Two-stage Study of Familial Prostate Cancer by Whole-exome Sequencing and Custom Capture Identifies 10 Novel Genes Associated with the Risk of Prostate Cancer

BackgroundFamily history of prostate cancer (PCa) is a well-known risk factor, and both common and rare genetic variants are associated with the disease.ObjectiveTo detect new genetic variants associated with PCa, capitalizing on the role of family history and more aggressive PCa.Design, setting, and participantsA two-stage design was used. In stage one, whole-exome sequencing was used to identify potential risk alleles among affected men with a strong family history of disease or with more aggressive disease (491 cases and 429 controls). Aggressive disease was based on a sum of scores for Gleason score, node status, metastasis, tumor stage, prostate-specific antigen at diagnosis, systemic recurrence, and time to PCa death. Genes identified in stage one were screened in stage two using a custom-capture design in an independent set of 2917 cases and 1899 controls.Outcome measurements and statistical analysisFrequencies of genetic variants (singly or jointly in a gene) were compared between cases and controls.Results and limitationsEleven genes previously reported to be associated with PCa were detected (ATM, BRCA2, HOXB13, FAM111A, EMSY, HNF1B, KLK3, MSMB, PCAT1, PRSS3, and TERT), as well as an additional 10 novel genes (PABPC1, QK1, FAM114A1, MUC6, MYCBP2, RAPGEF4, RNASEH2B, ULK4, XPO7, and THAP3). Of these 10 novel genes, all but PABPC1 and ULK4 were primarily associated with the risk of aggressive PCa.ConclusionsOur approach demonstrates the advantage of gene sequencing in the search for genetic variants associated with PCa and the benefits of sampling patients with a strong family history of disease or an aggressive form of disease.Patient summaryMultiple genes are associated with prostate cancer (PCa) among men with a strong family history of this disease or among men with an aggressive form of PCa.     

T‐cell receptor and B‐cell receptor repertoire profiling in adaptive immunity

B‐cell receptors and T‐cell receptors are the key molecules responsible for specific antigen recognition in adaptive immunity. The huge diversity of immune receptor repertoires constrained their comprehensive studies in the past. More recently, however, high‐throughput sequencing based techniques have revolutionized the field of immune receptor repertoire profiling enabling new insights into the development and function of the adaptive immune system. In this review we describe current methods for immune receptor profiling and software tools used for repertoire reconstruction from raw sequencing data. We also provide examples of how immune repertoire profiling can be used to study adaptive immunity in disease and in the course of organ and bone marrow transplantation.     

二代测序技术应用于脑脊液检测在结核性脑膜炎中的早期诊断价值

目的评价二代测序技术应用于脑脊液检测在结核性脑膜炎（TBM）患者中的早期诊断价值。 方法前瞻性纳入2018年2月2日至2018年8月2日于山东省胸科医院就诊的临床怀疑TBM的患者共50例,并跟踪随访其诊疗结局。送检脑脊液标本均进行二代测序,测序所得原始序列与病原微生物数据库进行对比得到最终结果。二代测序结果以检测到结核分枝杆菌复合群唯一比对序列为阳性,未检测到唯一比对序列为阴性。以符合脑脊液结核分枝杆菌培养阳性、涂片阳性、Xpert MTB/RIF检测阳性及结核分枝杆菌核酸检测阳性等4项中至少1项即为确诊TBM患者;临床可疑TBM且抗结核治疗有效为临床诊断患者;有其他病原学依据或临床排除TBM者为非TBM患者。分析二代测序在TBM早期诊断中的敏感性和特异度。 结果确诊为TBM患者22例中Xpert MTB/RIF检测阳性13例,培养阳性6例,结核分枝杆菌核酸PCR检测阳性5例,临床诊断为TBM患者12例,非TBM患者16例。在确诊及临床诊断患者中,二代测序技术检测到结核分枝杆菌复合群系列20例,敏感性为58.8%（20/34）,特异度为100%（16/16）。在确诊患者中,二代测序的敏感性为63.6%（14/22）;在同步进行结核分枝杆菌培养、Xpert MTB/RIF检测与二代测序的50例标本中,以临床诊断为标准,3种方法的特异度均为100%（16/16）;传统方法、Xpert MTB/RIF检测及二代测序的敏感性分别为29.4%（10/34）、38.2（13/24）和58.8（20/34）,前两种检测方法与二代测序敏感性差异均有统计学意义（McNemar检验：χ² = 8.333、P = 0.013,χ² = 8.333、P = 0.065）。传统方法与二代测序联合检测的敏感性高达82.4%（28/34）。 结论二代测序技术能够较快速地检测脑脊液中的结核分枝杆菌复合群,且其敏感性和特异度均较高,可作为TBM的早期诊断指标。二代测序联合传统检测方法可提高检出率。     

瘢痕疙瘩TNF受体Ⅱ基因1573位点突变的研究

目的 探讨瘢痕疙瘩患者TNF受体Ⅱ(TNF receptor Ⅱ,TNFR-Ⅱ)基因1573位点突变的情况. 方法收集22例自愿捐献的经临床及病理确诊的瘢痕疙瘩标本,其中男6例,女16例;年龄18～53岁.设患者自身外周静脉血标本为正常对照.提取基因组DNA,PCR扩增TNFR-Ⅱ基因1573位点片段,DNA测序,将测序结果 与GeneBank比较.结果 实验提取DNA浓度均＞0.5 μg/μL,纯度(A260/A280)均＞1.5,经琼脂糖凝胶电泳检测,与所设计DNA片段大小相近,符合实验要求.13例瘢痕疙瘩标本检测示不同程度突变,突变率为59.1%;9例1663编码子发生点突变,,占总数的40.9%.与外周静脉血比较,差异均有统计学意义(P＜0.01).突变类型主要为点突变、插入、缺失,为多位点、多类型,呈多态性. 结论 TNFR-Ⅱ基因1573位点突变与瘢痕疙瘩的发生有关.     

评价全血DNA二代测序共有序列在人类免疫缺陷病毒优势准种研究中的准确性

目的评价二代测序共有序列对人类免疫缺陷病毒(HIV)优势准种分析的代表性和准确性。方法共收集29个HIV感染病例的32份全血样本,其中3个病例在随访期间发生耐药,分别采集治疗前和耐药后的两份样本。提取样本DNA,分别用二代测序和一代测序方法测定HIV pol区扩增产物序列。利用软件Sequencher(4.10.1)和Bowtie 2(v2.2.5)处理一代和二代测序数据,并利用自建分析脚本确定二代测序共有序列,利用Mega软件进行聚类分析,比较二代测序共有序列与一代测序序列的差异。结果聚类分析结果表明,90.6%(29/32)来自于同一样本的序列被聚类在同一分支上,平均可信度为95.5%;与一代测序序列结果相比,二代测序平均每个碱基准确率为99.6%,完全不一致位点中碱基转换占81.6%(155/190),数量最多的为鸟嘌呤(G):腺嘌呤(A),共74个(占39.0%、74/190)。结论二代测序共有序列与一代测序序列具有较高的一致性,二代测序共有序列可用于HIV优势准种分析,具有较好的代表性和准确性。     

Performance of Sequencing Assays in Diagnosis of Prosthetic Joint Infection: A Systematic Review and Meta-Analysis

BackgroundA prompt, accurate diagnosis of prosthetic joint infection (PJI) allows early treatment, and with identification of the causative organism, sensitive antibiotics could be applied. However, routine methods cannot identify the causative organism under certain circumstances. Gene sequencing assays have unique superiority in promptness and broad coverage of pathogens, but evidence of its accuracy is quite limited.MethodsOf 247 citations identified for screening, 12 studies with 1965 patients in total were included. The diagnostic value of sequencing assays in PJI was systematically reviewed. Subgroup analysis was conducted to explore the source of heterogeneity.ResultsPooled sensitivity was 0.81 (95% confidence interval [CI], 0.73-0.87); pooled specificity was 0.94 (95% CI, 0.91-0.97); positive likelihood ratio was 14.2 (95% CI, 8.7-23.4); negative likelihood ratio was 0.20 (95% CI, 0.14-0.29); and the area under the curve was 0.94 (95% CI, 0.18-1.00). The results of subgroup analysis revealed that antibiotics reduced the sensitivity of sequencing-based diagnosis compared with withholding antibiotics before sampling (0.71 vs 0.94). In another subgroup analysis, sequencing by synthesis (Illumina sequencing) had better specificity than other next-generation sequencing methods (0.963 vs 0.829) and specificity similar to time-consuming and laborious Sanger sequencing (0.963 vs 0.967).ConclusionSequencing assays had favorable diagnostic accuracy of PJI. When sequencing assays were applied to diagnosing PJI, an antibiotic-free interval before sampling may enhance the ability to detect the causative organism and, among next-generation sequencing methods, sequencing by synthesis seemed to have advantages over other methods in specificity.     

Congenital anomalies of the kidney and urinary tract genetics in mice and men

The most common cause of paediatric end‐stage kidney disease results from congenital anomalies of the kidney and urinary tract (CAKUT). Genetic manipulation in mice has provided insight into the developmental events that give rise to the broad spectrum of malformations associated with CAKUT. Despite the increase in the number of identified CAKUT‐causing genes, the underlying genetic cause for the majority of patients with CAKUT remains unknown. In this mini‐review, we provide an overview of the genetic causes of CAKUT based on current mouse mutant models, as well as next‐generation sequencing approaches in humans that are helping to bridge the gaps in our understanding.     

Past and future impact of next‐generation sequencing in head and neck cancer

Progress in sequencing technology is intrinsically linked to progress in understanding cancer genomics. The purpose of this review was to discuss the development from Sanger sequencing to next‐generation sequencing (NGS) technology. We highlight the technical considerations for understanding reports using NGS. We discuss the findings of studies in head and neck cancer using NGS as well as The Cancer Genome Atlas. Finally we discuss future routes for research utilizing this methodology and the potential impact of this. © 2015 Wiley Periodicals, Inc. Head Neck 38 : E2395–E2402, 2016     

Molecular Genetics of Pancreatic Neoplasms

Pancreatic neoplasms have a wide range of histologic types with distinct clinical outcomes. Recent advances in high-throughput sequencing technologies have greatly deepened our understanding of pancreatic neoplasms. Now, the exomes of major histologic types of pancreatic neoplasms have been sequenced, and their genetic landscapes have been revealed. This article reviews the molecular changes underlying pancreatic neoplasms, with a special focus on the genetic changes that characterize the histologic types of pancreatic neoplasms. Emphasis is also made on the molecular features of key genes that have the potential for therapeutic targets.     

阴道微生物多样性与女性不孕症相关性研究进展

随着高通量测序技术的发展和成熟,其在临床诊疗中的作用和应用越来越广泛,其中对阴道微生物群的研究也逐渐展开。女性阴道微生物状况受自身内分泌状态、年龄、种族、生活习惯等多种因素影响,与女性生殖健康密切相关,并直接或间接影响女性妊娠率和妊娠结局。本文对不孕症妇女阴道微生物多样性进行了总结,并对阴道微生物的组成和功能进行探讨,有利于从微生物组学的角度对不孕症做更精确全面的认识,从而对女性不孕症的临床防治工作提供新的思考和方法。     

基于脑组织转录组整合分析探索大鼠氯胺酮的麻醉机制

目的:通过观察氯胺酮麻醉对大鼠大脑皮质转录组表达的影响,探讨其可能的麻醉机制。方法:选择5~6周龄雄性SD大鼠6只,采用完全随机法分为氯胺酮组（KET组）和对照组（Ctrl组）,每组3只。分别腹腔注射麻醉剂量氯胺酮（50 mg/kg）或等体积生理盐水后30 min,提取大脑皮质进行mRNA测序（mRNA sequenc...     

Robust Genetic Diagnosis of Split Hand–Foot Malformation by Exome Sequencing

Purpose The present study aimed to evaluate the genetic diagnostic yield and accuracy of exome sequencing in Chinese patients with split hand–foot malformation(SHFM),a severe heterogeneous congenital anomaly characterized by hypodevelopment of the central ray of the hands and feet.Methods A cohort of seven families and five sporadic patients with SHFM was investigated.Genomic DNA was prepared from the peripheral blood of affected as well as unaffected individuals.Whole exome sequencing(WES)was performed to identify the pathogenic mutations.Array-based comparative genomic hybridization(aCGH),CytoScan,quantitative polymerase chain reaction(qPCR),and Sanger sequencing were performed to validate the findings of WES.WES data of an additional cohort of 24 patients with non-SHFM congenital hand anomalies were analyzed as the control.Results Pathogenic variants of TP63,c.G956A p.R319H,and c.T602A:p.L201H,were identified in two families by WES.In the remaining patients,copy number analysis of the WES data by XHMM software identified pathogenic 10q 24 duplication in five individuals from three families,which was further validated via CytoScan and qPCR;however,WES could not detect duplication in 10q24 in an additional cohort of 24 individuals with non-SHFM congenital hand anomaly.Importantly,qPCR analysis of the 10q24 region copy number revealed a definite consistency with WES data in all individuals.Genotype–phenotype analysis did not present any unique feature that could differentiate between the families with TP63 mutation and 10q24 duplication.Conclusions Our study demonstrated that WES is an accurate and sensitive method to detect the pathogenic 10q24 duplication.Collectively,with TP63 mutation,a single WES testing could yield a diagnosis rate of about 40%(5/12)for the SHFM patients,at least in our cohort.As the genotype–phenotype correlation remains unclear,WES could be used as a cost-effective method for the genetic diagnosis of SHFM.     

Genetic diversity of equine herpesvirus 1 isolated from neurological,abortigenic and respiratory disease outbreaks

Equine herpesvirus 1 (EHV‐1) causes respiratory disease, abortion, neonatal death and neurological disease in equines and is endemic in most countries. The viral factors that influence EHV‐1 disease severity are poorly understood, and this has hampered vaccine development. However, the N752D substitution in the viral DNA polymerase catalytic subunit has been shown statistically to be associated with neurological disease. This has given rise to the term “neuropathic strain,” even though strains lacking the polymorphism have been recovered from cases of neurological disease. To broaden understanding of EHV‐1 diversity in the field, 78 EHV‐1 strains isolated over a period of 35 years were sequenced. The great majority of isolates originated from the United Kingdom and included in the collection were low passage isolates from respiratory, abortigenic and neurological outbreaks. Phylogenetic analysis of regions spanning 80% of the genome showed that up to 13 viral clades have been circulating in the United Kingdom and that most of these are continuing to circulate. Abortion isolates grouped into nine clades, and neurological isolates grouped into five. Most neurological isolates had the N752D substitution, whereas most abortion isolates did not, although three of the neurological isolates from linked outbreaks had a different polymorphism. Finally, bioinformatic analysis suggested that recombination has occurred between EHV‐1 clades, between EHV‐1 and equine herpesvirus 4, and between EHV‐1 and equine herpesvirus 8.     

e抗原阴性的慢性乙型肝炎患者病毒基因变异与临床转归

目的研究HBeAg阴性的慢性乙型肝炎病毒的基因变化及其与临床转归的关系。方法应用基因测序的方法检测HBeAg阴性的慢性乙型肝炎、肝硬化患者乙型肝炎病毒1762、1764、pre-18963个位点的基因突变,分析肝炎、肝硬化的发生与各位点变异的关系。结果135例HBeAg阴性的慢性乙型肝炎患者中发生1762位点变异者9例,pre-1896位点变异者36例,1762、1764位点联合变异者27例,1762、1764、pre-1896位点联合变异者63例。45例肝硬化患者中发生pre-1896位点变异者27例,1762、1764位点联合变异者9例,1762、1764、pre-1896位点联合变异者共9例。结论山东地区HBeAg阴性的慢性乙型肝炎患者乙型肝炎病毒以BCP1762、1764、pre-1896位点联合变异为主,其中pre-1896位点的变异对于肝硬化的发生有重要意义。     

Molecular Testing of Colorectal Cancer in the Modern Era: What Are We Doing and Why?

A plethora of tests are routinely ordered and interpreted by pathologists to assist the management of colorectal cancer patients. Many of these tests are immunohistochemistry assays using antibodies against prognostically relevant proteins, some of which predict therapeutic response. This review focuses on tissue DNA-based tests. It presents novel methodologies for assessing well-established biomarkers, updates the expanding spectrum of genetic alterations that are associated with resistance to inhibition of epidermal growth factor receptor signaling, and briefly discusses emerging actionable alterations that may translate into new therapeutic options for colorectal cancer patients. The utility of next-generation sequencing is emphasized.