Possible susceptibility of the HLA-DPB1*0402 and HLA-DPB1*04 alleles to unexplained recurrent abortion: analysis by means of polymerase chain reaction-restricted fragment length polymorphism method

PROBLEM: To clarify whether HLA-DP antigens are associated with patient population of unexplained recurrent abortion. METHOD OF STUDY: The frequency of HLA-DPB1 alleles in patients with unexplained recurrent abortion, and the compatibility of HLA-DPB1 alleles between patient couples, were studied using a polymerase chain reaction (PCR)-restricted fragment length polymorphism (RFLP) method. Thirty patients who had a history of unexplained primary recurrent abortion, and their husbands, were typed for HLA-DPB1 genotype. Two hundred and ninety-nine base pair fragments from the second exon of HLA-DPB1 genes were selectively amplified using the PCR-primers. After amplification, the DNAs were digested with restriction endonucleases, and subjected to electrophoresis in a 12% polyacrilamide gel to determine HLA-DPB1 genotype. RESULTS: The frequency of HLA-DPB1*0402 and DPB1*04 alleles in the patient group (n = 30) was significantly increased, as compared to that in the normal fertile women (n = 30). The frequency of HLA-DPB1*04 allele in the patient group was significantly increased, as compared to that in the general population (n = 112). No significant compatibility of HLA-DPB1 alleles could be observed between patient couples and normal fertile couples. CONCLUSION: These findings suggest a possible new class II association with patient population of unexplained recurrent abortion.     

中国江苏地区汉族人群肿瘤坏死因子β遗传多态性研究

目的　探讨中国江苏地区汉族人群肿瘤坏死因子 β遗传多态性分布。方法　收集江苏地区 16 8名无血缘关系健康个体的静脉血提取DNA ,应用聚合酶链反应限制性片段长度多态性 (PCR -RFLP) ,对TNFβ基因多态性进行分析。结果　TNFβ检测到三个等位基因 ,其基因型频率分别为TNFβ 1/ 1=2 1 4 % ,TNFβ 1/ 2 =4 7% ,TNFβ 2 / 2 =31 5 % ,基因型分布符合Hardy-Weinberg定律。统计分析显示 :江苏地区汉族人群TNFβ等位基因频率分布与欧美白种人均有显著性差异(P <0 0 5 )。结论　TNFβ等位基因频率分布具有种族差异     

Interleukin 6 −174G>C polymorphism and cancer risk: Meta-analysis reveals a site dependent differential influence in Ancestral North Indians

In our earlier studies, single nucleotide polymorphisms (SNPs) associated with anti-inflammatory cytokines were found to influence risk for breast cancer in western Indian women. Analysis of Interleukin 6 (IL-6) −174G>C polymorphism in this cohort (patients = 182; controls = 236) suggested a protective role for IL-6 −174C allele associated with the lower expression of the cytokine (OR = 0.54; 95% CI 0.32–0.89, dominant model). Together these observations suggested that in comparison to Caucasians, inflammation associated-cytokine gene polymorphisms may have higher influence on risk for cancer in this population. To examine this possibility we analyzed data assessing influence of Interleukin 6 (IL-6) −174G>C polymorphism on risk for various cancers. Overall, there was a marginally higher risk for rare allele homozygotes compared to wild type homozygotes (OR = 1.07; 95% CI 1.00–1.15). Increased risks for genitourinary cancers and for skin cancer were also indicated. The ethnicity based analysis indicated a protective effect of the minor allele in Ancestral North Indians (OR = 0.73; 95% CI 0.55–0.97). Site by ethnicity analysis once again revealed a significant protection against breast cancer (OR = 0.51; 95% CI = 0.37–0.70; dominant model) but an opposite influence on the risk of genitourinary malignancies (OR = 2.51; 95% CI 1.59–3.96; recessive model) in this population alone. The observations imply that contribution of IL-6 to inflammation or effector immunity may depend on the site of malignancy. Assessment of available data in relation to prognosis in breast cancer patients also revealed trends that are compatible with the observations of the meta-analysis. Thus, IL-6 −174G>C polymorphism clearly represents a potential modulator of risk for malignant disorders with ethnicity and site dependent trends. The results also support the possibility of higher influence of inflammation related cytokine gene polymorphisms on the risk for cancers in Ancestral North Indians.     

HSP65-PRA identification of non-tuberculosis mycobacteria from 4892 samples suspicious for mycobacterial infections

Various molecular methods have been used for the rapid identification of mycobacterial species. In this survey, evaluation of antibiotic resistance and PCR-restriction fragment length polymorphism analysis (PRA) of the hsp65 gene was carried out for identification of non-tuberculosis mycobacteria (NTM) isolates from different clinical specimens. Forty-eight different mycobacterial isolates were selected and followed by the conventional and PRA of hsp65 for species identification. The antibiotic susceptibility test was carried out according to standard methods. A 439 bp PCR product of hsp65 in all selected isolates was amplified and digested with the BstEII and HaeIII restriction enzymes. The restriction fragment length polymorphism (RFLP) patterns were analyzed for species identification. Using PRA for 48 mycobacterial selected isolates, including 15 M. tuberculosis, one M. bovis and all 32 isolates of NTM, revealed 11 different species among the NTM isolates. The most frequent NTM isolates were M. kansasii, M. gordonae III, M. marinum, M. chelonae, M. scrofluaceum and M. gastri. In most cases, the PRA results were perfectly in accordance with the classical biochemical method. Combination of resistance to rifampin and isoniazid was present among M. kansasi, M. gordoniae III, M. scrofluaceum, M. chelonae, M. marinum, M. gastri, M. gordoniae II and M. trivale isolates. A high incidence of co-resistance to six, five, four and three anti-TB drugs was observed in 18.5%, 9.1%, 6.6% and 11.7% of all NTM isolates, respectively. Our results showed that PRA, in comparison with classical methods, is rapid and accurate enough for the identification of mycobacterial species from LJ medium. Additionally, we found that in Iran we have a highly diverse population of NTM isolates among patients suspected of having TB.     

Identification and characterization of 31 isolates of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> (Spirochaetales,Spirochaetaceae) obtained from various hosts and vectors using PCR-RFLP and SDS-PAGE analysis

Borrelia burgdorferi sensu lato, the etiologic agent of Lyme borreliosis, circulates between ticks and vertebrate hosts. Two main genospecies typically occur in the Czech Republic Borrelia garinii and Borrelia afzelii, transmitted generally by Ixodes ricinus (L., 1758) ticks. The aim of our study was to identify spirochaete isolates focusing on Borrelia burgdorferi acquired from different sources: vectors (ticks), potential vectors (mosquitoes, small mites) and hosts (wild rodents). In the years 1996–2001 a total of 2398 ticks, 72 mites (from wild rodents), 2700 mosquito adults, 1798 mosquito larvae and organ parts (kidney and spleen) of 216 wild rodents were collected from seven localities in the Czech Republic. A total of 31 spirochaete strains were isolated: 13 strains from ticks, 1 strain from mite (Haemogamasus sp.), 15 strains from rodents, 1 strain from mosquito adults and 1 strain from mosquito larva. For the genospecies identification of these isolates PCR, PCR-RFLP was used and their characterization was also performed by SDS-PAGE. By nested PCR method all except one isolated strains were detected as Borrelia burgdorferi s.l. Following PCR-RFLP molecular analysis results, tick isolates were identified as B. garinii and B. afzelii, the strain isolated from the mite was identified as B. afzelii. This is the first isolated strain of B.b.s.l. from a different mite of infraorder Parasitiformes than tick. All of rodent isolates were identified as B. afzelii; mosquito adult isolate was identified as B. afzelii. Larval isolate from mosquito is spirochaete, but does not belong to Borrelia burgdorferi sensu lato group.     

Novel mutations in the cytochrome P450 2C19 gene: a pitfall of the PCR-RFLP method for identifying a common mutation

CYP2C19 is a clinically important enzyme involved in the metabolism of therapeutic drugs such as (S)-mephenytoin, omeprazole, proguanil, and diazepam. Individuals can be characterized as either extensive metabolizers (EM) or poor metabolizers (PM) on the basis of CYP2C19 enzyme activity. The PM phenotype occurs in 2–5% of Caucasian populations, but at higher frequencies (18–23%) in Asians. CYP2C19*2 and CYP2C19*3, which are single-nucleotide polymorphisms of CYP2C19, are the main cause of PM phenotyping in homozygotes or compound heterozygotes. We report two novel mutations in the CYP2C19 gene identified by direct sequencing and subcloning procedures. One of these mutations was considered to be CYP2C19*3 by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). This result suggests that mutations classed as CYP2C19*3 might include other mutations. Further studies are needed to clarify the relationship between these novel mutations and enzyme activity.The DDBJ accession number of the novel mutation is AB113829     

绞股蓝属植物及其混淆品乌蔹莓的PCR-RFLP鉴别研究

目的：寻找简便、可重复的分子标记方法对绞股蓝属Gynostemma植物及其混淆品乌蔹莓Cayratia japonica进行鉴别。方法：对7种常见药用绞股蓝属植物及其混淆品乌蔹莓的6个cpDNA片段进行PCR 扩增，再利用TaqⅠ，HpaⅡ，EcoRⅠ，RsaⅠ，HhaⅠ，HindⅢ等6种限制性内切酶分别对扩增片段进行消化。结果：36种DNA片段/内切酶组合中，trnK1f -trnK2r片段与RsaⅠ组合可将乌蔹莓从绞股蓝属植物中区分出来，产物清晰、结果稳定。结论：PCR-RFLP分析方法可有效区分常见绞股蓝属植物与其混淆品乌蔹莓。     

护骨素基因多态性对青春前期女童骨量的影响

目的:了解护骨素(osteoprotegerin,OPG)基因启动子区A163G、T245G和T950C位点多态性对青春前期汉族女童骨密度(BMD)的影响。方法:采用PCR-RFLP技术检测广州地区214名青春前期女童护骨素基因三个位点的基因型。采用双能X线骨密度仪测量全身、腰椎(L1-4)、股骨颈和Ward三角区骨矿含量(BMC)和骨密度。同时测定三种骨代谢生化指标。结果:OPG基因启动子区A163G、T245G和T950C位点基因型分布频率均符合Hardy-Weinberg遗传平衡定律,分布频率分别为:A163G:AA-74.8%,AG-21.5%,GG-3.7%;T245G:TT-77.1%,TG-21.5%,GG-1.4%;T950C:TT-41.1%,TC-50.5%,CC-8.4%。T950C位点CC基因型女童的全身骨量、骨密度及股骨颈和Ward三角区骨密度均显著高于TT、TC基因型女童(P<0.05),而TT和TC基因型女童未见显著差异(P>0.05)。A163G和T245G位点多态性与女童BMC和BMD的相关性未发现有统计学意义(P>0.05)。结论:护骨素基因T950C位点含有T等位基因的青春前期女童,其全身骨密度及股骨颈和Ward’s三角区骨密度均较低,该等位基因可能为我国青春期女童低骨量者筛选的遗传学标志提供了研究方向。     

中外六个猪种FUT1基因多态性研究

目的对6个中外猪种共245头猪的FUT1基因进行了研究。方法采用PCR-RFLP技术。结果与结论Hin6I位点上，大白猪、长白猪、杜洛克猪3个外来猪种均存在多态，且以敏感型（GG型和AG型）居多；山西黑猪、太原花猪、马身猪3个本地猪种的所有检测样品都表现为GG型。