Analysis of Heterozygous BRCA1 5382ins Founder Mutation in a Cohort of Egyptian Breast Cancer Female Patients Using Pyrosequencing Technique

Background: Up to half of the heritable mutations in breast cancer (BC) are attributed to BRCA1 and BRCA2 genes. The mutation prevalence is variable based on ethnicity and may be influenced by founder mutations. The aim of this pilot study is to determine for the first time, the prevalence of BRCA1 5382insC founder mutation in a cohort of Egyptian familial breast cancer patients (FBC). Methods: Female patients were selected to have familial type of breast cancer. Twenty healthy females were included as a control group. Peripheral blood samples were withdrawn from all studied females and were analyzed for BRCA1 5382insC founder mutation detection using pyrosequencing technique. Results: Eighty Egyptian FBC females were eligible to be enrolled in the study with a mean age of 48.31 ± 10.97years.We found a BRCA1 5382insC mutation carrier frequency of 5% of total studied FBC patients (4 out of 80 patients) with 95% confidence interval (1.61-12.99). There was a high statistical significant difference between carriers and non-carriers concerning the number of affected family members by BC, (p=0.001).  Conclusion: BRCA1 5382insC founder mutation is not uncommon among Egyptian FBC females. The carrier frequency is comparable to that reported worldwide; however it is lower than those from previous Egyptian studies using different molecular techniques. The strong association between the mutation and the number of affected family members suggest wider screening of the mutation among high risk families using the reliable pyrosequencing technique.     

Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .     

Evaluation of PyroMark Q24 pyrosequencing as a method for the identification of mycobacteria

We evaluated PyroMark Q24 (QIAGEN) pyrosequencing as a method for the identification of mycobacteria, with potential application in clinical practice. Sequence data from the hypervariable region A of the 16S rRNA gene (43 and 35 bp sequences) were obtained using PyroMark Q24, and a similarity search was performed automatically with PyroMark IdentiFire software. Of the 148 mycobacterial type strains tested, 138 (93.2%) were accurately identified to single or clade species level, including complex level. From the remaining 10 strains, 3 (Mycobacterium gilvum, Mycobacterium goodi, and Mycobacterium thermoresistible) showed poor sequencing quality of homopolymers. For 6 other strains (Mycobacterium cosmeticum, Mycobacterium flavescens, Mycobacterium pallens, Mycobacterium hodleri, Mycobacterium xenopi, and Mycobacterium crocinum), the sequences were unreadable from the middle, and Sanger sequencing indicated biallelic site. Finally, a 40 bp sequence for Mycobacterium gordonae could not be obtained despite repeated attempts. PyroMark Q24 provided accurate identification of multiple mycobacterial strains isolated from common clinical settings, but additional gene sequencing is required to distinguish species identified as a group or complex.     

Equine herpesvirus 1 (EHV-1) nucleotide polymorphism determination using formalin fixed tissues in EHV-1 induced abortions and myelopathies with real-time PCR and pyrosequencing

Equine herpesvirus-1 (EHV-1) strains with a single point mutation at the 2254 nucleotide position with a G₂₂₅₄ constitution within the DNA polymerase gene are associated strongly with equine myeloencephalopathies. Infections with non-neuropathogenic EHV-1 strains without the G₂₂₅₄ nucleotide but with an A₂₂₅₄ nucleotide are associated less frequently with equine neurologic disease. A retrospective study utilizing DNA extracted from formalin fixed paraffin embedded tissues was conducted with real time PCR and pyrosequencing, to determine the infecting EHV-1 strains. Infection with EHV-1 A₂₂₅₄ and or G₂₂₅₄ strain was detected with real time PCR, and was confirmed with a rapid pyrosequencing technique. Pyrosequencing was useful in at least 2 cases where real time PCR was equivocal in determining the infecting EHV-1 strain type. The strain with G₂₂₅₄ mutation was detected in 9.4% of 21 studied abortion cases, and in 86.6% of 15 neurologic cases.     

rtA181位点突变乙型肝炎病毒感染患者的临床特点及个体化再治疗效果

目的 研究rtA181位点突变慢性乙型肝炎(CHB)患者的用药史、临床特点及个体化治疗效果. 方法 核苷(酸)类似物(NUCs)治疗中病毒学突破并检出rtA181突变的54例CHHB及相关肝硬化患者,检测其血清HBV DNA、HBsAg定量及ALT水平,焦磷酸基因测序法定量检测HBV的P基因区10个NUCs相关耐药突变位点.回顾性分析不同用药史患者的病毒变异模式,比较病毒学突破时与基线期、rtA181单个与多个位点突变时的HBV DNA载量,分析发生病毒学突破时,含rtA181T与含rtA181V位点突变患者的血清学指标.前瞻性队列研究分析不同个体化干预措施的疗效.正态分布的计量资料用t检验进行比较,不符合正态分布的数据用Mann-Whitney检验分析,两组间计数资料的比较采用x2检验或Fisher's精确概率法. 结果 54例rtA181突变的患者中,35例(64.8％)为包含rtA181T的突变.既往用药主要为阿德福韦酯和拉米夫定.应用多种NUCs者,多位点突变占57.6％ (19/33);单一NUCs者,多位点突变占28.6％ (6/21),x2=4.342,P＜0.05.发生病毒学突破时,患者血清HBV DNA载量较初次NU Cs抗病毒治疗时低[(5.66±1.01)1og10拷贝/ml比(6.75±0.81)log10拷贝/ml,t=-4.210,P＜0.01],含rtA181T位点突变患者较含rtA181V位点突变患者HBsAg水平高[(3.80±0.45) log10 IU/ml比(3.46±0.60)1og10 IU/ml,t=2.109,P＜0.05].对患者分别给予加用或换用恩替卡韦和加用替比夫定治疗,随访满24周时,HBV DNA≥6 log10拷贝/ml的患者中,8例加用或换用恩替卡韦,4例加用替比夫定,其发生病毒学应答者分别为8例和3例,HBV DNA阴转者分别为3例和l例;HBV DNA＜6 log10拷贝/ml中,14例加用或换用恩替卡韦,7例加用替比夫定,其发生病毒学应答者分别为14例和5例,HBV DNA阴转者分别为12例和4例.结论 用药史与rtA181突变模式有一定关系,应用多种NUCs的患者,易出现多位点突变和多重耐药.加或换恩替卡韦干预治疗的疗效好于加用替比夫定方案.     

焦磷酸测序法快速检测胰腺癌K-ras基因点突变

目的 建立基于焦磷酸测序技术的胰腺癌K-ras基因点突变的检测方法,并与Sanger测序法作一比较.方法 用焦磷酸测序法(Pyrosequencing)和Sanger测序法(Sanger sequencing)分别在10名正常胰腺组织、49例胰腺癌、11例慢性胰腺炎、18例胰腺良性囊肿、7例胰岛素癌、9例壶腹癌、7例胆管癌及7例十二指肠乳头癌石蜡包埋组织的DNA中检测K-rag基因12密码子点突变.结果 采用上述两种方法在所有正常胰腺组织、慢性胰腺炎、胰腺良性囊肿、胰岛素癌、壶腹癌、胆管癌及十二指肠乳头癌均未见K-ras基因点突变,而采用焦磷酸测序法发现胰腺癌石蜡包埋组织K-ras基因点突变率为71.4%(35/49),显著高于采用Sanger测序法发现胰腺癌石蜡包埋组织K-rag基因点突变率(61.2%,30/49).结论 焦磷酸测序法较Sanger测序法更为敏感,且焦磷酸测序法准确、快速、高通量,适合临床标本的批量测定.     

拉米夫定治疗慢性乙型肝炎中变异病毒株的动态变化

目的:建立定量检测及监测乙肝病毒DNA的rtM204I/V(YMDD)及rtL180M变异的方法.方法:以克隆、测序鉴定及构建乙肝病毒DNA YMDD及变异(YIDD和YVDD)质粒作为标准,4例接受拉米夫定治疗(100 mg/d,疗程48 wk以上)的慢性乙型肝炎患者血清标本为对象,用焦磷酸测序的方法检测变异位点核苷酸的频率,并计算变异株的含量比例,通过检测不同变异株类型的标准质粒,确定测序峰图的背景信号.结果:通过检测标准质粒后,确定背景信号为5%,变异位点调整后的核苷酸频率转化为变异病毒株的比例后,4例患者治疗前均检测出变异的病毒株(4.5%-33%),并且随治疗时间延长呈增多的趋势.病毒载量及ALT分析提示基因型耐药发生后,将发生病毒学反跳及临床耐药.结论:焦磷酸测序可以定量检测及动态监测变异病毒株的含量.拉米夫定耐药突变株在拉米夫定治疗前已存在,并随治疗时间的增加而增长,出现病毒学反弹及临床耐药.     

Subclinical infections of cardiac implantable electronic devices: Insights into the host–bacteria dialog from blood and pocket tissue with pyrosequencing

While bacteria exist in CIED patients without clinical signs of infection, the underlying bacterial community structure and diversity in the bloodstream and pocket tissue of asymptomatic CIED patients remain unknown. In this study, we performed high-throughput 454 pyrosequencing of bacterial 16S rDNA of blood and pocket tissue from 54 asymptomatic CIED patients as well as blood from 30 normal individuals (normal controls). Firstly, we observed a significant increase of blood bacterial diversity in patients as compared with blood of normal subjects or patient tissues. We also found significant differences in 13 blood-associated bacterial genera between patients and normal subjects, and 14 bacteria genera between blood and tissues within patients. Secondly, we found that the serum levels of four inflammatory markers (CRP, IL-1β, IL-6, and MCP-1) in CIED patients were significantly higher than those in normal subjects. Thirdly, we found that there were significant correlations between 43 bacterial species and these inflammatory markers. Taken together, our results reveal a high diversity in the microbial community in CIED patients, and suggest the potential roles of multiple bacteria co-occurrence in the CIED subclinical infections.     

江苏部分地区慢性乙型肝炎病毒感染者天然耐药的调查

目的 通过连续监测江苏部分地区乙型肝炎病毒（HBV）感染者发生天然耐药基因变异的频度及耐药位点的分布特点,为合理选择抗病毒治疗的药物提供依据.方法 回顾性分析2004～2010年未服用核苷类似物抗病毒药物的352例慢性乙型肝炎（CHB）患者临床资料.应用焦磷酸测序技术及双脱氧链终止法基因测序的方法进行耐药基因的检测.结果 352例CHB患者中,345例基因测序成功,7例测序失败.8例有耐药基因突变,天然耐药突变检出率为2.32％.主要耐药位点为rtV173L、rtL180M、rtA181T、rtM204V、rtV207L、rtS213T、rtV214I、rtN236T.2004～2006年间145例CHB患者均未检测出耐药突变,2007、2008、2009及2010年耐药突变的检出率分别为2.63％、3.92％、4.69％和4％.多因素Logistic回归分析显示耐药突变的发生与感染者年龄相关.结论 江苏部分地区CHB患者有天然耐药基因突变,耐药突变检出率为2.32％.2004～2010年耐药突变检出率呈上升趋势.年龄超过30岁是耐药突变发生的相关因素.     

A comparison of methylation levels in HPV18, HPV31 and HPV33 genomes reveals similar associations with cervical precancers

BackgroundHigh risk human papillomavirus (HR-HPV) infection is common and only a small minority of infections become persistent and lead to cervical cancers. Women positive for HR-HPV usually require a second test to avoid unnecessary colposcopies and over treatment. Elevated DNA methylation of HR-HPV L1 and L2 genes in high grade disease has emerged as a promising molecular triage tool.ObjectivesOur aim was to accurately measure methylation levels at selected CpG positions in the HPV18, HPV31 and HPV33 genomes. We focused on the L2, L1, URR and E6 regions because these were previously shown to be interesting areas for study.Study designPyrosequencing was used to measure methylation in 208 HPV18, 207 HPV31, and 126 HPV33 positive women selected from a London colposcopy referral population.ResultsAfter adjustment for multiple testing, at FDR 5%, elevated methylation was significantly associated with cervical intraepithelial neoplasia grades 2 or worse (CIN2+) in all investigated CpGs in HPV18 L2 and L1. Two of 6 L2 and 12 of 15 L1 sites in HPV31 and 6 of 8 L2 and 3 of 13 L1 sites in HPV33 showed significantly elevated methylation in CIN2+. Methylation of CpG sites in the URR and E6 region of the HPV types was low and most differences were not significant.ConclusionElevated methylation of CpG sites in the L1 and L2 regions of HPV18, HPV31 and HPV33 is associated with CIN2+ and a panel test may be useful for triage of women with HR-HPV infections.