Clinical exome sequencing—Mistakes and caveats Corominas, Jordi; Smeekens, Sanne P.; Nelen, Marcel R. ...
Human mutation,
August 2022, 2022-08-00, 20220801, Letnik:
43, Številka:
8
Journal Article
Recenzirano
Odprti dostop
Massive parallel sequencing technology has become the predominant technique for genetic diagnostics and research. Many genetic laboratories have wrestled with the challenges of setting up genetic ...testing workflows based on a completely new technology. The learning curve we went through as a laboratory was accompanied by growing pains while we gained new knowledge and expertise. Here we discuss some important mistakes that have been made in our laboratory through 10 years of clinical exome sequencing but that have given us important new insights on how to adapt our working methods. We provide these examples and the lessons that we learned to help other laboratories avoid to make the same mistakes.
Lessons learned from performing clinical exome sequencing for over 10 years.
Objective
To identify novel causes of recessive ataxias, including spinocerebellar ataxia with saccadic intrusions, spastic ataxias, and spastic paraplegia.
Methods
In an international collaboration, ...we independently performed exome sequencing in 7 families with recessive ataxia and/or spastic paraplegia. To evaluate the role of VPS13D mutations, we evaluated a Drosophila knockout model and investigated mitochondrial function in patient‐derived fibroblast cultures.
Results
Exome sequencing identified compound heterozygous mutations in VPS13D on chromosome 1p36 in all 7 families. This included a large family with 5 affected siblings with spinocerebellar ataxia with saccadic intrusions (SCASI), or spinocerebellar ataxia, recessive, type 4 (SCAR4). Linkage to chromosome 1p36 was found in this family with a logarithm of odds score of 3.1. The phenotypic spectrum in our 12 patients was broad. Although most presented with ataxia, additional or predominant spasticity was present in 5 patients. Disease onset ranged from infancy to 39 years, and symptoms were slowly progressive and included loss of independent ambulation in 5. All but 2 patients carried a loss‐of‐function (nonsense or splice site) mutation on one and a missense mutation on the other allele. Knockdown or removal of Vps13D in Drosophila neurons led to changes in mitochondrial morphology and impairment in mitochondrial distribution along axons. Patient fibroblasts showed altered morphology and functionality including reduced energy production.
Interpretation
Our study demonstrates that compound heterozygous mutations in VPS13D cause movement disorders along the ataxia–spasticity spectrum, making VPS13D the fourth VPS13 paralog involved in neurological disorders. Ann Neurol 2018
Summary
Objective
Epilepsy is highly prevalent among patients with intellectual disability (ID), and seizure control is often difficult. Identification of the underlying etiology in this patient ...group is important for daily clinical care. We assessed the diagnostic yield of whole exome sequencing (WES). In addition, we evaluated which clinical characteristics influence the likelihood of identifying a genetic cause and we assessed the potential impact of the genetic diagnosis on (antiepileptic) treatment strategy.
Methods
One hundred patients with both unexplained epilepsy and (borderline) ID (intelligence quotient ≤ 85) were included. All patients were evaluated by a clinical geneticist, a (pediatric) neurologist, and/or a specialist ID physician. WES analysis was performed in two steps. In step 1, analysis was restricted to the latest versions of ID and/or epilepsy gene panels. In step 2, exome analysis was extended to all genes (so‐called full exome analysis). The results were classified according to the American College of Medical Genetics and Genomics guidelines.
Results
In 58 patients, the diagnostic WES analysis reported one or more variant(s). In 25 of the 100 patients, these were classified as (likely) pathogenic, in 24 patients as variants of uncertain significance, and in the remaining patients the variant was most likely not related to the phenotype. In 10 of 25 patients (40%) with a (likely) pathogenic variant, the genetic diagnosis might have an impact on the treatment strategy in the future.
Significance
This study illustrates the clinical diagnostic relevance of WES for patients with both epilepsy and ID. It also demonstrates that implementing WES diagnostics might have impact on the (antiepileptic) treatment strategy in this population. Confirmation of variants of uncertain significance in (candidate) genes may further increase the yield.
To identify candidate genes for intellectual disability, we performed a meta-analysis on 2,637 de novo mutations, identified from the exomes of 2,104 patient-parent trios. Statistical analyses ...identified 10 new candidate ID genes: DLG4, PPM1D, RAC1, SMAD6, SON, SOX5, SYNCRIP, TCF20, TLK2 and TRIP12. In addition, we show that these genes are intolerant to nonsynonymous variation and that mutations in these genes are associated with specific clinical ID phenotypes.
Implementation of novel genetic diagnostic tests is generally driven by technological advances because they promise shorter turnaround times and/or higher diagnostic yields. Other aspects, including ...impact on clinical management or cost-effectiveness, are often not assessed in detail prior to implementation.
We studied the clinical utility of whole-exome sequencing (WES) in complex pediatric neurology in terms of diagnostic yield and costs. We analyzed 150 patients (and their parents) presenting with complex neurological disorders of suspected genetic origin. In a parallel study, all patients received both the standard diagnostic workup (e.g., cerebral imaging, muscle biopsies or lumbar punctures, and sequential gene-by-gene-based testing) and WES simultaneously.
Our unique study design allowed direct comparison of diagnostic yield of both trajectories and provided insight into the economic implications of implementing WES in this diagnostic trajectory. We showed that WES identified significantly more conclusive diagnoses (29.3%) than the standard care pathway (7.3%) without incurring higher costs. Exploratory analysis of WES as a first-tier diagnostic test indicates that WES may even be cost-saving, depending on the extent of other tests being omitted.
Our data support such a use of WES in pediatric neurology for disorders of presumed genetic origin.Genet Med advance online publication 23 March 2017.
Objectives
The majority of people with suspected genetic dystonia remain undiagnosed after maximal investigation, implying that a number of causative genes have not yet been recognized. We aimed to ...investigate this paucity of diagnoses.
Methods
We undertook weighted burden analysis of whole‐exome sequencing (WES) data from 138 individuals with unresolved generalized dystonia of suspected genetic etiology, followed by additional case‐finding from international databases, first for the gene implicated by the burden analysis (VPS16), and then for other functionally related genes. Electron microscopy was performed on patient‐derived cells.
Results
Analysis revealed a significant burden for VPS16 (Fisher's exact test p value, 6.9 × 109). VPS16 encodes a subunit of the homotypic fusion and vacuole protein sorting (HOPS) complex, which plays a key role in autophagosome‐lysosome fusion. A total of 18 individuals harboring heterozygous loss‐of‐function VPS16 variants, and one with a microdeletion, were identified. These individuals experienced early onset progressive dystonia with predominant cervical, bulbar, orofacial, and upper limb involvement. Some patients had a more complex phenotype with additional neuropsychiatric and/or developmental comorbidities. We also identified biallelic loss‐of‐function variants in VPS41, another HOPS‐complex encoding gene, in an individual with infantile‐onset generalized dystonia. Electron microscopy of patient‐derived lymphocytes and fibroblasts from both patients with VPS16 and VPS41 showed vacuolar abnormalities suggestive of impaired lysosomal function.
Interpretation
Our study strongly supports a role for HOPS complex dysfunction in the pathogenesis of dystonia, although variants in different subunits display different phenotypic and inheritance characteristics. ANN NEUROL 2020;88:867–877
Maternal cell contamination (MCC) poses a risk for misdiagnosis in prenatal genetic testing, and is examined in accredited diagnostic laboratories However, the awareness of possible MCC in ...perinatal/postnatal genetic testing, mainly of umbilical cord blood (CB), is lower.
We investigated the rate of MCC in DNA from both umbilical CB samples and umbilical cord samples that were sent to our diagnostic laboratory for diagnostic testing between 1995 and 2021 (n = 236).
MCC was detected in 4% of umbilical CB samples, and in one umbilical cord sample. Particularly tests enriching for a specific variant are very sensitive for low amounts of MCC, as we emphasize here with a false positive diagnosis of myotonic dystrophy type 1 in a newborn.
Overall, with appropriate collection and use, umbilical CB and umbilical cord samples are suitable for genetic testing based on the low rates of MCC and misdiagnosis. These findings do however underline the importance of routine MCC testing in umbilical CB samples and umbilical cord samples for both requesting clinicians and diagnostic genetic laboratories.
Copy-number variation is a common source of genomic variation and an important genetic cause of disease. Microarray-based analysis of copy-number variants (CNVs) has become a first-tier diagnostic ...test for patients with neurodevelopmental disorders, with a diagnostic yield of 10-20%. However, for most other genetic disorders, the role of CNVs is less clear and most diagnostic genetic studies are generally limited to the study of single-nucleotide variants (SNVs) and other small variants. With the introduction of exome and genome sequencing, it is now possible to detect both SNVs and CNVs using an exome- or genome-wide approach with a single test.
We performed exome-based read-depth CNV screening on data from 2,603 patients affected by a range of genetic disorders for which exome sequencing was performed in a diagnostic setting.
In total, 123 clinically relevant CNVs ranging in size from 727 bp to 15.3 Mb were detected, which resulted in 51 conclusive diagnoses and an overall increase in diagnostic yield of ~2% (ranging from 0 to -5.8% per disorder).
This study shows that CNVs play an important role in a broad range of genetic disorders and that detection via exome-based CNV profiling results in an increase in the diagnostic yield without additional testing, bringing us closer to single-test genomics.Genet Med advance online publication 27 October 2016.
PDF
