Application Of Exome Sequencing Research Articles

EP146: Application of exome sequencing in patients of congenital anomalies with or without intellectual disability

Congenital anomalies (CA) and intellectual disability (ID) both comprise a heterogeneous spectrum of diseases. They affect about 3% of live birth worldwide. The causes of CA can be divided into genetic and nongenetic. About a quarter of CA has been estimated to have a genetic cause previously, with the two most common causes as single gene defects and chromosome abnormalities. ID has highly variable severity and frequently combines with multiple congenital anomalies (MCA). Exome sequencing (ES) is not only massive parallel but also high throughput sequencing.

The role of chromosomal microarray and exome sequencing in prenatal diagnosis.

Advancements in technologies have revolutionized prenatal diagnosis. Chromosomal microarray analysis (CMA) became a proven method and was implemented to detect gains and losses of DNA and absence of heterozygosity across the genome. Next-generation sequencing technologies have brought opportunities and challenges to genetic testing. Exome sequencing detects single-nucleotide variants (SNVs) across the exome and its prenatal application is an emerging field. We reviewed the literature to define the role of CMA and exome sequencing in prenatal diagnosis. The application of exome sequencing in genetic diagnosis shows increased diagnostic yield and could be potentially implemented for prenatal diagnosis of fetuses with one or more ultrasound structural abnormalities or suspected monogenetic conditions. Although CMA is a gold standard for copy number variant (CNV) detection, large clinical cohort studies emphasized integrated CNV and SNV analyses for precise molecular diagnosis. Recent studies also suggest low-pass genome sequencing-based CNV detection can identify genome-wide imbalances at higher resolutions. Data suggest exome sequencing for SNVs and CMA for CNV detection are the most effective approach for prenatal genetic diagnosis. Emerging evidences show genome sequencing has the potential to replace CMA and even exome sequencing to become a comprehensive genetic test in the clinical diagnostic laboratory.

Movement disorders in 2020: clinical trials, genetic discoveries, and COVID-19

With 2020 hindsight, this has been an extraordinary year not just for patients with movement disorders but also for every individual around the world, due to the impact of the COVID-19 pandemic. Several studies investigating the effect of COVID-19 on individuals with Parkinson's disease have suggested that parkinsonian symptoms worsened among those who were infected, that those with more advanced Parkinson's disease were at increased risk of pulmonary compromise, that hospitalised patients with Parkinson's disease and COVID-19 appeared to have a heightened mortality rate, and that patients with Parkinson's disease showed more stress, depressive symptoms, and anxiety during the lockdown period than before this period.1Tan EK Albanese A Chaudhuri K et al.Adapting to post-COVID19 research in Parkinson's disease: lessons from a multinational experience.Parkinsonism Relat Disord. 2020; (published Oct 7.)https://doi.org/10.1016/j.parkreldis.2020.10.009Summary Full Text Full Text PDF Scopus (7) Google Scholar A case report linking severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection with new-onset Parkinson's disease has led to an increased awareness of this potential association and also its underlying biological plausibility.2Cohen ME Eichel R Steiner-Birmanns B et al.A case of probable Parkinson's disease after SARS-CoV-2 infection.Lancet Neurol. 2020; 19: 804-805Summary Full Text Full Text PDF PubMed Scopus (112) Google Scholar Amid the pandemic, several clinical safety and proof-of-concept trials have taken small steps towards identifying disease-modifying therapies. Targeting α-synuclein, either through active or passive immunisations, gained considerable attention in the past few years for its potential for slowing down neurodegeneration, as dysfunction of the protein is a major driver of neurodegeneration. In a first-in-human phase 1 randomised study of subcutaneous immunisations with PD01A (ie, an active immunotherapy using a short peptide directed at oligomeric α-synuclein), 21 of the original 24 patients completed all the initial and booster immunisations over more than 4 years of follow-up.3Volc D Poewe W Kutzelnigg A et al.Safety and immunogenicity of the α-synuclein active immunotherapeutic PD01A in patients with Parkinson's disease: a randomised, single-blinded, phase 1 trial.Lancet Neurol. 2020; 19: 591-600Summary Full Text Full Text PDF PubMed Scopus (64) Google Scholar The immunisation led to an increase in antibody titres with neither imaging evidence of neuroinflammation nor significant clinical adverse effects, providing impetus for investigators to proceed to the next phase of the study. Loss-of-function mutations in the glucocerebrosidase gene that encodes lysosomal β-glucocerebrosidase (GCase) represent an important risk factor for Parkinson's disease, and can lead to α-synuclein accumulation. Ambroxol, a secretolytic agent used in the treatment of respiratory diseases, increases GCase activity and hence reduces α-synuclein. In an open-label study using an escalating dose of oral ambroxol, 18 of the 23 patients with moderately severe Parkinson's disease completed the study.4Mullin S Smith L Lee K et al.Ambroxol for the treatment of patients with Parkinson disease with and without glucocerebrosidase gene mutations: a nonrandomized, noncontrolled trial.JAMA Neurol. 2020; 77: 427-434Crossref PubMed Scopus (144) Google Scholar Ambroxol was well tolerated and detected in the CSF, with an increase in CSF GCase activity. Although an improvement in the clinical motor scores was suggested with the drug, future randomised controlled trials will be required to show its effect on motor progression. Subcutaneous injection or infusion of apomorphine (a non-selective dopamine agonist) via mini pumps has been used in patients with advanced Parkinson's disease to relieve off states. Alternative routes of delivery of apomorphine have now been investigated. A multicentre, randomised, double-blind study in patients with Parkinson's disease and more than 2 h off time per day compared an effective dose of apomorphine sublingual film individually titrated to 10–35 mg with a placebo (54 vs 55 patients).5Olanow CW Factor SA Espay AJ et al.Apomorphine sublingual film for off episodes in Parkinson's disease: a randomised, double-blind, placebo-controlled phase 3 study.Lancet Neurol. 2020; 19: 135-144Summary Full Text Full Text PDF PubMed Scopus (59) Google Scholar There was a significantly better improvement in the Movement Disorder Society-Unified Parkinson Disease Rating Scale motor scores (from pre-dose to 30 min post-dose) at week 12 in participants receiving apomorphine than in those receiving placebo. 31% of participants who were on the drug reported mild-to-moderate oropharyngeal side-effects, with 17% discontinuing the treatment. The long-term safety and efficacy of this apomorphine formulation as an on-demand therapy for off episodes in patients with Parkinson's disease still needs to be further evaluated. Repurposing of drugs continues to attract interest as this strategy can potentially reduce the time frame and cost and improve regulatory support for successful clinical translation. Nilotinib has been used to treat chronic myeloid leukaemia. In a single-centre, double-blind, placebo-controlled phase 2 trial, 75 patients with moderately severe Parkinson's disease were randomised into placebo, nilotinib (150 mg), or nilotinib (300 mg) groups (daily oral administration for a year).6Pagan FL Hebron ML Wilmarth B et al.Nilotinib effects on safety, tolerability, and potential biomarkers in Parkinson disease: a phase 2 randomized clinical trial.JAMA Neurol. 2020; 77: 309-317Crossref PubMed Scopus (68) Google Scholar Although there were more adverse side effects in the nilotinib groups than in the placebo group, the drug was generally safe and detectable in CSF. The concentrations of some metabolic markers and specific proteins (such as α-synuclein oligomers and hyperphosphorylated tau) in CSF were altered in those on nilotinib, raising the possibility that these can be used as potential biomarkers of drug response in future phase 3 trials. Next generation sequencing approaches to unravel underlying genetic causes of rare movement disorders can provide definitive diagnosis and facilitate pathophysiological studies to identify novel therapeutic targets. To unravel the monogenic causes of dystonia (a clinically and genetically heterogeneous disease), whole exome sequencing was done in 764 patients with dystonia and 346 healthy parents in an international collaborative study involving 33 centres.7Zech M Jech R Boesch S et al.Monogenic variants in dystonia: an exome-wide sequencing study.Lancet Neurol. 2020; 19: 908-918Summary Full Text Full Text PDF PubMed Scopus (74) Google Scholar The authors managed to identify causative or probable causative gene variants in 135 (19%) of the 728 families, and diagnostic variants in 11 genes not previously linked to dystonia. Most of the identified variants are in genes that are linked to neurodevelopmental disorders. In essence, the application of exome sequencing in a collective large number of patients with dystonia improves diagnosis, facilitates genotype–phenotype correlation, potentially guides genetic testing, and improves the clinical management of patients with dystonia. Repeat expansion disorders have been linked to a wide clinical phenotype. One study examined 100 genetically confirmed carriers of cerebellar ataxia with neuropathy and vestibular areflexia syndrome, which is due to biallelic repeat expansions in RFC1 (replication factor complex subunit 1).8Cortese A Tozza S Yau WY et al.Cerebellar ataxia, neuropathy, vestibular areflexia syndrome due to RFC1 repeat expansion.Brain. 2020; 143: 480-490Crossref PubMed Scopus (86) Google Scholar The investigators found that a dry spasmodic cough is commonly associated with repeat expansion disorders, and that sensory neuropathy was the only manifestation in some patients,8Cortese A Tozza S Yau WY et al.Cerebellar ataxia, neuropathy, vestibular areflexia syndrome due to RFC1 repeat expansion.Brain. 2020; 143: 480-490Crossref PubMed Scopus (86) Google Scholar although other investigators have reported RFC1 mutations in multiple system atrophy.9Wan L Chen Z Wan N et al.Biallelic intronic AAGGG expansion of RFC1 is related to multiple system atrophy.Ann Neurol. 2020; (published online Sept 16.)https://doi.org/10.1002/ana.25902Crossref PubMed Scopus (29) Google Scholar Similarly, GGC repeat expansions in NOTCH2NLC, which causes neuronal intranuclear inclusion disease, have now been reported in patients with Parkinson's disease and essential tremor who do not show cognitive dysfunction or imaging evidence of intranuclear inclusion disease.10Ma D Tan YJ Ng ASL et al.Association of NOTCH2NLC repeat expansions with Parkinson disease.JAMA Neurol. 2020; (published online Aug 24.)https://doi.org/10.1001/jamaneurol.2020.3023Crossref Scopus (39) Google Scholar A high index of suspicion and awareness is needed for appropriate genetic testing in patients who present with movement disorders. I declare that I have received honoraria from Elsevier and Wiley for editorial duties.

COngenital heart disease and the Diagnostic yield with Exome sequencing (CODE) study: prospective cohort study and systematic review.

To determine the incremental yield of antenatal exome sequencing (ES) over chromosomal microarray analysis (CMA) or conventional karyotyping in prenatally diagnosed congenital heart disease (CHD). A prospective cohort study of 197 trios undergoing ES following CMA or karyotyping owing to CHD identified prenatally and a systematic review of the literature were performed. MEDLINE, EMBASE, CINAHL and ClinicalTrials.gov (January 2000 to October 2019) databases were searched electronically for studies reporting on the diagnostic yield of ES in prenatally diagnosed CHD. Selected studies included those with more than three cases, with initiation of testing based upon prenatal phenotype only and that included cases in which CMA or karyotyping was negative. The incremental diagnostic yield of ES was assessed in: (1) all cases of CHD; (2) isolated CHD; (3) CHD associated with extracardiac anomaly (ECA); and (4) CHD according to phenotypic subgroup. In our cohort, ES had an additional diagnostic yield in all CHD, isolated CHD and CHD associated with ECA of 12.7% (25/197), 11.5% (14/122) and 14.7% (11/75), respectively (P = 0.81). The corresponding pooled incremental yields from 18 studies (encompassing 636 CHD cases) included in the systematic review were 21% (95% CI, 15-27%), 11% (95% CI, 7-15%) and 37% (95% CI, 18-56%), respectively. The results did not differ significantly when subanalysis was limited to studies including more than 20 cases, except for CHD associated with ECA, in which the incremental yield was greater (49% (95% CI, 17-80%)). In cases of CHD associated with ECA in the primary analysis, the most common extracardiac anomalies associated with a pathogenic variant were those affecting the genitourinary system (23/52 (44.2%)). The greatest incremental yield was in cardiac shunt lesions (41% (95% CI, 19-63%)), followed by right-sided lesions (26% (95% CI, 9-43%)). In the majority (68/96 (70.8%)) of instances, pathogenic variants occurred de novo and in autosomal dominant (monoallelic) disease genes. The most common (19/96 (19.8%)) monogenic syndrome identified was Kabuki syndrome. There is an apparent incremental yield of prenatal ES in CHD. While the greatest yield is in CHD associated with ECA, consideration could also be given to performing ES in the presence of an isolated cardiac abnormality. A policy of routine application of ES would require the adoption of robust bioinformatic, clinical and ethical pathways. Copyright © 2020 ISUOG. Published by John Wiley & Sons Ltd.

[A consensus recommendation for the interpretation and reporting of exome sequencing in prenatal genetic diagnosis].

With the rapid development and adaptation of high-throughput sequencing in clinical settings, application of exome sequencing (ES) has been gradually expanded from pediatric to prenatal diagnosis in recent years. There is an urgent need to establish criteria for clinical grade ES in order to facilitate such a complex testing. The standardization of pre- and post-test consultation, quality control for sample processing process and validation of bioinformatics data analysis, and more importantly data interpretation and reporting, as well as appropriate reporting scope, is of great importance for health care stakeholders. To achieve this, a committee composed of a wide range of healthcare professionals has proposed an ES standard for prenatal diagnosis. This has provided expert opinion on the genetic counseling and reporting standards of prenatal ES for the purpose of applying ES technology in prenatal setting.

Leukodystrophies and Genetic Leukoencephalopathies in Children Specified by Exome Sequencing in an Expanded Gene Panel.

The overlapping clinical and neuroimaging phenotypes of leukodystrophies pose a diagnostic challenge to both clinicians and researchers alike. Studies on the application of exome sequencing in the diagnosis of leukodystrophies are emerging. We used targeted gene panel sequencing of 6440 genes to investigate the genetic etiology in a cohort of 50 children with neuroimaging diagnosis of leukodystrophy/genetic leukoencephalopathy of unknown etiology. These 50 patients without a definite biochemical or genetic diagnosis were derived from a cohort of 88 patients seen during a 2.5-year period (2015 January-2017 June). Patients who had diagnosis by biochemical or biopsy confirmation (n = 17) and patients with incomplete data or lack of follow-up (n = 21) were excluded. Exome sequencing identified variants in 30 (60%) patients, which included pathogenic or likely pathogenic variants in 28 and variants of unknown significance in 2. Among the patients with pathogenic or likely pathogenic variants, classic leukodystrophies constituted 13 (26%) and genetic leukoencephalopathies 15 (30%). The clinical and magnetic resonance imaging (MRI) findings and genetic features of the identified disorders are discussed.

Exome sequencing of fetal anomaly syndromes: novel phenotype\u2013genotype discoveries

The monogenic etiology of most severe fetal anomaly syndromes is poorly understood. Our objective was to use exome sequencing (ES) to increase our knowledge on causal variants and novel candidate genes associated with specific fetal phenotypes. We employed ES in a cohort of 19 families with one or more fetuses presenting with a distinctive anomaly pattern and/or phenotype recurrence at increased risk for lethal outcomes. Candidate variants were identified in 12 families (63%); in 6 of them a definite diagnosis was achieved including known or novel variants in recognized disease genes (MKS1, OTX2, FGFR2, and RYR1) and variants in novel disease genes describing new fetal phenotypes (CENPF, KIF14). We identified variants likely causal after clinical and functional review (SMAD3, KIF4A, and PIGW) and propose novel candidate genes (PTK7, DNHD1, and TTC28) for early human developmental disease supported by functional and cross-species phenotyping evidence. We describe rare and novel fetal anomaly syndromes and highlight the diagnostic utility of ES, but also its contribution to discovery. The diagnostic yield of the future application of prenatal ES will depend on our ability to increase our knowledge on the specific phenotype–genotype correlations during fetal development.

Abstract 18895: A Novel Gene Involved in Severe Neonatal Cardiomyopathy

Cardiomyopathies (CMP) in children are heterogeneous disorders of cardiac muscle with a relatively high incidence in infancy (8/100.000) as compared to older children (0.7/100.000). Beyond infancy, CMP is the most important indication for heart transplantation and transplantation free survival at 5 years after initial diagnosis is only 54%. Mutations in over 65 genes encoding sarcomeric, cytoskeletal and calcium-metabolizing proteins, cell-signaling molecules, and mitochondrial enzymes have been shown to cause CMPs. This has led to new recommendations for diagnostic evaluation and family screening incorporating genetic testing. However, present (targeted/custom based) diagnostic tests do not represent the whole spectrum of genetic etiologies and are leading to a correct diagnosis in just a subset of pediatric CMP patients. We present four children from two consanguineous families of Dutch and Moroccan descent with severe neonatal onset of CMP, ranging from lethal cardiomegaly with reduced contractility in three to severe left ventricular hypertrophy that remained stable in one child. A combined approach using homozygosity mapping and exome sequencing in both families identified a homozygous splice site variant resulting in exon skipping in the Dutch family, and a homozygous nonsense mutation in the Moroccan family within the same gene. This novel disease causing gene is implicated in the early differentiation of cardiomyocytes. Both variants were confirmed with Sanger sequencing and absent in large control populations. Previously published studies in a knockout mouse model demonstrated the functional role of this gene in CMPs. Additionally, we investigated the effect of the homozygous splice site mutation on the expression of different proteins at myocardial cell-cell junctions and myofibrils. These results point towards a role in intercalated disk remodeling. Electron microscopy- and protein expression studies on heart tissue of the patients are ongoing. Our findings add a new autosomal recessive gene to the list of genes already implicated in pediatric CMP, underscoring its genetic heterogeneity. Furthermore, our study indicates that the application of exome sequencing can improve the diagnostic yield in pediatric CMP.

Nuclear-mitochondrial proteins: too much to process?

This scientific commentary refers to ‘ PMPCA mutations cause abnormal mitochondrial protein processing in patients with non-progressive cerebellar ataxia’, by Jobling et al. (doi:10.1093/brain/awv057). In the not too distant past, non-progressive focal neurological deficits emerging during child development were all labelled as ‘cerebral palsy’, and assumed to be caused by an insult during pregnancy or in the perinatal period. This view has changed dramatically over the last two decades, in large part as a result of advances in structural brain imaging, which failed to identify the characteristic patterns of hypoxic-ischaemic brain injury or perinatal infection in every case. Metabolic and genetic studies subsequently unravelled a diverse and unexpected range of mechanisms responsible for these ‘static’ neurological deficits, and the pace has accelerated with the application of exome sequencing (Srivastava et al. , 2014). Patients with non-progressive cerebellar ataxia have been particularly difficult to explain. The number of known causes has remained limited, and includes extreme rarities such as congenital disorder of glycosylation type Ia, caused by phosphomannomutase 2 deficiency (Boddaert et al. , 2010). However, the differential diagnosis and mechanisms responsible for this phenotype have now broadened with the findings of Jobling et al. (2015), described in this issue of Brain . Initially studying a family from the Bekaa valley in north east Lebanon, Jobling et al. looked for regions of shared homozygosity using microsatellite markers in 12 individuals with non-progressive ataxia, spasticity, and intellectual disability (Delague et al. , 2002). All shared a 12.1-cM homozygous region on chromosome 9q34-qter between markers D9S67 and D9S312. Exome sequencing identified homozygous mutations in two genes within the mapped candidate region: PMPCA and CAMSAP1 , neither previously associated with disease. These findings presented a …

Exome Sequencing in Today's Lab: Shifting the Paradigm in Translational and Clinical Research

Next generation sequencing technologies have enabled a rapid expansion toward the understanding of inherited disorders, cancer biology, drug development, and treatment resistance. Exome sequencing has been increasingly and successfully applied in the clinical research setting for identifying common single nucleotide variants, copy number variations, and small insertions or deletions as well as rare de novo mutations that may explain Mendelian, complex, and rare genetic disorders. Recent advancement in rapid and low-cost exome sequencing make it an attractive alternative to traditional targeted gene panel sequencing for clinical research, while maintaining the possibility of discovering mutations in genes previously not associated with a disorder. Furthermore, the exome encompasses approximately 1% of the genome, yet contains approximately 85% of disease-causing mutations, making exome sequencing easier and cheaper than whole genome sequencing for identifying disease-causing variants in research. Researchers are now applying proband-father-mother (trio) exome sequencing to uncover variants that potentially either cause or modify the condition under study. In this webinar, the audience will learn about the current applications of exome sequencing in clinical research and its impact on the future of health care.

The genetic landscape of immune-competent and HIV lymphoma

Burkitt lymphoma (BL) and diffuse large B cell lymphoma (DLBCL) are aggressive forms of lymphoma in adults and demonstrate overlapping morphology, immunophenotype and clinical behavior. The risk of developing these tumors increases ten to hundred-fold in the setting of HIV infection. The genetic causes and the role of specific mutations, especially in the setting of HIV, are largely unknown. The decoding of the human genome and the advent of high-throughput sequencing have provided rich opportunities for the comprehensive identification of the genetic causes of cancer. In order to comprehensively identify genes that are recurrently mutated in immune-competent DLBCL and BL, we obtained a total of 92 cases of DLBCLs and 40 cases of BL. These cases were compared to a set of 5 DLBCLs and BL tumors derived from patients with HIV. The DLBCL cases were divided into a discovery set (N=34) and a prevalence set (N=61). The Burkitt cases were also divided into discovery and prevalence sets (N=15, N=45 respectively). For each of the discovery set cases we also obtained paired normal tissue. We performed whole-exome sequencing for all of these using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ~700 human miRNAs from miRBase (v13). In all, we generated over 6 GB of sequencing data using high throughput sequencing on the Illumina platform. We identified a total of 432 genes that were recurrently mutated in DLBCL and BL. We found that each tumor had an average of 20 gene alterations, which is fewer than most other solid tumors sequenced to date. Commonly implicated biological processes comprising these genes included signal transduction (e.g. PIK3CD, PDGFRA), immune response (e.g. B2M, CD83, IRF8) and chromatin modification (e.g. MLL3, SETD2). We found that lymphomas that arose in the setting of HIV had fewer mutations overall and had a paucity of mutations related to immune response. These data implicate the depressed immune response by HIV as a contributing risk factor for the development of lymphomas and suggest that HIV lymphomas are genetically less complex than their immune competent counterparts. This study represents one of the largest applications of exome sequencing in cancer, and provides early clues to the genetic causes of HIV-lymphomas.

Application of exome sequencing in the search for genetic causes of rare disorders of copper metabolism

The genetic defect in a number of rare disorders of metal metabolism remains elusive. The limited number of patients with these disorders impedes the identification of the causative gene through positional cloning, which requires numerous families with multiple affected individuals. However, with next-generation sequencing all coding DNA (exomes) or whole genomes of patients can be sequenced to identify genes that are consistently mutated in patients. With this strategy only a limited number of patients and/or pedigrees is needed, bringing the elucidation of the genetic cause of even very rare diseases within reach. The main challenge associated with whole exome sequencing is the identification of the disease-causing mutation(s) among abundant genetic candidate variants. We describe several strategies to manage this data wealth, including comparison with control databases, increasing the number of patients and controls, and reducing the genomic region under investigation through homozygosity mapping. In this review we introduce a number of rare disorders of copper metabolism, with a suspected but yet unknown monogenetic cause, as an attractive target for this strategy. We anticipate that use of these novel techniques will identify the basic defect in the disorders described in this review, as well as in other genetic disorders of metal metabolism, in the next few years.