰＦ＝０．３１）和＊０．０６０１（３．９％，ＲＲ＝０．２７，Ｐ＜０．０５，ＰＦ＝０．０９）显著下降。排除ＤＱＡｌ＊０４０１的影响后，＊０１０２频率的升高表现得更加明显（ＲＲ＝２．８４，Ｐ＜０．０１）。上述发现显示：ＤＱＡｌ＊０１０２及＊０４０１对ＳＬＥ有遗传易感作用，而ＤＱＡｌ＊０５０１和＊０６０１有遗传抵抗作用，并提出了有关可能的单体型。     

PCR-RFLP-based Mamu-DQB1 typing of rhesus monkeys: characterization of two novel alleles

Up to now 19 allelic sequences of the rhesus monkey DQB1 locus have been published. Referring to these sequences, we have developed a typing protocol for Mamu-DQB1 alleles which was verified by additional cloning, sequence analysis and segregation studies. The protocol is based on the amplification of the second exon with only one specific primer pair followed by the digestion of the PCR products with up to 10 different restriction endonucleases. The alleles can be identified in homozyous and heterozygous combinations since most amplified second exon sequences give unique band patterns after digestion with at least one of the selected restriction endonucleases. By the use of this protocol we analyzed DNA-samples from 182 rhesus monkeys. Among these samples two novel Mamu-DQB1 alleles were detected, subsequently cloned and their nucleic sequence determined. Since we typed four complete breeding groups consisting of two generations we were able to identify several DQ haplotypes by segregation analysis using the previously developed typing protocol for DQA1.     

Chronological difference in walking impairment among Japanese group A xeroderma pigmentosum (XP-A) patients with various combinations of mutation sites

Almost all Japanese group A xeroderma pigmentosum (XP-A) patients have nonsense and/or nonsense codon-leading mutations in the XP group A (XPA) gene, and develop neurological abnormalities. Walking ability is one of the most important neuromuscular functions of the patients, because it determines their daily activities. We studied the correlation between the various combinations of mutations found by PCR-RFLP in Japanese XP-A patients and their chronological walking impairment. We classified these patients into six groups. Group I: A patient who was homozygous for the mutation at codon 116 in exon 3 (Type 1 mutation) could never walk unaided. Group III: Typical patients who were homozygous for the mutation at intron 3 (Type 2 mutation) could walk unaided till 7–16 years of age. Group V: Patients who were compound heterozygous for Type 2 mutation and for the mutation at codon 228 in exon 6 (Type 3 mutation) began to develop some walking difficulty at 5–13 years of age and became unable to walk at 25–28 years of age. Group VI: A patient who was homozygous for Type 3 mutation could walk unaided without any difficulty till the age of 21. The walking ability of group II and IV patients is not known yet.     

Analysis on allelic variation of the HLA-DMB gene in Japanese by PCR-RFLP as well as direct DNA sequencing and identification of a new DMB allele, DMB*0105

In the HLA-D region, one of the class II genes, DMA and DMB have been identified between the DQ and DP genes, and four allelic polymorphisms in each of the DMA (DMA*0101–0104) and DMB (DMB*0101–0104) genes have been so far recognized. Several recent studies suggested that the DM molecule is required for class II antigen presentation pathway especially by promoting the binding of antigenic peptides to the classical HLA class II molecule. In this study, we have analyzed genetic polymorphism and allelic variation of the DMB gene in a Japanese population by the direct DNA sequencing technique and also by the polymerase chain reaction — restriction fragment length polymorphism (PCR-RFLP) method, and could recognize DMB*0101 (49.3%), DMB*0102 (23.2%), DMB*0103 (23.2%), and DMB*0104 (0.4%). Further, a new DMB allele, DMB*0105 characterized by the presence of Val and Iie at two polymorphic sites, codons 144 and 179, respectively was identified. Strong linkage disequilibria were found between DMB*0101 and DRB1*0101, DPB1*0402 and DRB1*1502, and also between DMB*0103 and DRB1*1501 and DQB1*0602. HLA-DMB genotyping using the PCR-RFLP method established here will provide accurate evaluation of the effects of sequence allelism in the DMB gene on the HLA class II disease associations.     

Apolipoprotein E gene polymorphism in cerebrovascular disease: a case-control study

The association between apolipoprotein E (apo E) polymorphism and stroke has been controversial. So far there are no studies reported on the polymorphism of apolipoprotein E in cerebrovascular diseases in the Asian Indians. A blinded case-control study was therefore undertaken and the apo E genotypes and lipid profile of a total of 120 subjects (63 stroke patients and 57 healthy controls) were done. The frequency distribution of apo E alleles and genotypes were assessed and their relation with the occurrence of stroke in Asian Indian subjects was determined. A significantly high frequency of apo epsilon4 allele (30%) was observed in the stroke patients than the controls (11%) (p < 0.005), and patients with epsilon4 allele had a fourfold higher odds to develop stroke OR (95%CI) 4.2 (1.8-10.1) (p < 0.005). On multivariate analysis, after adjusting for age, triglycerides and hypertension, the association of epsilon4 allele with stroke was found to be no longer statistically significant, OR (95%CI) 1.2 (0.4-4.5) (p = NS). On multiple logistic regression analysis age, OR (95%CI) 1.1 (1.1-1.2) (p < 0.001), and hypertension OR (95%CI) 15.1 (2.6-89.1) (p < 0.005) were found to be independent risk factors for development of stroke. This is the first report to have examined the association of apo E gene polymorphism with stroke in the Asian Indians. This study suggests that apo epsilon4 allele, triglycerides, age and hypertension are the predictors for stroke development.     

The apolipoprotein CIII T2854G variants are associated with postprandial triacylglycerol concentrations in normolipidemic Korean men

This study investigated associations between the apolipoprotein (apo) CIII polymorphism and triacylglycerol (TAG) concentrations in fasting and postprandial plasma. Polymerase chain reaction followed by a restriction fragment length genotyping was conducted to assess the allele frequency of the apo CIII T2854G variants in healthy and normolipidemic Korean men (n=262). Waist circumference, body mass index (kilograms per meter squared), fasting plasma concentrations of TAG, total cholesterol, high-density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol (LDLC), glucose, and insulin were compared across the genotypes. Compared to TT homozygotes and TG heterozygotes, GG homozygotes had 22% higher fasting TAG concentrations, respectively (p<0.05). A subgroup of 60 subjects (TT homozygotes=20, TG heterozygotes=22, GG homozygotes= 18) were further invited to participate in a high-fat meal test to assess postprandial TAG concentrations. During the high-fat meal test, the GG homozygotes had 21% higher TAG area under the curve (AUC) than the TT homozygotes (p<0.05) and 22% higher TAG AUC than the TG heterozygotes (p<0.05). In conclusion, this is the first study to show that the apo CIII T2854G variants are associated with elevated postprandial TAG concentrations in the study population of Korean men.     

Comprehensive typing of DQB1 alleles by PCR-RFLP

Abstract: The protocols represented in this report can resolve all 22 DQB1 alleles. The second exon of DQB1 was subjected to PCR using two group-specific primers to obtain DQB1 group 1 (DQ5 and DQ6) and group 2 (DQ2, DQ3, DQ4) specific amplified products, respectively. Three endo-nucleases, Apal, BssHII and Ncil, can provide typing of DQ5 and DQ6 based on easy-to-read uncleaved, cleaved and a combination of uncleaved/cleaved patterns. Similarly, two endonucleases, FokI and BgII can define the specificities DQ2, DQ3 and DQ4. Moreover, all 13 group 1 DQB1 alleles and all but one of their 78 possible heterozygotes can be unambiguously resolved using an extended panel of 10 endonucleases. The remaining pair of heterozygotes, DQB1*05031/0603 and 05032/0608, can however be resolved by double digestion with BsmFI and SfaNI. RsaI splits the previously unresolved alleles DQB1*0602 and 0603 in the amplified products of the modified primer SDQ-01. Fnu4HI can resolve DQB1*0606 from 0605. DQB1*0603, 0607 and 0608 can be resolved by SfaNI and the new endonuclease BsmFI. The comprehensive typing of group 2 DQB1 alleles can be achieved using five endonucleases. All 9 group 2 DQB1 alleles and all but one pair (DQB1*0301/0302 from DQB1*03032/0304) of 36 possible heterozygotes can be resolved. Thus, PCR-RFLP remains a simple, inexpensive and reliable method for DQB1 typing. The PCR-RFLP can be used for comprehensive DQB1 typing either independently or to complement the PCR-SSP and PCR-SSOP methods.     

Identification of tuna species (Thunnini tribe) by PCR-RFLP analysis of mitochondrial DNA fragments

Tunas are highly priced and the Atlantic bluefin tuna is most endangered trade fish in the world. For effective fishery management and protection of consumers’ rights, it is important to develop a molecular method to identify the species of tuna products. In this study, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was developed to identify 10 tuna or tuna-like species. Primers were designed to amplify six mitochondrial DNA fragments from each specimen, and three restriction enzymes including AfaI, DdeI, and AluI were used to analyze the short length fragments produced from ATPase and control region (CR) genes. Besides these, the phylogenetic relationships among tested fish species were analyzed based on phylogenetic trees constructed using the amplified COI and CR sequences. The results indicated that the developed PCR-RFLP really provided a useful and academic technique to identify high-priced tuna species.     

Contribution of Toll like receptor polymorphisms to dengue susceptibility and clinical outcome among eastern Indian patients

Dengue infection has been one of the major public health concerns in India causing simple dengue fever (DF) to severe dengue infection. In the present study, contribution of TLR3, 7 and 8 polymorphisms towards dengue disease susceptibility and severity among Eastern Indian patients was analysed. Genomic DNA was extracted from blood of 201 dengue infected patients and 157 healthy individuals, followed by genotyping of eight polymorphisms of TLR3 (rs3775290), TLR7 (rs5741880, rs3853839, rs179008 and rs179010) and TLR8 (rs3764879, rs3764880 and rs5744080) genes by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Functional analyses of the polymorphisms were predicted. Genotypic association of polymorphisms, alone and in combination, with dengue disease susceptibility and development of WHO-defined warning signs among patients was calculated by using SPSS software. TLR7-rs179008 & TLR8-rs3764880 were implicated to be non-synonymous polymorphisms. Specific genotypes of majority of the analysed TLR polymorphisms exhibited significant positive association with disease susceptibility. CC/C and AA/A of TLR7-rs179008 (p < 0.0001) and TLR8-rs3764880 (p < 0.00001) respectively were significantly associated with development of warning signs among dengue infected patients. Particular genotypic combinations of rs3853839-rs5744080 and rs179008-rs3764880 increased the risk of dengue infectivity, whereas, presence of last combination was more prevalent among dengue patients with warning signs. Thus these polymorphic variants of TLR3, 7 and 8 might act as potential prognostic biomarkers for predicting disease severity among dengue virus infected patients.