A novel 56-bp variable tandem repeat polymorphism in the human deoxyribonuclease I gene and its population data

This study confirms the presence of a novel variable number of tandem repeats polymorphism, designated as HumDN1, in intron 4 of the human deoxyribonuclease I (DNase I) gene. Genotyping was performed without difficulty by PCR-amplification and separation by agarose gel electrophoresis in 423 Japanese, originating from four geographically diverse areas in Japan, and 89 Germans. The HumDN1 allele variability was due to different numbers of 56-bp repeat sequences, and five different alleles were distinguished with apparent size between 364 and 588 bp. Although there was a general uniformity for the polymorphism in the Japanese population, significant differences in genotype distribution were found between the Japanese and German populations. Furthermore, linkage disequilibrium between the HumDN1 and DNase I protein polymorphisms was revealed.     

Lack of technical specificity in the molecular diagnosis of toxoplasmosis

The polymerase chain reaction amplification of a fragment of the B1 gene of Toxoplasma gondii coupled to hybridization was performed in 42 patients from Rio de Janeiro, Brazil. The results showed 50% of positivity in the IgM positive toxoplasmosis group, and 12.5% in the positive IgG and negative IgM individuals. The data presented here revealed a lack of specificity of the molecular approach, clearly indicating that the primers used may co-amplify human sequences.     

Sequence determination of exp- 1 Gene of Plasmodium Falciparum Isolate FCC1／HN

目的 测定恶性疟原虫FCCl／HN株exp-1基因序列。方法 根据exp-1基因已知序列设计合成1对引物，用PCR技术从FCCl／HN株基因组DNA中扩增exp-1基因；将exp-1基因克隆入pMD-18T载体，转化大肠杆菌JMl09感受态细胞，铺x—gal LB平板；挑取阳性菌落，用酶切，PCR扩增进行鉴定。以正确的重组质粒为模板，用双脱氧链末端终止法测定exp-1基因序列。结果 从恶性疟原虫FCCl／HN株基因组DNA中获取exp-1基因，成功克隆入pMD-18T载体；测序表明FCCl／HN株exp-1基因全长937bp，编码162个氨基酸。结论 克隆了恶性疟原虫FCCl／HN株exp-1基因，并测定了其核苷酸序列，为进-步研究其功能奠定基础。     

Nucleotide sequence of the cellulase gene celF of Clostridium thermocellum

The nucleotide sequence of the celF gene of Clostridium thermocellum was determined. The open reading frame extended over 2217 bp. The encoded 739-aa polypeptide, CelF, with a M_w = 82,015, was an endoglucanase with activity against carboxymethylcellulose. The N terminus showed a typical signal peptide, and a cleavage site after Ala-27 was predicted. From residues 28 to 470, the sequence of CelF was related to the catalytic domains of type E₂ endoglucanases, with a strong homology to the endoglucanases CelZ of Clostridium stercorarium and CenB of Cellulomonas fimi. The catalytic region was followed by a 134-aa segment also present in C. stercorarium CelZ and in C. fimi CenB, and belonging to the family of non-catalytic, presumably cellulose-binding domains first identified in Bacillus subtilis endoglucanase. A 21-aa segment rich in Pro/Thr/Ser residues separated the putative cellulose-binding region from the COOH-terminal region, which contained two conserved stretches of 24 amino acids closely similar to those previously described in endoglucanases CelA, CelB, CelD, CelE, CelH and CelX, and xylanase XynZ of C. thermocellum.     

华北地区汉族人群Rh(D)抗原弱表现型个体的分子遗传机制研究

目的探讨汉族人群非血缘关系Rh(D)抗原弱阳性个体的血清学表型及分子遗传机制。方法采用常规血清学技术从非血缘关系随机献血者中筛检Rh(D)抗原弱阳性个体(包括弱D型、部分D型),对其进行Rh D、C、c、E、e抗原表型的检测;采用序列特异性引物-聚合酶链反应(PCR-SSP)方法同时检测其RHD基因和RHCE基因;测序分析RHD基因全长编码区序列;同时通过特异性PCR技术测定其RHD合子型。结果血清学试验证实为D抗原弱阳性表型的有32例个体,占无关供者人群比率为0.015%,其中18例个体为弱D15型(845G>A),1例为弱D12型(830G>A),1例为携带DEL等位基因(1227G>A)的弱D型,8例为部分D表型中的DⅥⅢ型(RHD-CE(3-6)-D),1例为部分D表型中的DⅤa(Hus)(RHD-CE(5)-D),3例标本10个外显子检测均未见异常。Rh小因子检测有3种表型CcEe(4例)、Ccee(10例)、ccEe(18例),其血清学与分子生物学检测一致。RHD杂合性试验鉴定显示仅4例标本为纯合型RHD+/RHD+,其余为杂合型RHD+/RHD-。结论汉族人群D抗原弱阳性比率明显少于高加索人,汉族人群D弱表现型中,弱D15型频率最高;部分D的弱表现型中,DⅥⅢ型占主要比例。     

乙型肝炎病毒前C区及基本核心启动子变异检测方法的比较研究

目的 建立错配聚合酶链反应-限制性片段长度多态性（mPCR-RFLP）检测乙型肝炎病毒（HBV）前C区A1896、基本核心启动子（BCP）T1762/A1764双变异的方法,与直接测序法比较,评估其应用价值.方法 利用错配PCR的原理扩增HBV前C区长194 bp、C启动子区长184 bp的基因片段,扩增产物分别经限制性内切酶Bsu36I、BclI酶切,琼脂糖凝胶电泳,根据酶切图谱多态性,建立检测HBV前C A1896、BCP T1762/A1764双变异的方法,对127份HBsAg、HBV DNA阳性的HBV感染者血清进行分析,酶切同时测序.2份标本用克隆测序以验证酶切鉴定变异株、野株混合感染的准确性.结果 127份血清中125（98.42%）份能用酶切、121（95.21%）份能用测序分析HBV前CA1896、BCP T1762/A1764状况.酶切与测序均成功的119份血清中,16份为前C区A1896变异株,34份为BCP T1762/A1764双变异株,21份为前C区A1896、BCP T1762/A1764联合变异株,48份为前C区G1896、BCP A1762/G1764野株,酶切与测序的结果完全一致.1份酶切鉴定为前C区A1896、G1896混合感染标本的5个克隆子中,1个为前C区A1896变异株,另外4个为前C区G1896野株;另1份酶切鉴定为单纯前C区A1896变异标本的5个克隆子均为前C区A1896变异株,酶切分析结果与克隆测序结果完全相符.结论 与测序法相比,本方法较简便、特异性强,能区分野毒株、变异株混合感染,适合较大样本分析,可用于流行病学调查及临床筛查.     

Clinical,immunological and genomic characteristics of children with X-linked agammaglobulinemia from Kerala,South India

X-linked agammaglobulinemia (XLA) is an X-linked recessive primary immunodeficiency disorder caused due to a pathogenic variant in the Bruton tyrosine (BTK) gene with an incidence of 1:379,000 live births and 1:190,000 male births. Patients affected with XLA present with recurrent infections of the gastrointestinal and respiratory tracts. Here we report the first case series of 17 XLA patients of 10 South Indian families with a wide spectrum of clinical and genetic features. In our cohort, patients presented mainly with recurrent pneumonia, gastrointestinal infection, otitis media, pyoderma, abscesses, empyema, arthritis, and osteomyelitis. Using next-generation and Sanger sequencing we have identified 10 unique pathogenic and likely pathogenic variants in 17 patients. This encompasses three nonsynonymous, two stop-gain, two frameshifts, two structural, and one splicing variant, out of which two of them are novel. Based on the type of variant, patients had variable clinical features and treatment responses. We have also evaluated Btk protein expression for six patients in comparison to the healthy individuals and determined mosaic Btk expression patterns in four mothers. We have also performed family screening in 6 families using Sanger sequencing and identified 19 carriers for the variant. The diagnosis for the patients led to the proper treatment i.e. 15 patients were on intravenous immunoglobulin (IVIG) and the other two had successful hematopoietic stem cell transplantation (HSCT). Unfortunately, two of our patients died due to sepsis, while on IVIG. We envision the present study could help in better understanding of patients with XLA and help in family screening and prenatal diagnosis. To the best of our knowledge, this is the largest case series of patients affected with XLA from South India.     

Allelic Polymorphisms of Killer Immunoglobulin-Like Receptor Genes in Malay and Orang Asli Populations of Peninsular Malaysia

Next-generation DNA sequencing (NGS) technology advancements provide new insight into the level of variation in killer immunoglobulin-like receptor (KIR) genes. High resolution allele genotyping of seven KIR genes was conducted among 94 unrelated Malay and Orang Asli (OA) individuals of Peninsular Malaysia. A manual bioinformatics analysis is performed and optimised by Sanger sequencing method. The Malays expressed a total of 22 alleles, as compared to only 15 alleles in the OA population. In total, 12 centromeric and 9 telomeric allelic haplotypes were identified in the Malays, whereas 8 centromeric and 5 telomeric allelic haplotypes were identified in the OA. The KIR2DL1, KIR2DL3, and KIR2DS4 genes exhibited a high degree of variation and balanced distribution in the Malay and OA populations. On the other hand, KIR2DL4, KIR3DL1, KIR3DL2 and KIR3DL3 genes exhibited a high degree of conservation, with less number of alleles identified and the dominance of a single allele at high frequency. High-resolution KIR allele genotyping has revealed unique sequence variations and allelic haplotypes between individuals and populations. The distributions of KIR alleles and haplotypes are useful for genetic population studies and serve as a baseline for future transplantation matching and disease association research.     

结核分枝杆菌CFP10诊断抗原基因的克隆及序列特性分析

目的扩增结核分枝杆菌培养滤液蛋白10基因（CFP10）,并将其克隆至质粒pGEMT中进行核苷酸序列特性分析,为研究结核病候选诊断抗原基因奠定基础。方法以结核菌标准菌株H37Rv为模板,通过PCR技术扩增出结核分枝杆菌CFP10抗原基因,将其重组到pGEM-T载体后进行酶切鉴定及序列测定和生物信息学分析。结果成功扩增出结核分枝杆菌CFP10抗原基因,测序表明该片段开放阅读框由303bp组成,与已发表基因核苷酸序列相比,同源性为100%,推导编码氨基酸序列同源性为100%。结论成功克隆CFP10基因,经DNA测序证实,该片段阅读框完整,为其原核表达及相关研究奠定了基础。     

广东珠海市2007年登革病毒分子流行病学分析

目的了解2007年珠海市登革热暴发期间登革Ⅰ型（ZH1067/07）及Ⅱ型（ZH1340/07）病毒序列特征和可能的传播来源。方法以GenBank公布的病毒AB178 040和M29 095为参考,分别设计PCR引物,扩增病毒ZH1067/07和ZH1340/07全基因组的不同片段并测序,通过末端重叠序列进行拼接组成病毒全基因组序列。序列GenBank登录号分别为Eu359 008和Eu359 009。参考已公布的登革Ⅰ/Ⅱ型病毒全基因组序列进行进化分析。结果拼接后的登革Ⅰ型病毒基因组全长10 735个核苷酸,经序列进化分析,属登革Ⅰ型中的第1群（基因Ⅰ型）,与2004年分离自日本的登革Ⅰ型毒株AB178 040及分离自福建的登革Ⅰ型毒株Fj231/04核苷酸同源性最高,达99%（10 638/10735）,经流行病学证据支持福建输入病例导致珠海本地暴发;拼接后的登革Ⅱ型病毒基因组全长10 723个核苷酸,经序列的进化分析,属登革Ⅱ型病毒中的第4群（基因Ⅳ型）,与2005年分离自文莱的登革Ⅱ型EU179 857和EU179858两毒株距离最近,核酸同源性99%,（10 609/10 705）。流行病学调查证明2例均为澳门输入病例。结论2007年珠海登革Ⅰ型病毒ZH1 067/07可能来源于福建登革Ⅰ型毒株Fj231/04,与来源于密克罗尼西亚的AB178 040有很近的亲缘关系;珠海登革Ⅱ型病毒ZH1340/07可能由澳门输入,与文莱EU179 857有很近的亲缘关系,未导致珠海本地暴发。