Large-scale genotyping of single nucleotide polymorphisms by Pyrosequencingtrade mark and validation against the 5'nuclease (Taqman((R))) assay

Here we present the first large-scale effort at genotyping using a novel sequencing method, Pyrosequencingtrade mark, as a method for genotyping of single nucleotide polymorphisms (SNPs). Pyrosequencingtrade mark genotypes were validated through duplicate analysis of 1,022 genotypes using the PSQ96trade mark instrument for pyrosequencing and TaqMan((R)) for 5'nuclease assays. Identical results were obtained using both methods. In a small pilot study, a pooling strategy using Pyrosequencingtrade mark was successfully tested. We conclude that Pyrosequencingtrade mark is highly efficient and accurate in the analysis of SNPs and represents a promising solution to high-throughput genotyping of large sample populations.     

Detecting low-quality markers using map expanders

Genetic marker data play a crucial role in gene mapping, and genotyping errors may have substantial influence on the power to detect and the precision to locate disease loci. Statistical methods can identify individuals, markers, or pedigrees with a high likelihood of containing genotyping errors, and the putative erroneous genotypes can then be rechecked and either verified, removed, or corrected to reduce the loss of power introduced by errors. We present a method to identify genetic markers with a high genotyping error rate. Genotyping errors are likely to appear as double recombinations which expand the genetic map around the marker. Markers flagged as map expanders (i.e., having an excessive number of double recombinations) can then be reread or regenotyped, or a replacement marker of higher quality can be used instead. The proposed method can be applied to any type of pedigree. Simulation studies of nuclear pedigrees and sib-pairs show that the proposed method generally has high power to identify map expanders when the set of markers is reasonably dense (average intermarker distance of 5 cM), even when the nominal genotyping error rate is low (2%). Not surprisingly, the power to detect map expanders increases with marker heterozygosity and genotyping error rate, and is reduced with increasing intermarker distance. When the method was applied to a real dataset consisting of 56 nuclear pedigrees genotyped for 20 microsatellite markers on chromosome 4, the method diagnosed three markers as map expanders. Subsequent examination of these markers proved that they all had high genotyping error frequencies.     

Phenotypic and genotypic relationship between Campylobacter spp isolated from humans and chickens in Northern Ireland--a comparison of three phenotyping and two genotyping schemes

Human campylobacteriosis is currently the most common cause of acute bacterial gastroenteritis on the island of Ireland, accounting for over 3,000 laboratory reports per year, where circa 2,000 reports originate from the Republic of Ireland and circa 1,000 reports from Northern Ireland. Elsewhere, consumption of contaminated poultry has been associated with the zoonotic transmission of disease, therefore it was the aim of this study to examine the phenotypic and genotypic relatedness of campylobacters isolated from chickens and humans locally. Sixty isolates were subtyped using phenotyping techniques (biotyping, phage-typing), as well as genotyping techniques (multilocus enzyme electrophoresis (MEE), ribotyping) and the data compared. The frequency of shared phenotypes and genotypes between poultry and humans varied depending on the typing technique employed ranging from 98.2% of human isolates sharing a similar resistotyping (MAST) disc type with poultry strains to 20% similarity with MEE typing. Overall, this small study is the first report on phenotypic and genotypic relatedness between human and poultry campylobacters in Northern Ireland, isolated under controlled conditions. The study demonstrated an association between chicken and human sub-species types, taken from a relatively contained epidemiological environment. Further work is required with larger numbers of isolates coupled with typing schemes, which are able to reliably cluster strains from chicken and humans, which share high degrees of clonality, before local poultry can be conclusively proven to be a significant source of human campylobacteriosis.     

RHD gene polymorphism among RhD-negative Han Chinese

TheRhbloodgroupsystemwasfirstdescribed 60 yearsagointhecontextofacaseofhemolyticdiseaseinanewborn (HDN ) 1　ItisnowunderstoodthatthepathogenesisofHDNinvolvesmaternalalloimmunizationbyRhantigens TheRhbloodgroupsystemisthemostpolymorphicofhumanbloodgroupsconsistingofatleast45independentantigens.2 　ExceptfortheABOsystem ,Rhisthemostclinicallysignificantintransfusionmedicine Rhantigensareassociatedwithincompatiblehemolytictransfusionreactions,HDN ,andautoimmunehemolyticanemia Individualsa…     

陕西省麻疹病毒基因分型及疫苗免疫因素分析

目的确定引起陕西省麻疹流行的病原毒株,探讨相关的疫苗免疫因素。方法采集麻疹病人咽拭子标本分离病毒,用酚-氯仿抽提法提取病毒悬液中的RNA,逆转录-聚合酶链反应（RT-PCR）扩增N基因碳末端的核苷酸450个（bp）片段,对扩增产物进行序列测定和基因分型。用微量中和试验测定健康人群麻疹病人急性期血清中和麻疹疫苗株和野毒株的中和抗体水平。结果从82份咽拭子中分离出19株H1基因型麻疹病毒。麻疹疫苗免疫血清和有免疫史麻疹急性期病人血清对麻疹野毒株的中和抗体几何平均滴度（GMT）分别为26.36和26.90,明显低于疫苗株,GMT分别为49.30和56.18。结论陕西省近年麻疹流行是由H1基因型麻疹野病毒引起,不排除疫苗对目前流行株保护性不足的因素。     

The Q/E27 polymorphism in the beta2-adrenoceptor gene is associated with increased body weight and dyslipoproteinaemia involving triglyceride-rich lipoproteins

OBJECTIVES: To investigate whether a substitution of glutamine by glutamic acid at amino acid position 27 (Q/E27) and an arginine to glycine transition at amino acid 16 (R/G16) in the beta2-adrenoceptor gene are associated with lipid and lipoprotein disturbances and/or increased body weight in men. DESIGN: Population-based study. SETTING: Department of medicine at a university hospital. SUBJECTS: A total of 180 healthy men, aged 30-45 years, were recruited at random from a register containing all permanent residents in Stockholm County (response rate of 70%). MAIN OUTCOME MEASURES: Frequency of beta2-adrenoceptor genotypes and alleles in relation to plasma lipid and lipoprotein levels and body mass index. RESULTS: Individuals carrying the E27 allele and/or the G16 allele had significantly higher body mass index (BMI). Furthermore, carriers of the E27 allele had significantly higher plasma concentrations of cholesterol, triglycerides, VLDL cholesterol and VLDL triglycerides than did subjects homozygous for the Q allele. CONCLUSION: The E27 allele of the beta2-adrenoceptor gene is associated with slightly to moderately elevated BMI and dyslipoproteinaemia involving triglyceride-rich lipoproteins in healthy younger and middle-aged men.     

检测水产品食物中副溶血弧菌基因分型方法的建立

目的建立副溶血弧菌的基因分型技术,应用于广州市水产品食物中毒居前列的致病菌——副溶血弧菌的检测,寻找一种简便快速准确的检验方法。方法分析副溶血性弧菌分离株的16S-23S rDNA IGS的相关信息(电泳图谱、序列分析等),找出合适的16S-23S rDNA IGS作为基因分型依据,建立副溶血性弧菌的基因分型技术。结果通过克隆和测序,分析了副溶血性弧菌的16S-23S rDNA IGS片段,初步确定了以16S-23S rDNA IGS为分子标记的副溶血性弧菌基因分型技术。结论本检测方法不但快速、灵敏,还对深入了解副溶血性弧菌的发病机制,研究其分子流行病学特征具有重要的意义。     

Identification of oral mitis group streptococci by arbitrarily primed polymerase chain reaction

"Mitis group" streptococci are commensal but may play some role in dental caries, septicemia or endocarditis. Rapid genotypic identification would aid studies of dental plaque ecology, or diagnostic use. AP-PCR with 58 unpaired arbitrary primers was used to characterize 7 Streptococcus gordonii, 11 Streptococcus sanguis, 2 Streptococcus crista, 5 Streptococcus parasanguis, 18 Streptococcus oralis, and 36 Streptococcus mitis (22 biovar 1 and 14 biovar 2). S. parasanguis 16S rRNA variable region primer RR2 produced species-specific bands with all S. gordonii and S. sanguis. Human V beta 1 T-cell receptor primer 434 yielded concordant genotypic identification of all phenotypically defined S. crista and S. parasanguis, 83% of S. oralis, and 74% of S. mitis biovar 1. Amplicon patterns for S. mitis biovar 2 were heterogeneous. Findings suggest that primers RR2 and 434 in succession will allow rapid identification of genotypic groups corresponding closely to mitis group species established by phenotype.     

p16 Immunoreactivity in unusual types of cervical adenocarcinoma does not reflect human papillomavirus infection

Houghton O, Jamison J, Wilson R, Carson J & McCluggage W G
(2010) Histopathology 57, 342–350
p16 Immunoreactivity in unusual types of cervical adenocarcinoma does not reflect human papillomavirus infection Aims: The association between human papillomavirus (HPV) and cervical carcinoma is well known, with HPV being identifiable in almost all cervical squamous carcinomas and most adenocarcinomas. However, the prevalence of HPV in unusual morphological types of cervical adenocarcinoma has not been investigated extensively. The aim was to determine HPV status in a series of primary cervical adenocarcinomas, enriched for unusual morphological types. The relationship between HPV and p16 immunoreactivity in these neoplasms was also investigated, as it is generally assumed that in cervical neoplasms diffuse p16 expression is predictive of the presence of high‐risk HPV. Methods and results: Sixty‐three cervical adenocarcinomas, comprising those of usual type (n = 43), minimal deviation type (n = 4), gastric type (n = 3), intestinal type (n = 3), mesonephric type (n = 3), clear cell type (n = 4), serous type (n = 2) and hepatoid type (n = 1) underwent linear array HPV genotyping and immunohistochemistry for p16. Overall, HPV was identified in 32 of 56 cases (57%) in which sufficient DNA was present for analysis. The most common HPV types were 16 and 18, with these being identified in 20 and 18 cases, respectively, either alone or in combination. Seventy‐eight per cent of usual‐type adenocarcinomas were HPV‐positive, as was the single serous carcinoma in which there was sufficient DNA for analysis. In contrast, all minimal deviation adenocarcinomas and those of gastric, intestinal, mesonephric and clear cell types were HPV‐negative, as was the single hepatoid carcinoma. All usual‐type adenocarcinomas exhibited p16 immunoreactivity (diffuse staining in all but one case), as did 11 of 20 of those of unusual morphological type (five focal, six diffuse). Conclusions: Most, but not all, cervical adenocarcinomas of usual type contain HPV, but those of unusual morphological type are almost always HPV‐negative. This has implications for the efficacy of HPV vaccination in the prevention of cervical adenocarcinoma. A significant proportion of cervical adenocarcinomas are p16‐positive in the absence of HPV, illustrating that in these neoplasms diffuse p16 immunoreactivity is not a reliable surrogate marker of the presence of high‐risk HPV.     

Analysis of hepatitis C virus strains circulating in Republic of the Congo

The aim of this study was to assess the seroprevalence, viremia, genotype distribution, and demographic history of hepatitis C virus (HCV) in the Republic of the Congo. Testing was carried out on sera samples collected in 2005 from 807 Bantus belonging to the Kongo, Teke, and Ngala subgroups and 80 Pygmies. Positive HCV serology was found in 50 (5.6%) individuals including 31 (60%) who were viremic. Seroprevalence increased with age with a cutoff at 50 years: 2.8% <50 versus 12% >50. Twenty‐one strains belonged to four described subtypes, that is, 4c in eight cases, 4h in two, 4k in three, and 4r in eight. Ten strains could not be assigned to any known subtype and may represent six new variants, that is, subtype 4 in five cases and subtype 2 in one. Evolutionary analysis of subtype 4c and 4r sequences indicated a period of enhanced transmission in the mid‐twentieth century probably due to iatrogenic causes. This study underlines the high genetic diversity of strains in the Republic of the Congo with nine subtypes 4 and one subtype 2. J. Med. Virol. 82:562–567, 2010. © 2010 Wiley‐Liss, Inc.