A Post‐Hoc Comparison of the Utility of Sanger Sequencing and Exome Sequencing for the Diagnosis of Heterogeneous Diseases

The advent of massive parallel sequencing is rapidly changing the strategies employed for the genetic diagnosis and research of rare diseases that involve a large number of genes. So far it is not clear whether these approaches perform significantly better than conventional single gene testing as requested by clinicians. The current yield of this traditional diagnostic approach depends on a complex of factors that include gene‐specific phenotype traits, and the relative frequency of the involvement of specific genes. To gauge the impact of the paradigm shift that is occurring in molecular diagnostics, we assessed traditional Sanger‐based sequencing (in 2011) and exome sequencing followed by targeted bioinformatics analysis (in 2012) for five different conditions that are highly heterogeneous, and for which our center provides molecular diagnosis. We find that exome sequencing has a much higher diagnostic yield than Sanger sequencing for deafness, blindness, mitochondrial disease, and movement disorders. For microsatellite‐stable colorectal cancer, this was low under both strategies. Even if all genes that could have been ordered by physicians had been tested, the larger number of genes captured by the exome would still have led to a clearly superior diagnostic yield at a fraction of the cost.     

Contact investigations for outbreaks of Mycobacterium tuberculosis: advances through whole genome sequencing

The control of tuberculosis depends on the identification and treatment of infectious patients and their contacts, who are currently identified through a combined approach of genotyping and epidemiological investigation. However, epidemiological data are often challenging to obtain, and genotyping data are difficult to interpret without them. Whole genome sequencing (WGS) technology is increasingly affordable, and offers the prospect of identifying plausible transmission events between patients without prior recourse to epidemiological data. We discuss the current approaches to tuberculosis control, and how WGS might advance public health efforts in the future.     

Identification and pathogenicity analysis of a novel non-tuberculous mycobacterium clinical isolate with nine-antibiotic resistance

With mycobacteriosis increasing, the study of non-tuberculous mycobacteria is imperative for clinical therapy and management. Nontuberculous mycobacteria are naturally resistant to most anti-tuberculosis drugs. Accordingly, it is important to decipher the biology of the novel non-tuberculous mycobacteria through complete genomic analysis of novel pathogenic mycobacteria. We describe Mycobacterium sinense JDM601, a novel, slow-growing mycobacterium of the Mycobacterium terrae complex resistant to nine antibiotics, by clinical presentation, cultural and biochemical characteristics, minimal inhibitory concentrations, and genome-sequencing analysis. JDM601 is closest to Mycobacterium nonchromogenicum according to mycolic acid composition, but closest to Mycobacterium algericum sp. nov according to 16S rDNA. JDM601 is resistant to isoniazid, streptomycin, rifampin, euteropas, protionamide, capromycin, ciprofloxacin, amikacin and levofloxacin but not ethambutol. The clinical information, mycolic acid composition, and virulence genes indicate that JDM601 is an opportunistic pathogen.     

药物重定位候选药物筛选路径

相对传统新药研发模式,药物重定位策略发现药物新用途具有显著的成本效益优势,能加快药物上市步伐,满足恶性肿瘤、罕见病、个性化医疗等特定领域药物临床用药需求,因而被各界关注。本文主要介绍了药物重定位的一般流程与候选药物筛选路径,如从非理性设计方法向基于相似性、基于结构虚拟筛选、推理与机器学习等理性设计方法发现重定位药物的系统性转变。     

植入前胚胎非整倍体筛查的临床应用研究进展

研究证明基于囊胚期活检和全基因组分析技术的"第二代植入前遗传学筛查(PGS)"可以显著提高IVF治疗的临床结局。目前认为囊胚期活检是PGS活检的最佳时间,也是第二代PGS采用的活检方法。具体为通过单细胞全基因组扩增技术使样本达到可检测的量,再利用微阵列技术、高通量测序技术完成全基因组检测。除了技术方面的改进,第二代PGS还应在适用人群的选择方面综合考虑不孕者胚胎非整倍体的发生率和可用于移植的囊胚数2个方面,PGS的最适宜人群应该是排除了内膜因素的影响后其染色体异常发生率较高、同时可移植胚胎数较多者。     

A simplified Sanger sequencing method for full genome sequencing of multiple subtypes of human influenza A viruses

BackgroundFull genome sequencing of influenza A viruses (IAV), including those that arise from annual influenza epidemics, is undertaken to determine if reassorting has occurred or if other pathogenic traits are present. Traditionally IAV sequencing has been biased toward the major surface glycoproteins haemagglutinin and neuraminidase, while the internal genes are often ignored. Despite the development of next generation sequencing (NGS), many laboratories are still reliant on conventional Sanger sequencing to sequence IAV.ObjectivesTo develop a minimal and robust set of primers for Sanger sequencing of the full genome of IAV currently circulating in humans.Study designA set of 13 primer pairs was designed that enabled amplification of the six internal genes of multiple human IAV subtypes including the recent avian influenza A(H7N9) virus from China. Specific primers were designed to amplify the HA and NA genes of each IAV subtype of interest. Each of the primers also incorporated a binding site at its 5′-end for either a forward or reverse M13 primer, such that only two M13 primers were required for all subsequent sequencing reactions.ResultsThis minimal set of primers was suitable for sequencing the six internal genes of all currently circulating human seasonal influenza A subtypes as well as the avian A(H7N9) viruses that have infected humans in China.ConclusionsThis streamlined Sanger sequencing protocol could be used to generate full genome sequence data more rapidly and easily than existing influenza genome sequencing protocols.     

The future is here: Integrating genetics into the pediatric pulmonary clinic

Recognition of underlying genetic etiologies of disease is increasing at an exponential rate, likely due to greater access to and lower cost of genetic testing. Monogenic causes of disease, or conditions resulting from a mutation or mutations in a single gene, are now well recognized in every subspecialty, including pediatric pulmonary medicine; thus, it is important to consider genetic conditions when evaluating children with respiratory disease. In the pediatric pulmonary clinic, genetic testing should be considered when multiple family members present with similar or related clinical features and when individuals have unusual clinical presentations, such as early‐onset disease or complex, syndromic features. This review provides a practical guide for genetic diagnosis in the pediatric pulmonary setting, including a review of genetic concepts, considerations for test selection and results in interpretation, as well as an overview of genetic differential diagnoses for common pediatric pulmonary phenotypes. Genetic conditions that commonly present to the pediatric pulmonary clinic are reviewed in a companion article by Yonker et al.     

An ORF1‐rearranged hepatitis E virus derived from a chronically infected patient efficiently replicates in cell culture

Hepatitis E is an increasingly reported disease in industrialized countries. Studies on the replication cycle of hepatitis E virus (HEV) are hampered due to the lack of efficient and robust cell culture systems for this virus. We describe the successful isolation of HEV derived from a chronically infected kidney transplant patient held under immunosuppressive therapy. Inoculation of serum sample 47832 onto the human lung carcinoma cell line A549 resulted in the replication of the virus as shown by RT‐qPCR. This novel human‐derived HEV strain is closely related to a wild boar‐derived genotype 3 strain, which did not replicate in A549 cells. It carries a 186 nucleotide insertion in the hypervariable ORF1‐region, derived from two parts of its ORF1. By passaging of the infected cells, a cell line continuously producing HEV particles was generated as demonstrated by RT‐qPCR, immuno‐electron microscopy, density gradient centrifugation and immunohistochemistry. Replication of the produced virus was demonstrated after its inoculation onto fresh A549 cells and two consecutive passages, whereas heating at 65 °C for 2 min abolished its infectivity. Several point mutations scattered along the whole genome were present in the HEV strain from the second passage; however, the ORF1 insertion was still present. Previously, cell culture isolation of two other HEV strains carrying insertions in their hypervariable regions, but originating from human ribosomal protein genes, has been described. The findings may indicate that cell culture adaptation of is mostly dependent on the length and position of the insertion, rather than from the sequence itself.