基因变异检测技术在恶性肿瘤精准医疗中的应用

恶性肿瘤是严重威胁人类健康和社会发展的重大疾病,寻找科学的方法对其进行诊断、治疗和评估已成为近年来全球最重要的公共卫生问题之一。随着医疗行业的不断发展,传统的肿瘤筛查、防治和预后评估手段在近几年发展迅速。但是考虑到肿瘤异质性和患者个体化的特征,精准化的疾病筛查、诊断、治疗等医疗模式将成为未来医疗发展的趋势。肿瘤领域的基因变异检测是精准医疗中重要的组成部分,其应用可涉及早期筛查、复发监测、靶向用药指导、疗效及预后评价等众多环节,但在临床运用中仍存在诸多局限,有待更深入的研究来推动肿瘤精准医疗的发展。本文就近年来基因变异检测技术在恶性肿瘤精准医疗中的发展历程以及应用进展作一介绍。

脊髓性肌萎缩症并发希特林蛋白缺乏症患儿及家系经基因变异检测和生物信息学结果分析

目的研究脊髓性肌萎缩症并发希特林蛋白缺乏症患儿及家系经基因变异检测和生物信息学结果。方法将医院收治的1例脊髓性肌萎缩症并发希特林蛋白缺乏症患儿纳入研究。采集患儿及其双亲的外周血,进行全基因组脱氧核糖核酸(Deoxyribonucleic acid,DNA)的提取,同时进行全外显子组测序。在明确先证者的致病基因型后,采用桑格(Sanger)测序法完成验证,并对患儿双亲的变异情况予以检测。采用多重连接探针扩增杂交技术(Multiplex ligation-dependent probe amplification,MLPA)完成患儿运动神经元存活基因(survival motor neuron,SMN)基因的检测。结果 Sanger测序验证结果显示:线粒体聚合酶γ(mitochondrial DNA polymeraseγ,POLG)基因和复合杂合变异相符,且变异c·948G>A(p·K316K)源自父亲,c·2693T>C(p·I898T)源自母亲;其中SCL25A13基因变异位点c·1737G>A(p·W579X)和二代测序结果存在明显差异,属于纯合变异,且未在患儿双亲的相同位点发现变异。IVSI6ins3kb变异检测结果显示SLC25A13基因存在3 kb的插入,且主要来源于患儿父亲。由于患儿体格检查结果显示为颈部柔软,因此对患儿及其双亲同时开展脊髓性肌萎缩检测,结果发现患儿SMN1基因存在第7、8外显子的纯合缺失,患儿双亲在同一外显子均表现为杂合缺失,且与孟德尔遗传规律相符,提示患儿存在脊髓性肌萎缩症。结论 SLC25A13基因变异与脊髓性肌萎缩症患儿并发希特林蛋白缺乏症密切相关,且POLG基因变异c·948G>A以及c·2693G>A复合杂合变异可能引起患儿发生线粒体DNA缺失综合征4A型,值得临床重点关注。

基因拷贝数变异检测在胚胎流产物遗传学检测中的应用

目的探讨基于高通量测序技术的基因拷贝数变异检测(NGS-CNVs)在胚胎流产物遗传学诊断中的价值。方法选取2015年6月至2016年2月在唐山市妇幼保健院产前诊断中心确诊为稽留流产,流产原因不明,要求遗传学检测的样本105例。在知情同意的前提下行绒毛细胞培养染色体核型分析检查和NGS-CNVs检测。分析两种诊断方法的检测结果。结果 (1)NGS-CNVs成功检测101例(96.0%),失败4例(4.0%)。诊断染色体异常53例(50.5%),其中包括数目异常36例(34.3%)、结构异常(微缺失或重复)17例(16.2%)、48例未见异常(45.7%)。(2)染色体核型分析成功检测89例(84.8%),失败16例(15.2%)。诊断染色体异常38例(36.2%),其中包括数目异常32例(30.5%)、结构异常(平衡易位及多态性)6例(5.7%)、51例未见异常(48.6%)。(3)两种技术相比,NGS-CNVs检测成功率明显高于绒毛细胞培养染色体核型分析,差异具有统计学意义(P=0.005);NGS-CNVs检测出更多的染色体结构异常,差异具有统计学意义(P=0.015)。NGS-CNVs检测染色体数目异常结果与绒毛细胞培养染色体核型分析结果一致。结论 NGS-CNVs检测用于胚胎流产物遗传学检测切实可行。NGS-CNVs可以快速准确的检测出染色体数目异常,同时能够检测出常规染色体核型分析无法发现的大量微缺失或重复,其临床意义有待进一步证实。

数字PCR技术在动物疫苗研究中的应用前景

数字PCR(digital PCR,dPCR)技术是基于“有限稀释”和“泊松分布”原理迅速发展的第三代PCR技术,无需标准品即可实现样品的绝对定量,灵敏度高、操作简单,在疫病检测、转基因鉴定、食品检测等方面得到快速发展,而在动物疫苗研究领域也被越来越多用于质控、筛查和定量。本文从动物疫苗研究的角度,阐述了数字PCR在动物疫苗基因变异检测、基因定量、效价测定、疫苗效果评价等方面的发展潜力。该技术将有助于解决动物疫苗研究过程中的病毒生长曲线和疫苗效价测定以及基因变异分析等重点难点问题,未来将成为疫苗研发的有力工具。

Genetic Variation Analysis of 3D Gene and Molecular Detection of Porcine Kobuvirus in 2013-2014

[Objective] This study aimed to investigate the prevalence and variation of porcine kobuvirus （PKV） in suckling piglets in China. [Method] In 2013-2014, 224 feces samples from suckling piglets with diarrhea in 27 pig farms of five provinces in China were collected to detect 3D genes of PKV with RT-PCR method; the sequences and genetic variation of 29 PKV 3D genes were analyzed. [Result] Total positive rate of PKV in feces samples from suckling piglets with diarrhea was 65.18% （146/224）; total positive rate of PKV in pig farms was 85,2% （23/27）; nucleotide sequences and the deduced amino acid sequences of 29 PKV 3D genes shared 87.0%-100% and 92.7%-100% homologies with six PKV-related 3D sequences, respectively. [Conclusion] PKV infection is prevalent in suckling piglets in China; PKV 3D genes exhibit high diversity.

Importance of early diagnosis of pancreaticobiliary maljunction without biliary dilatation

AIM:To clarify the strategy for early diagnosis of pancreaticobiliary maljunction(PBM) without biliary dilatation and to pathologically examine gallbladder before cancer develops.METHODS:The anatomy of the union of the pancreatic and bile ducts was assessed by using endoscopic retrograde cholangiopancreatography(ERCP).Patients with a long common channel in which communication between the pancreatic and bile ducts was maintained even during sphincter contraction were diagnosed as having PBM.Of these,patients in which the maximal diameter of the bile duct was less than 10 mm were diagnosed with PBM without biliary dilatation.The process of diagnosing 54 patients with PBM without biliary dilatation was retrospectively investigated.Histopathological analysis of resected gallbladder specimens from 8 patients with PBM without biliary dilatation or cancer was conducted.RESULTS:Thirty-six PBM patients without biliary dilatation were diagnosed with gallbladder cancer after showing clinical symptoms such as abdominal or back pain(n = 16) or jaundice(n = 12).Radical surgery for gallbladder cancer was only possible in 11 patients(31%) and only 4 patients(11%) survived for 5 years.Eight patients were suspected as having PBM without biliary dilatation from the finding of gallbladder wall thickening on ultrasound and the diagnosis was confirmed by ERCP and/or magnetic resonance cholangiopancreatography(MRCP).The median age of these 8 patients was younger by a decade than PBM patients with gallbladder cancer.All 8 patients underwent prophylactic cholecystectomy and bile duct cancer has not occurred.Wall thickness and mucosal height of the 8 resected gallbladders were significantly greater than controls,and hyperplastic changes,hypertrophic muscular layer,subserosal fibrosis,and adenomyomatosis were detected in 7(88%),5(63%),7(88%) and 5(63%) patients,respectively.Ki-67 labeling index was high and K-ras mutation was detected in 3 of 6 patients.CONCLUSION:To detect PBM without biliary dilatation before onset of gallbladder cancer,we should perform MRCP for individuals showing increased gallbladder wall thickness on ultrasound.

A consensus linkage map of common carp (Cyprinus carpio L.) to compare the distribution and variation of QTLs associated with growth traits

The ability to detect and identify quantitative trait loci (QTLs) in a single population is often limited. Analyzing multiple populations in QTL analysis improves the power of detecting QTLs and provides a better understanding of their functional allelic variation and distribution. In this study, a consensus map of the common carp was constructed, based on four populations, to compare the distribution and variation of QTLs. The consensus map spans 2371.6 cM across the 42 linkage groups and comprises 257 microsatellites and 421 SNPs, with a mean marker interval of 3.7 cM/marker. Sixty-seven QTLs affecting four growth traits from the four populations were mapped to the consensus map. Only one QTL was common to three populations, and nine QTLs were detected in two populations. However, no QTL was common to all four populations. The results of the QTL comparison suggest that the QTLs are responsible for the phenotypic variability observed for these traits in a broad array of common carp germplasms. The study also reveals the different genetic performances between major and minor genes in different populations.

Impact of the next-generation sequencing data depth on various biological result inferences

Next-generation sequencing(NGS) technologies have revolutionized the field of genomics and provided unprecedented opportunities for high-throughput analysis at the levels of genomics,transcriptomics and epigenetics.However,the cost of NGS is still prohibitive for many laboratories.It is imperative to address the trade-off between the sequencing depth and cost.In this review,we will discuss the effects of sequencing depth on the detection of genes,quantification of gene expression and discovering of gene structural variants.This will provide readers information on choosing appropriate sequencing depth that best meet the needs of their particular project.

97例产前诊断染色体结构异常的遗传学分析

目的探讨胎儿染色体结构异常核型发生的类型、频率及遗传率,分析其产前诊断指征及妊娠结局,为优生优育提供科学依据。方法对8600例具有产前诊断指征的孕妇,行介入性产前诊断,羊水或脐血细胞培养,染色体核型分析,诊断结果为胎儿染色体结构异常的,胎儿父母行外周血染色体核型分析。结果胎儿染色体结构异常共97例,包含平衡易位30例,倒位24例,衍生11例,罗伯逊易位11例,缺失9例,标记染色体7例,未知来源附加片段3例,插入2例。97例病例中,遗传性结构异常64例,新发结构异常33例。其主要产前诊断指征唐氏筛查高风险、胎儿超声异常、高龄及夫妻染色体异常携带。将97例病例分成遗传组和新发组,将两组的染色体异常类型分布进行比较,P<0.05,差异有统计学意义;97例病例按照性别分为男女两组,染色体异常类型分布相比,P>0.05,差异无统计学意义。结论胎儿染色体结构异常类型多种多样,导致胎儿畸形的风险与其密切相关,遗传咨询过程中应该结合超声检查、染色体微阵列序列分析或者高通量全基因组拷贝数变异检测等的结果综合分析,给予妊娠指导。