临床基因检测报告规范与基因检测行业共识探讨CSCD

二代测序技术（next generation sequencing,NGS）在临床的应用为广泛开展遗传病的基因检测、病因诊断、治疗及预防奠定了基础,但同时也伴随着一系列的问题,其中很重要的一个环节就是基因检测报告缺乏统一或基本的标准。首届“临床基因检测标准与规范专题研讨会”于2017年10月28日在深圳召开,来自全国138家机构的250多位遗传学专家、临床专家及第三方检测机构的代表共同探讨了遗传病基因检测报告的标准和规范问题。本文根据此次研讨会专家的意见和建议,就遗传病基因检测报告的原则、规范以及基因检测行业的发展进行了讨论,并发布了临床基因检测报告规范共识,以促进检测报告的规范化和标准化,推进我国基因检测行业的健康有序地发展。     

遗传病二代测序临床检测全流程规范化共识探讨(3)——数据分析流程

基因组测序数据的生物信息学分析以及变异的临床相关性解读是基于二代测序的遗传病诊断全流程的核心内容。本文探讨了生物信息学分析、数据存储和数据解读环节的流程、功能、主要参数指标及建议等,经行业内临床医师、检测实验室等相关专家讨论,对符合孟德尔遗传规律的胚系突变的遗传病规范化检测达成了共识,以期为提髙基于二代测序的遗传病诊断的同质性和可靠性提供指导。     

福尔马林固定石蜡包埋标本二代测序终文库去除引物二聚体的方法

二代测序(next generation sequencing,NGS)技术能够同时对上百万乃至数十亿个DNA分子进行测序,相较于传统测序技术,其速度较快、一次可检测大量靶基因,因而广泛应用于染色体非整倍体无创产前筛查、肿瘤靶向治疗基因突变检测、遗传性肿瘤检测、病原微生物及宏基因组检测等领域。影响NGS结果的限制性因素包括:平台类别、靶区域富集方法、文库构建及扩增效率、测序数据量、生物信息学分析流程等。NGS流程主要包括3个部分:文库制备、测序和数据分析,建库是NGS实验第一步,终文库质量的好坏直接影响后续测序仪上机实验及数据分析。     

结合MLPA技术与高通量测序技术对DMD患者进行基因检测

目的探讨多重连接探针扩增技术(MLPA)与高通量测序技术(NGS)对假性肥大型肌营养不良(DMD)患者基因检测的临床应用价值,为进一步创建经济可靠的、高效的DMD诊断技术奠定技术平台。方法使用MLPA技术对66名假性肥大型肌营养不良患者进行DMD基因的缺失或重复检测,然后应用NGS技术对未检测出DMD基因缺失或重复的患者进行DMD基因测序。结果 49例为DMD基因外显子缺失,3例为DMD基因外显子重复,14例为DMD基因微小突变。结论结合MLPA技术与高通量测序技术可高效检测DMD基因缺失、重复及微小突变,更好地为DMD患者做出临床诊断。     

一例假肥大型肌营养不良家系DMD基因分析及产前诊断

目的对1例临床症状疑似假肥大型肌营养不良(DMD/BMD)的患儿进行基因诊断,并进行携带者筛查及产前诊断。方法使用多重链接探针扩增技术(MLPA)进行DMD基因79个外显子缺失或重复检测,同时应用高通量测序技术(NGS)对神经肌肉相关基因进行靶向测序,并通过Sanger测序对先证者及家系成员进行验证。结果 MLPA检测未发现患儿DMD基因外显子发生缺失或者重复突变,NGS测序发现DMD基因第39号外显子c.5544-5548delGATAA半合子突变。Sanger测序验证发现患儿母亲、弟弟(胎儿)均存在c.5544-5548delGATAA突变。结论本研究发现一种尚未病例报道的新发突变,该缺失突变是导致患儿发病的原因,MLPA结合NGS技术可有效提高疾病的诊断效率,为产前诊断提供准确信息。     

中国人重要遗传病临床实践指南专辑导读

随着新一代测序技术(next-generation sequencing,NGS)在人类遗传病研究和临床应用中的推广,遗传检测在遗传性疾病临床诊断上的作用和需求更为突出,遗传变异的信息解读也面临更多的挑战。临床上如何针对疾病对象选择合理的检测方法,如何正确解读实验室发出的基因型检测报告,并依据遗传检测结果与临床表型的关系准确诊断遗传病,是临床上广受关注的核心问题,也是目前在我国医院体系中还没有设立独立医学遗传学专科的背景下,本学科所面临的重要任务。     

下一代测序技术在弥漫大B细胞淋巴瘤基因重排检测中的应用

目的探究下一代测序技术(next generation sequencing, NGS)在弥漫大B细胞淋巴瘤(diffuse large B cell lymphoma, DLBCL)辅助诊断中的应用价值。方法收集陕西省人民医院病理科存档的20例组织蜡块,其中15例诊断为DLBCL,5例诊断为淋巴组织反应性增生(reactive lymphoid hyperplasia, RLH)。采用天根公司石蜡包埋组织基因组DNA提取试剂盒进行DNA提取,安捷伦4200评价DNA质量。采用LymphoTrack IGH FR2 Assay和LymphoTrack Dx TCRG Assay进行组织克隆性测定。结果 5例RLH均为多克隆性,11例DLBCL出现NGS IgH FR2单克隆重排(阳性率73.3%,11/15),另有4例未检测出单克隆性重排(2、5、8、15号)。15例中有3例出现TCRG重排性扩增(6、7、14号)。经PCR凝胶电泳法进行验证:4例NGS检测的IgH FR2多克隆性病例均检测到IgH单克隆性基因重排;3例NGS检测到TCRG单克隆性重排病例中,PCR凝胶电泳法只检测到1例呈TCRG克隆性重排。结论高通量测序方法特异性较高,但由于其检测过程复杂,影响因素较多,检测方法的敏感性受到一定影响。虽然该方法可以准确得到基因重排序列,但由于其检测成本较高,对技术平台和人员要求更高,其在临床上的应用尚需时日。     

二代测序技术检测伴神经内分泌分化乳腺癌的基因突变

目的探讨高通量测序技术在伴神经内分泌分化乳腺癌(breast carcinomas with neuroendocrine differentiation,BCND)分子遗传学中的应用,提高对该肿瘤生物学特性的认识水平。方法使用二代测序(next-generation sequencing,NGS)技术对20例BCND的石蜡包埋组织进行基因突变检测。使用Sanger测序对NGS检测的阳性结果进行验证,并通过qPCR检测对肿瘤中KRAS、PIK3CA、EGFR、BRAF和NRAS基因突变进行对比分析。结果 NGS和qPCR检测发现2例BCND分别存在PIK3CA、H1047R和E542K突变,其中H1047R突变也通过Sanger测序证实。2例PIK3CA突变肿瘤HER-2均阳性。结论高通量NGS技术可用于BCND石蜡标本的基因突变检测,BCND具有不同于非特殊型浸润性乳腺癌的基因突变谱,其PIK3CA突变可能与HER-2阳性相关。     

自闭症的基因检测研究进展

自闭症是一种多基因遗传、神经系统发育失调引起的广泛性发育障碍,其发病机制不祥,且具有高度临床异质性。自闭症的实验室检测从传统的核型分析到目前的高通量测序技术,同时不断的发现许多相关的致病基因,使得自闭症的检出率有所增高。DNA测序技术的不断发展,为自闭症的潜在遗传病因鉴定奠定了良好的基础,且对未来自闭症的临床诊断及个性化治疗至关重要。     

基因芯片联合二代测序技术在产前耳聋基因筛查中的应用价值

目的探究基因芯片(GC)联合二代测序技术(NGS)检测在产前耳聋基因筛查中的应用价值。方法选取2016年1月到2018年1月在我院产前筛查中心行孕妇外周血耳聋基因芯片及二代测序技术检测的孕妇及其新生儿资料。使用自身对照法,回顾性分析GC、NGS筛查和两者联合使用三种方法之间的准确率、灵敏度以及特异度等指标。结果全部病例420例,有效随访者360例,最终确诊为携带耳聋突变基因孕妇为18例,发病率为5.0%。其中GJB2基因突变11例(包括6例235delC和5例299-300delAT位点突变),携带率为3.1%(11/360);GJB3基因突变3例(包括2例538CT和1例1555AG位点突变),携带率为0.8%(3/360);SLC26A4(PDS基因突变4例(均为IVS7-2AG位点突变),携带率为1.1%(4/360)。结论 GC联合NGS法应用于产前耳聋基因的筛查,可提高其诊断准确率和特异度,具有较高的临床应用价值,值得进一步推广使用。     

Next-generation sequencing-based genome diagnostics across clinical genetics centers: implementation choices and their effects

Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found ‘no causal variant'', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of ‘greediness'' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care.     

Next-generation sequencing for inborn errors of immunity

Inborn errors of immunity (IEIs) include several hundred gene defects affecting various components of the immune system. As with other constitutional disorders, next-generation sequencing (NGS) is a powerful tool for the diagnosis of these diseases. While NGS can provide molecular confirmation of disease in a patient with a suspected or classic phenotype, it can also identify new molecular defects of the immune system, expand gene-disease phenotypes, clarify mechanism of disease, pattern of inheritance or identify new gene-disease associations. Multiple clinical specialties are involved in the diagnosis and management of patients with IEI, and most have no formal genetic training or expertise. To effectively utilize NGS tools and data in clinical practice, it is relevant and pragmatic to obtain a modicum of knowledge about genetic terminology, the variety of platforms and tools available for high-throughput genomic analysis, the interpretation and implementation of such data in clinical practice. There is considerable variability not only in the technologies and analytical tools used for NGS but in the bioinformatics approach to variant identification and interpretation. The ability to provide a molecular basis for disease has the potential to alter therapeutic management and longer-term treatment of the disease, including developing personalized approaches with molecularly targeted therapies. This review is intended for the clinical specialist or diagnostic immunologist who works in the area of inborn errors of immunity, and provides an overview of the need for genetic testing in these patients (the “why” aspect), the various technologies and analytical approaches, bioinformatics tools, resources, and challenges (the “how” aspect), and the clinical evidence for identifying which patients might be best served by such testing (the “when” aspect).     

Best Practice Guidelines for the Use of Next‐Generation Sequencing Applications in Genome Diagnostics: A National Collaborative Study of Dutch Genome Diagnostic Laboratories

Next‐generation sequencing (NGS) methods are being adopted by genome diagnostics laboratories worldwide. However, implementing NGS‐based tests according to diagnostic standards is a challenge for individual laboratories. To facilitate the implementation of NGS in Dutch laboratories, the Dutch Society for Clinical Genetic Laboratory Diagnostics (VKGL) set up a working group in 2012. The results of their discussions are presented here. We provide best practice guidelines and criteria for implementing and validating NGS applications in a clinical setting. We introduce the concept of “diagnostic yield” as the main performance characteristic for evaluating diagnostic tests. We recommend that the laboratory procedures, including the tested genes, should be recorded in a publicly available document describing the complete “diagnostic routing.” We also propose that laboratories should use a list of “core disease genes” for specific genetic diseases. This core list contains the essential genes for each disease, and they should all be included in a diagnostic test to establish a reliable and accurate molecular diagnosis. The guidelines will ensure a clear and standardized quality of care provided by genetic diagnostic laboratories. The best practice guidelines and criteria that are presented here were adopted by the VKGL in January 2013.     

Economic evaluation of next-generation sequencing techniques in diagnosis of genetic disorders: A systematic review

In recent years, massively parallel sequencing or next generation sequencing (NGS) has considerably changed both the research and diagnostic fields, and rapid developments have led to the combination of NGS techniques in clinical practice, ease of analysis, and detection of genetic mutations. This article aimed at reviewing the economic evaluation studies of the NGS techniques in the diagnosis of genetic diseases. In this systematic review, scientific databases (PubMed, EMBASE, Web of Science, Cochrane, Scopus, and CEA registry) were searched from 2005 to 2022 to identify the related literature on the economic evaluation of NGS techniques in the diagnosis of genetic diseases. Full-text reviews and data extraction were all performed by two independent researchers. The quality of all the articles included in this study was evaluated using the Checklist of Quality of Health Economic Studies (QHES). Out of 20 521 screened abstracts, 36 studies met the inclusion criteria. The mean score of the QHES checklist for the studies was 0.78 (high quality). Seventeen studies were conducted based on modeling. Cost-effectiveness analysis, cost-utility analysis, and cost-minimization analysis were done in 26 studies, 13 studies, and 1 study, respectively. Based on the available evidence and findings, exome sequencing, which is one of the NGS techniques, could have the potential to be used as a cost-effective genomic test to diagnose children with suspected genetic diseases. The results of the present study support the cost-effectiveness of exome sequencing in diagnosing suspected genetic disorders. However, the use of exome sequencing as a first- or second-line diagnostic test is still controversial. Most studies have been conducted in high-income countries, and research on the cost-effectiveness of NGS methods is recommended in low- and middle-income countries.     

遗传病二代测序临床检测全流程规范化共识探讨(1)——遗传检测前流程

遗传检测前的准备工作是基因检测的基础和出发点,其流程包括临床信息收集、检测方案拟定、检测前遗传咨询以及知情同意书和检测委托书的填写等。临床若能有效识别出遗传性疾病,可极大提高二代测序的诊断率,从而降低医疗成本,提高临床诊疗的功效。二代测序结果的分析在很大程度上依赖于对基因型-表型相关性的了解,全面准确地采集和评估临床表型,并用统一的标准术语来描述和记录尤为重要。不同类型的遗传病或突变种类需要用特定的检测手段方能事半功倍。检测前遗传咨询能够帮助患者及其亲属了解相关基因检测的意义并协助制定个体化的检测策略,为后续的跟踪随访奠定基础。     

Genetic counselors' (GC) knowledge,awareness, understanding of clinical next‐generation sequencing (NGS) genomic testing

Genomic tests are increasingly complex, less expensive, and more widely available with the advent of next‐generation sequencing (NGS). We assessed knowledge and perceptions among genetic counselors pertaining to NGS genomic testing via an online survey. Associations between selected characteristics and perceptions were examined. Recent education on NGS testing was common, but practical experience limited. Perceived understanding of clinical NGS was modest, specifically concerning tumor testing. Greater perceived understanding of clinical NGS testing correlated with more time spent in cancer‐related counseling, exposure to NGS testing, and NGS‐focused education. Substantial disagreement about the role of counseling for tumor‐based testing was seen. Finally, a majority of counselors agreed with the need for more education about clinical NGS testing, supporting this approach to optimizing implementation.     

Challenges in the application of NGS in the clinical laboratory

Next-generation sequencing (NGS), also known as massively parallel sequencing, has revolutionized genomic research. The current advances in NGS technology make it possible to provide high resolution, high throughput HLA typing in clinical laboratories. The focus of this review is on the recent development and implementation of NGS in clinical laboratories. Here, we examine the critical role of NGS technologies in clinical immunology for HLA genotyping. Two major NGS platforms (Illumina and Ion Torrent) are characterized including NGS library preparation, data analysis, and validation. Challenges of NGS implementation in the clinical laboratory are also discussed, including sequencing error rate, bioinformatics, result interpretation, analytic sensitivity, as well as large data storage. This review aims to promote the broader applications of NGS technology in clinical laboratories and advocate for the novel applications of NGS to drive future research.     

Detection of structural variation using target captured next-generation sequencing data for genetic diagnostic testing

PurposeStructural variation (SV) is associated with inherited diseases. Next-generation sequencing (NGS) is an efficient method for SV detection because of its high-throughput, low cost, and base-pair resolution. However, due to lack of standard NGS protocols and a limited number of clinical samples with pathogenic SVs, comprehensive standards for SV detection, interpretation, and reporting are to be established.MethodsWe performed SV assessment on 60,000 clinical samples tested with hereditary cancer NGS panels spanning 48 genes. To evaluate NGS results, NGS and orthogonal methods were used separately in a blinded fashion for SV detection in all samples.ResultsA total of 1,037 SVs in coding sequence (CDS) or untranslated regions (UTRs) and 30,847 SVs in introns were detected and validated. Across all variant types, NGS shows 100% sensitivity and 99.9% specificity. Overall, 64% of CDS/UTR SVs were classified as pathogenic/likely pathogenic, and five deletions/duplications were reclassified as pathogenic using breakpoint information from NGS.ConclusionThe SVs presented here can be used as a valuable resource for clinical research and diagnostics. The data illustrate NGS as a powerful tool for SV detection. Application of NGS and confirmation technologies in genetic testing ensures delivering accurate and reliable results for diagnosis and patient care.     

The Challenge for the Next Generation of Medical Geneticists

Next‐generation sequencing (NGS) has allowed a tremendous progress in the characterization of the molecular bases of genetic diseases and the last annual American Society of Human Genetics meeting has highlighted the implementation of whole exome sequencing in medical genetics. Several investigators suggest that it should be medically relevant for each individual to have the exome sequenced. These perspectives do not take into account the complexity of genetic variation interpretation and genetic determinism of human diseases: an important limiting step of targeted analyses of gene(s) involved in Mendelian diseases is already the interpretation of variants of unknown significance; most of the 20,000 single nucleotide variations present in each exome, even those having a very low allelic frequency, are not deleterious; the genetic determinism of the majority of human diseases involves either a combination of numerous genetic variations, each conferring a slightly increased risk, or rare genetic variations with a strong effect, but the demonstration of their involvement in diseases is particularly challenging. The challenge for the next generation of medical geneticists will be to integrate the technological power of NGS technologies, the complexity of genome interpretation, the importance of phenotyping before genotyping, and the guidelines of medical genetics raised in the pre‐NGS era.     

Clinical applications of high-throughput genetic diagnosis in inherited retinal dystrophies: Present challenges and future directions

The advent of next generation sequencing (NGS) techniques has greatly simplified the molecular diagnosis and gene identification in very rare and highly heterogeneous Mendelian disorders. Over the last two years, these approaches, especially whole exome sequencing (WES), alone or combined with homozygosity mapping and linkage analysis, have proved to be successful in the identification of more than 25 new causative retinal dystrophy genes. NGS-approaches have also identified a wealth of new mutations in previously reported genes and have provided more comprehensive information concerning the landscape of genotype-phenotype correlations and the genetic complexity/diversity of human control populations. Although whole genome sequencing is far more informative than WES, the functional meaning of the genetic variants identified by the latter can be more easily interpreted, and final diagnosis of inherited retinal dystrophies is extremely successful, reaching 80%, particularly for recessive cases. Even considering the present limitations of WES, the reductions in costs and time, the continual technical improvements, the implementation of refined bioinformatic tools and the unbiased comprehensive genetic information it provides, make WES a very promising diagnostic tool for routine clinical and genetic diagnosis in the future.