Era Of Next-generation Sequencing Research Articles

The Diagnostic Approach to Mitochondrial Disorders in Children in the Era of Next-Generation Sequencing: A 4-Year Cohort Study.

Mitochondrial diseases (MDs) are a large group of genetically determined multisystem disorders, characterized by extreme phenotypic heterogeneity, attributable in part to the dual genomic control (nuclear and mitochondrial DNA) of the mitochondrial proteome. Advances in next-generation sequencing technologies over the past two decades have presented clinicians with a challenge: to select the candidate disease-causing variants among the huge number of data provided. Unfortunately, the clinical tools available to support genetic interpretations still lack specificity and sensitivity. For this reason, the diagnosis of MDs continues to be difficult, with the new “genotype first” approach still failing to diagnose a large group of patients. With the aim of investigating possible relationships between clinical and/or biochemical phenotypes and definitive molecular diagnoses, we performed a retrospective multicenter study of 111 pediatric patients with clinical suspicion of MD. In this cohort, the strongest predictor of a molecular (in particular an mtDNA-related) diagnosis of MD was neuroimaging evidence of basal ganglia (BG) involvement. Regression analysis confirmed that normal BG imaging predicted negative genetic studies for MD. Psychomotor regression was confirmed as an independent predictor of a definitive diagnosis of MD. The findings of this study corroborate previous data supporting a role for neuroimaging in the diagnostic approach to MDs and reinforce the idea that mtDNA sequencing should be considered for first-line testing, at least in specific groups of children.

Abstract 2211: The landscape and implications of kinase-intergenic fusions in a large real-world lung cancer cohort

Abstract Background: Kinase gene fusions are strong driver mutations in neoplasia and have provided fundamental insights into the disease mechanisms that are involved in tumorigenesis. Into the era of next generation sequencing (NGS), numerous studies have described fusion events with breakpoints occurring at two intragenic positions that result in in-frame gene-gene fusions, but fusions that occur with intergenic regions are not uncommon. The clinical significance of such fusions needs to be carefully examined. Methods: We retrospectively reviewed the mutation profiles of over 30 000 lung cancer patients whose tissue and/or plasma samples underwent targeted NGS from June 2016 to July 2019. Clinical data of patients who had kinase-intergenic fusions were retrieved for analysis. RNA sequencing and immunohistochemical (IHC) assays were performed to verify chimeric fusion products on the transcriptional or protein level. Results: A total of 624 kinase-intergenic fusion events were identified in 538 unique lung cancer patients. Approximately 75% (404/538) of the entire cohort were non-small cell lung cancer (NSCLC), and 93% (375/404) of NSCLC were adenocarcinoma. The most frequently identified kinase genes included ALK, RET, ROS1, ERBB2/3, EGFR, FGFR1/2/3, and NTRK1/2/3. Our data showed that most (67%) kinase-intergenic gene rearrangements occurred on the same chromosome and kinase domains remained intact at 3' end. About 3% (19/624) of the kinase-intergenic fusions had one genomic breakpoint located in promoter regions, including 9 events involving ALK, RET, ROS1, EGFR, ERBB2, and FGFR3, which were fused to different gene promoters that may result in changes in kinase gene transcription. Importantly, 371 patients (69%) had concurrent oncogenic driver mutations or intragenic fusions, but the remaining 167 patients (31%) had kinase-intergenic fusions detected solely without other known oncogenic aberrations. Noteworthy, three patients who had ALK-intergenic fusions, but no other known oncogenic aberrations, were confirmed of EML4-ALK chimeric fusion product by RNA sequencing and/or ALK IHC results. Among them, one patient achieved a durable response of 14-month on crizotinib. An additional two patients who had ROS1 intergenic fusions demonstrated clinical benefit of 11 and 19 months from crizotinib, respectively, but the underlying functional fusion products remained to be further determined. Conclusion: This study investigated the role of kinase-intergenic fusions on a large-scale lung cancer population and revealed the potential oncogenic function of kinase-intergenic fusions. Our data also suggested that in addition to DNA sequencing, additional validation testing using RNA and/or protein assay should be performed in intergenic-breakpoint fusion cases to guide optimal treatment. Citation Format: Yutong Ma, Qiuxiang Ou, Xue Wu, Yang Shao. The landscape and implications of kinase-intergenic fusions in a large real-world lung cancer cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2211.

Diagnostic Approach to the Suspected Cases of Hereditary Spastic Paraplegia

Hereditary spastic paraplegia (HSP) is a heterogeneous group of monogenic neurodegenerative disorders characterized by progressive spasticity of the lower limbs. The clinical features and imaging abnormalities vary greatly according to the affected genes. HSP is classified clinically as pure and complex forms, depending upon the presence or absence of additional neurological defects other than spastic lower limbs. Despite the recent advances in next-generation sequencing technology and its wide availability, a genetic diagnosis of HSP is still not made in more than half of all suspected cases of HSP. In this review, we summarized the various phenotypes of relatively common HSP in clinical practice according to the inheritance pattern, highlighting their clinical, radiological, and neurophysiological features. We further discussed the practical approach to patients with suspected HSP in the current era of next-generation sequencing. Keywords: Spastic paraplegia, hereditary, Diagnosis

Synchronized Tissue Acquisition Techniques for Novel Biomarker Discovery: Are You Ready to Waltz?

Synchronized Tissue Acquisition Techniques for Novel Biomarker Discovery: Are You Ready to Waltz?

Revisiting the Role of γδ T Cells in Anti-CMV Immune Response after Transplantation

Gamma delta (γδ) T cells form an unconventional subset of T lymphocytes that express a T cell receptor (TCR) consisting of γ and δ chains. Unlike conventional αβ T cells, γδ T cells share the immune signature of both the innate and the adaptive immunity. These features allow γδ T cells to act in front-line defense against infections and tumors, rendering them an attractive target for immunotherapy. The role of γδ T cells in the immune response to cytomegalovirus (CMV) has been the focus of intense research for several years, particularly in the context of transplantation, as CMV reactivation remains a major cause of transplant-related morbidity and mortality. Therefore, a better understanding of the mechanisms that underlie CMV immune responses could enable the design of novel γδ T cell-based therapeutic approaches. In this regard, the advent of next-generation sequencing (NGS) and single-cell TCR sequencing have allowed in-depth characterization of CMV-induced TCR repertoire changes. In this review, we try to shed light on recent findings addressing the adaptive role of γδ T cells in CMV immunosurveillance and revisit CMV-induced TCR reshaping in the era of NGS. Finally, we will demonstrate the favorable and unfavorable effects of CMV reactive γδ T cells post-transplantation.

Focused Revision: ACMG practice resource: Genetic evaluation of short stature

Focused Revision: ACMG practice resource: Genetic evaluation of short stature

Towards population-specific pharmacogenomics in the era of next-generation sequencing

Pharmacogenomics (PGx) has essential roles in identifying optimal drug responders, optimizing dosage regimens and avoiding adverse events. Population-specific therapeutic interventions that tackle the genetic root causes of clinical outcomes are an important precision medicine strategy. In this perspective, we discuss next-generation sequencing genotyping and its significance for population-specific PGx applications. We emphasize the potential of NGS for preemptive pharmacogenotyping, which is crucial to population-specific clinical studies and patient care. We also provide examples that use publicly available population-based genomics data for population-specific PGx studies. Last, we discuss the remaining challenges and regulatory efforts towards improvements in this field.

Congenital Cataract and Its Genetics: The Era of Next-Generation Sequencing

Congenital cataract is a challenging ophthalmological disorder which can cause severe visual loss. It can be diagnosed at birth or during the first year of life. Early diagnosis and treatment are crucial for the visual prognosis. It can be associated with various ocular and systemic abnormalities. Determining whether congenital cataract is isolated or associated with other pathology is an indispensable step for the prediction of potential vision as well as early diagnosis and treatment of conditions that can cause morbidity or mortality. Many genes have been identified in the molecular etiology of congenital cataract. Most mutations have been reported in the crystallin genes. Determination of the genetic cause may not only enable individualized genetic counseling but also help to identify concomitant ocular and/or systemic disorders depending on the characteristics of the genetic test used. Recently, next-generation sequencing in particular has become an evolving technology for determining the molecular etiology of congenital cataract and furthering our knowledge of the disease.

Impact of Integrated Genetic Information on Diagnosis and Prognostication for Myeloproliferative Neoplasms in the Next-Generation Sequencing Era.

Since next-generation sequencing has been widely used in clinical laboratories, the diagnosis and risk stratification of hematologic malignancies are greatly dependent on genetic aberrations. In this study, we analyzed the genomic landscapes of 200 patients with myeloproliferative neoplasms (MPNs) and evaluated the impact of the genomic landscape on diagnosis and risk stratification. Mutations in JAK2, CALR and MPL were detected in 76.4% of MPNs. The proportion of patients with clonal genetic markers increased up to 86.4% when all detectable genetic aberrations were included. Significant co-occurring genetic aberrations potentially associated with phenotype and/or disease progression, including those in JAK2/SF3B1 and TP53/del(13q), del(5q), −7/del(7q) and complex karyotypes, were detected. We also identified genetic aberrations associated with patient outcomes: TP53 and −7/del(7q) were associated with an inferior chance of survival, RUNX1, TP53 and IDH1/2 were associated with leukemic transformation and SF3B1, IDH1/2, ASXL1 and del(20q) were associated with fibrotic progression. We compared risk stratification systems and found that mutation-enhanced prognostic scoring systems could identify lower risk polycythemia vera, essential thrombocythemia and higher risk primary myelofibrosis. Furthermore, the new risk stratification systems showed a better predictive capacity for patient outcome. These results collectively indicate that integrated genetic information can enhance diagnosis and prognostication in patients with myeloproliferative neoplasms.

Genetic Evaluation of the Parents Following Demise of the Index Case: Report of a Family with Fucosidosis.

It is common in obstetric practice to encounter couples who seek prenatal genetic counseling and testing in view of history of known or suspected genetic disorders in the previous offspring or in other family members. Recent advances in genetic testing techniques, especially the availability of the next-generation sequencing (NGS) technology, have greatly facilitated genetic evaluation of the proband and/or the consultand couple and enabled provision of accurate genetic counseling and prenatal genetic testing in such clinical scenarios. However, even in this era of NGS, comprehensive clinical history taking and detailed phenotype characterization through clinical examination and thorough perusal of available medical records, are very important and essential for accurate diagnosis, as reiterated by this report of a 30-year-old third gravida, who was referred for prenatal genetic counseling and testing, in view of history of death of the first offspring due to a suspected neurogenetic disorder. Retrospective clinical diagnosis for the deceased index child with the help of available medical records and reports, followed by relevant NGS-based clinical exome sequencing of the couple, helped to arrive at a definitive diagnosis of fucosidosis, based on which accurate prenatal genetic testing could be done.

A decade of next-generation sequencing in genodermatoses: the impact on gene discovery and clinical diagnostics.

Discovering the genetic basis of inherited skin diseases is fundamental to improving diagnostic accuracy and genetic counselling. In the 1990s and 2000s, genetic linkage and candidate gene approaches led to the molecular characterization of several dozen genodermatoses, but over the past decade the advent of next-generation sequencing (NGS) technologies has accelerated diagnostic discovery and precision. This review examines the application of NGS technologies from 2009 to 2019 that have (i) led to the initial discovery of gene mutations in known or new genodermatoses and (ii) identified involvement of more than one contributing pathogenic gene in individuals with complex Mendelian skin disorder phenotypes. A comprehensive review of the PubMed database and dermatology conference abstracts was undertaken between January 2009 and December 2019. The results were collated and cross-referenced with OMIM. We identified 166 new disease-gene associations in inherited skin diseases discovered by NGS. Of these, 131 were previously recognized, while 35 were brand new disorders. Eighty-five were autosomal dominant (with 43 of 85 mutations occurring de novo), 78 were autosomal recessive and three were X-linked. We also identified 63 cases harbouring multiple pathogenic mutations, either involving two coexisting genodermatoses (n = 13) or an inherited skin disorder in conjunction with other organ system phenotypes (n = 50). NGS technologies have accelerated disease-gene discoveries in dermatology over the last decade. Moreover, the era of NGS has enabled clinicians to split complex Mendelian phenotypes into separate diseases. These genetic data improve diagnostic precision and make feasible accurate prenatal testing and better-targeted translational research.

Integrative bioinformatics and omics data source interoperability in the next-generation sequencing era—Editorial

Integrative bioinformatics and omics data source interoperability in the next-generation sequencing era—Editorial

Next-Generation Sequencing-Based Biomarkers in Breast Cancer.

For the realization of precision medicine in cancer treatment, discovery, and validation of clinically useful biomarker is the most important prerequisite. Biomarkers are needed and used for evaluation of cancer susceptibility, cancer screening (early detection), cancer subtyping, prediction of prognosis, decision of appropriate adjuvant therapy and duration of therapy, and for monitoring of recurrence. Biomarkers are also needed for decision of target therapy in metastatic cancer and monitoring of their response during follow-up. Now is the era of Next-Generation Sequencing (NGS). NGS technology can detect almost all kind of genomic changes that occur in cancer that is different from normal condition. The cost also is now reasonably low to use in routine clinical practice.This chapter will review four kinds of NGS-based biomarkers that are already being used in clinical practice although the routine use is controversial, and that are promising and under active investigation focusing on studies done in Seoul National University Hospital (SNUH).

Ovarian cancer risk assessment in the era of next-generation sequencing.

Ovarian cancer is one of the cancers most influenced by hereditary factors. Testing for hereditary susceptibility genes is recommended for every woman with epithelial ovarian cancer (EOC). Pathogenic germline variants in BRCA1 and BRCA2 genes are responsible for a substantial fraction of hereditary ovarian cancer. However, alterations in other genes, such as BRIP1, RAD51C, RAD51D, and mismatch repair genes, also enhance ovarian cancer risk. Other genes may also participate in ovarian carcinogenesis, but their role as ovarian cancer susceptibility genes still needs to be clarified. With several genes involved, the complexity of genetic testing increases. In this context, next-generation sequencing (NGS) allows testing for multiple genes simultaneously, with rapid turn-around time. However, the incorporation of this technology into clinical practice faces some challenges. In this review, we will discuss the ovarian cancer risk assessment in the era of NGS.

Examination of Clinical and Molecular Characteristics and Treatment Patterns of Adolescent and Young Adult (AYA) Patients with Chronic Lymphocytic Leukemia

Examination of Clinical and Molecular Characteristics and Treatment Patterns of Adolescent and Young Adult (AYA) Patients with Chronic Lymphocytic Leukemia

Correlating Neuroimaging and CNVs Data: 7 Years of Cytogenomic Microarray Analysis on Patients Affected by Neurodevelopmental Disorders.

The aim of this study was to evaluate the relationship between neurodevelopmental disorders, brain anomalies, and copy number variations (CNVs) and to estimate the diagnostic potential of cytogenomical microarray analysis (CMA) in individuals neuroradiologically characterized with intellectual developmental disorders (IDDs) isolated or associated with autism spectrum disorders (ASDs) and epilepsy (EPI), all of which were identified as a "synaptopathies." We selected patients who received CMA and brain magnetic resonance imaging (MRI) over a 7-year period. We divided them into four subgroups: IDD, IDD + ASD, IDD + EPI, and IDD + ASD + EPI. The diagnostic threshold of CMA was 16%. The lowest detection rate for both CMA and brain anomalies was found in IDD + ASD, while MRI was significantly higher in IDD and IDD + EPI subgroups. CMA detection rate was significantly higher in patients with brain anomalies, so CMA may be even more appropriate in patients with pathological MRI, increasing the diagnostic value of the test. Conversely, positive CMA in IDD patients should require an MRI assessment, which is more often associated with brain anomalies. Posterior fossa anomalies, both isolated and associated with other brain anomalies, showed a significantly higher rate of CMA positive results and of pathogenic CNVs. In the next-generation sequencing era, our study confirms once again the relevant diagnostic output of CMA in patients with IDD, either isolated or associated with other comorbidities. Since more than half of the patients presented brain anomalies in this study, we propose that neuroimaging should be performed in such cases, particularly in the presence of genomic imbalances.

The vaginal microbiome of sub-Saharan African women: revealing important gaps in the era of next-generation sequencing.

Accurate characterization of the vaginal microbiome remains a fundamental goal of the Human Microbiome project (HMP). For over a decade, this goal has been made possible deploying high-throughput next generation sequencing technologies (NGS), which indeed has revolutionized medical research and enabled large-scale genomic studies. The 16S rRNA marker-gene survey is the most commonly explored approach for vaginal microbial community studies. With this approach, prior studies have elucidated substantial variations in the vaginal microbiome of women from different ethnicities. This review provides a comprehensive account of studies that have deployed this approach to describe the vaginal microbiota of African women in health and disease. On the basis of published data, the few studies reported from the African population are mainly in non-pregnant post pubertal women and calls for more detailed studies in pregnant and postnatal cohorts. We provide insight on the use of more sophisticated cutting-edge technologies in characterizing the vaginal microbiome. These technologies offer high-resolution detection of vaginal microbiome variations and community functional capabilities, which can shed light into several discrepancies observed in the vaginal microbiota of African women in an African population versus women of African descent in the diaspora.

Novel NGS pipeline for virus discovery from a wide spectrum of hosts and sample types

The study of the microbiome data holds great potential for elucidating the biological and metabolic functioning of living organisms and their role in the environment. Metagenomic analyses have shown that humans, along with for example, domestic animals, wildlife and arthropods, are colonized by an immense community of viruses. The current Coronavirus pandemic (COVID-19) heightens the need to rapidly detect previously unknown viruses in an unbiased way. The increasing availability of metagenomic data in this era of next-generation sequencing (NGS), along with increasingly affordable sequencing technologies, highlight the need for reliable and comprehensive methods to manage such data. In this article, we present a novel bioinformatics pipeline called LAZYPIPE for identifying both previously known and novel viruses in host associated or environmental samples and give examples of virus discovery based on it. LAZYPIPE is a Unix-based pipeline for automated assembling and taxonomic profiling of NGS libraries implemented as a collection of C++, Perl, and R scripts.

Epidemiological data analysis of viral quasispecies in the next-generation sequencing era.

The unprecedented coverage offered by next-generation sequencing (NGS) technology has facilitated the assessment of the population complexity of intra-host RNA viral populations at an unprecedented level of detail. Consequently, analysis of NGS datasets could be used to extract and infer crucial epidemiological and biomedical information on the levels of both infected individuals and susceptible populations, thus enabling the development of more effective prevention strategies and antiviral therapeutics. Such information includes drug resistance, infection stage, transmission clusters and structures of transmission networks. However, NGS data require sophisticated analysis dealing with millions of error-prone short reads per patient. Prior to the NGS era, epidemiological and phylogenetic analyses were geared toward Sanger sequencing technology; now, they must be redesigned to handle the large-scale NGS datasets and properly model the evolution of heterogeneous rapidly mutating viral populations. Additionally, dedicated epidemiological surveillance systems require big data analytics to handle millions of reads obtained from thousands of patients for rapid outbreak investigation and management. We survey bioinformatics tools analyzing NGS data for (i) characterization of intra-host viral population complexity including single nucleotide variant and haplotype calling; (ii) downstream epidemiological analysis and inference of drug-resistant mutations, age of infection and linkage between patients; and (iii) data collection and analytics in surveillance systems for fast response and control of outbreaks.

Differential diagnosis of vacuolar myopathies in the NGS era.

Altered autophagy accompanied by abnormal autophagic (rimmed) vacuoles detectable by light and electron microscopy is a common denominator of many familial and sporadic non‐inflammatory muscle diseases. Even in the era of next generation sequencing (NGS), late‐onset vacuolar myopathies remain a diagnostic challenge. We identified 32 adult vacuolar myopathy patients from 30 unrelated families, studied their clinical, histopathological and ultrastructural characteristics and performed genetic testing in index patients and relatives using Sanger sequencing and NGS including whole exome sequencing (WES). We established a molecular genetic diagnosis in 17 patients. Pathogenic mutations were found in genes typically linked to vacuolar myopathy (GNE, LDB3/ZASP, MYOT, DES and GAA), but also in genes not regularly associated with severely altered autophagy (FKRP, DYSF, CAV3, COL6A2, GYG1 and TRIM32) and in the digenic facioscapulohumeral muscular dystrophy 2. Characteristic histopathological features including distinct patterns of myofibrillar disarray and evidence of exocytosis proved to be helpful to distinguish causes of vacuolar myopathies. Biopsy validated the pathogenicity of the novel mutations p.(Phe55*) and p.(Arg216*) in GYG1 and of the p.(Leu156Pro) TRIM32 mutation combined with compound heterozygous deletion of exon 2 of TRIM32 and expanded the phenotype of Ala93Thr‐caveolinopathy and of limb‐girdle muscular dystrophy 2i caused by FKRP mutation. In 15 patients no causal variants were detected by Sanger sequencing and NGS panel analysis. In 12 of these cases, WES was performed, but did not yield any definite mutation or likely candidate gene. In one of these patients with a family history of muscle weakness, the vacuolar myopathy was eventually linked to chloroquine therapy. Our study illustrates the wide phenotypic and genotypic heterogeneity of vacuolar myopathies and validates the role of histopathology in assessing the pathogenicity of novel mutations detected by NGS. In a sizable portion of vacuolar myopathy cases, it remains to be shown whether the cause is hereditary or degenerative.