Diagnostic Utility Of Exome Sequencing Research Articles

Looks Can Be Deceiving: Diagnostic Power of Exome Sequencing in Debunking 15q11.2 Copy Number Variations

Background/Objectives: The pathogenetic role of 15q11.2 Copy Number Variations (CNVs) remains contentious in the scientific community, as microdeletions and microduplications in this region are linked to neurodevelopmental disorders with variable expressivity. This study aims to explore the diagnostic utility of Exome Sequencing (ES) in a cohort of pediatric patients with 15q11.2 CNVs. Methods: We enrolled 35 probands with 15q11.2 microdeletions or microduplications from two genetic centers between January 2021 and January 2023. Chromosomal Microarray Analysis (CMA) and ES were performed with written consent obtained from all parents. Pathogenic variants were classified according to ACMG guidelines. Results: CMA identified additional pathogenic CNVs in 3 of 35 children (9%). Subsequent ES revealed likely pathogenic or pathogenic variants in 11 of 32 children (34%). Notably, a higher percentage of isolated autism spectrum disorder (ASD) diagnoses was observed in patients without other CNVs or point mutations (p = 0.019). Conclusions: The ES analysis provided a diagnostic yield of 34% in this pediatric cohort with 15q11.2 CNVs. While the study does not dismiss the contribution of the CNV to the clinical phenotype, the findings suggest that ES may uncover the underlying causes of neurodevelopmental disorders. Continuous monitoring and further genetic testing are recommended for all 15q11.2 CNV carriers to optimize clinical management and familial counseling.

Diagnostic utility of exome sequencing followed by research reanalysis in human brain malformations.

This study aimed to determine the diagnostic yield of singleton exome sequencing and subsequent research-based trio exome analysis in children with a spectrum of brain malformations seen commonly in clinical practice. We recruited children ≤ 18 years old with a brain malformation diagnosed by magnetic resonance imaging and consistent with an established list of known genetic causes. Patients were ascertained nationally from eight tertiary paediatric centres as part of the Australian Genomics Brain Malformation Flagship. Chromosome microarray was required for all children, and those with pathogenic copy number changes were excluded. Cytomegalovirus polymerase chain reaction on neonatal blood spots was performed on all children with polymicrogyria with positive patients excluded. Singleton exome sequencing was performed through a diagnostic laboratory and analysed using a clinical exome sequencing pipeline. Undiagnosed patients were followed up in a research setting, including reanalysis of the singleton exome data and subsequent trio exome sequencing. A total of 102 children were recruited. Ten malformation subtypes were identified with the commonest being polymicrogyria (36%), pontocerebellar hypoplasia (14%), periventricular nodular heterotopia (11%), tubulinopathy (10%), lissencephaly (10%) and cortical dysplasia (9%). The overall diagnostic yield for the clinical singleton exome sequencing was 36%, which increased to 43% after research follow-up. The main source of increased diagnostic yield was the reanalysis of the singleton exome data to include newly discovered gene-disease associations. One additional diagnosis was made by trio exome sequencing. The highest phenotype-based diagnostic yields were for cobblestone malformation, tubulinopathy and lissencephaly and the lowest for cortical dysplasia and polymicrogyria. Pathogenic variants were identified in 32 genes, with variants in 6/32 genes occurring in more than one patient. The most frequent genetic diagnosis was pathogenic variants in TUBA1A. This study shows that over 40% of patients with common brain malformations have a genetic aetiology identified by exome sequencing. Periodic reanalysis of exome data to include newly identified genes was of greater value in increasing diagnostic yield than the expansion to trio exome. This study highlights the genetic and phenotypic heterogeneity of brain malformations, the importance of a multidisciplinary approach to diagnosis and the large number of patients that remain without a genetic diagnosis despite clinical exome sequencing and research reanalysis.

Diagnostic Utility of Exome Sequencing Among Israeli Children With Kidney Failure

Genetic etiologies are estimated to account for a large portion of chronic kidney diseases (CKD) in children. However, data are lacking regarding the true prevalence of monogenic etiologies stemming from an unselected population screen of children with advanced CKD. We conducted a national multicenter prospective study of all Israeli pediatric dialysis units to provide comprehensive "real-world" evidence for the genetic basis of childhood kidney failure in Israel. We performed exome sequencing and assessed the genetic diagnostic yield. Between 2019 and 2022, we recruited approximately 88% (n= 79) of the children on dialysis from all 6 Israeli pediatric dialysis units. We identified genetic etiologies in 36 of 79 (45%) participants. The most common subgroup of diagnostic variants was in congenital anomalies of the kidney and urinary tract causing genes (e.g., EYA1, HNF1B, PAX2, COL4A1, and NFIA) which together explain 28% of all monogenic etiologies. This was followed by mutations in genes causing renal cystic ciliopathies (e.g., NPHP1, NPHP4, PKHD1, and BBS9), steroid-resistant nephrotic syndrome (e.g., LAGE3, NPHS1, NPHS2, LMX1B, and SMARCAL1) and tubulopathies (e.g., CTNS and AQP2). The genetic diagnostic yield was higher among Arabs compared to Jewish individuals (55% vs. 29%) and in children from consanguineous compared to nonconsanguineous families (63% vs. 29%). In 5 participants (14%) with genetic diagnoses, the molecular diagnosis did not correspond with the pre-exome diagnosis. Genetic diagnosis has a potential influence on clinical management in 27 of 36 participants (75%). Exome sequencing in an unbiased Israeli nationwide dialysis-treated kidney failure pediatric cohort resulted in a genetic diagnostic yield of 45% and can often affect clinical decision making.

Clinical Integration of Genomic Testing in Kidney Transplantation Clinics.

In 2019, Groopman et al set out to assess the diagnostic utility of exome sequencing in a diverse, all-cause, chronic kidney disease cohort of over 3000 individuals and demonstrated the existence of a monogenic form of kidney disease in approximately 10% of cases.1 Furthermore, the diagnostic yield was even higher (17.1%) among individuals with kidney disease of unknown etiology. This groundbreaking study brought to light how the high genetic and phenotypic heterogeneity of monogenic diseases, coupled with our limited appreciation for the full spectrum of their clinical manifestations, contributes to diagnosis delays and disease misclassifications. Today, with greater clinical access to molecular tests, we are increasingly able to establish the molecular diagnosis for a growing number of monogenic kidney diseases. Recent studies have demonstrated the clinical utility of genomic testing as part of the diagnostic evaluation of patients with kidney disease, which supports personalized management by guiding choice of therapy, informing targeted disease surveillance, and identifying at-risk family members for cascade screening.2-5 However, genomic tests are still relatively new diagnostic tools for most subspecialties of medicine and surgery and not widely used in routine care. Although various factors likely contribute to their limited clinical use, it is important to understand that broader implementation of genomic testing in the clinical domain relies largely on providers’ appreciation for a breadth of core knowledge specific to genomic medicine.6 This includes understanding specialized terminology (eg, penetrance, phenotype, carrier frequency, etc), various sequencing approaches (eg, single-gene tests, gene panels, exome and genome sequencing, etc), categories of genomic results (eg, polygenic risk scores, risk alleles, primary results and otherwise medically actionable secondary findings, variants of uncertain significance, etc), and an awareness of the complex ethical, legal, and technical considerations that come with genomic sequencing. Given the complexity of using genomic data in medical decision-making, as well as kidney experts, limited experience diagnosing monogenic diseases and using genomic technologies, a multidisciplinary approach is often needed to guide interpretation of raw genomic data, clinical correlation, and the integration of diagnostic findings in patient care.2-5,7-9 In this issue of Transplantation, El Ters et al share their experience using molecular testing in the evaluation of individuals presenting to Mayo’s adult kidney transplant clinic. In total, 30 prospective transplant recipients (the majority [n = 24, 80%] who had histopathologic evidence of focal segmental glomerulosclerosis pattern lesions on biopsy) plus an additional 5 prospective living donors underwent clinical-grade exome sequencing to try to identify an underlying monogenic form of kidney disease. In their study, they used a semi-masked approach to analyze the exome data by focusing their search to deleterious variants in a subset of 344 genes associated with hereditary forms of kidney disease. They considered potentially diagnostic variants to be those that met the American College of Medical Genetics and Genomics’ standards for classification as either pathogenic or likely pathogenic.10 The authors then reviewed these identified variants with a multidisciplinary team of molecular and clinical genetic specialists to ensure agreement in the adjudication of each variant’s classification and ensure the findings were consistent with the patients’ clinical presentations before deeming the findings diagnostic. They then reported that among the 30 prospective transplant recipients who underwent sequencing, 13 (43%) individuals were found to have a diagnostic variant in one of these known kidney disease genes. The most common findings they reported were deleterious variants in the COL4A3/4/5 genes, which were identified in 7 of these 13 individuals, and are diagnostic for type IV collagen-associated nephropathies, which encompasses Alport syndrome, thin basement membrane disease, hereditary nephritis, and some hereditary forms of focal segmental glomerulosclerosis. In addition, the authors report that 1 of the 5 prospective living donors that underwent sequencing was found to have a pathogenic/likely pathogenic variant in a gene associated with a hereditary form of kidney disease and was excluded from donation. Overall, this study reinforces the clinical utility of using molecular testing in the diagnostic evaluation of patients with kidney disease, specifically within the workflow of a kidney transplant clinic. Although the authors do not describe a novel approach in their implementation of genomic testing in the context of kidney disease patients, they demonstrate how diagnostic findings can inform the selection of prospective living kidney donors among at-risk family members, underscoring the important role genomic testing can play in transplant clinics. Moreover, their study reminds us of the precaution providers must take when adopting clinical genomics in patient care and the value added by employing a multidisciplinary approach that includes genomic experts. Studies like this one offer institution-specific experiences and lessons learned that can inform future genomic implementation initiatives. Further study is needed to identify innovative approaches that effectively advance the operationalization of precision nephrology care that can be scaled for use across distinct workflows and institutions.

Diagnostic utility of exome sequencing for inherited peripheral neuropathies

Introduction. Hereditary motor and sensory neuropathies, a highly genetic heterogeneous group of disorders, have a phenotype caused by peripheral nerve damage.Purpose of the study – to assess the extent of genetic heterogeneity of hereditary motor and sensory neuropathies in Russian patients and to evaluate the diagnostic effectiveness of using full-exome research methods to find the genetic cause of hereditary motor and sensory neuropathies.Materials and methods. The material for the study was DNA samples from 51 patients and their family members referred for whole exome sequencing to the DNA-diagnostics laboratory of Research Centre for Medical Genetics in 2017–2019. Methods: whole exome sequencing, Sanger sequencing, restriction fragment length polymorphism.Results. Whole exome sequencing in combination with segregation analysis of the pathogenic variants in families allowed to determine the cause of the disease in 41 % of cases. In another 16 % of cases, candidate genetic variants as a possible cause of the disease were revealed, but additional studies are needed to confirm it. The most frequently mutated gene was MFN2 caused neuropathy in 6 unrelated families. MPZ gene mutations were detected in two families, AARS gene mutations were revealed in another two families, and mutations in GJB1, HINT1, INF2, LRSAM1, LITAF, MME, NEFL, WWOX were detected once. Among the causal variants, mutations in B4GALNT1 caused spastic paraplegia, in COL6A1 led to Bethlem’s congenital muscular dystrophy, and in SYT2 caused congenital myasthenic syndrome indicating difficulties in differential diagnosis of inherited neuromuscular disorders. A PMP22 duplication was detected in 2 families prior to whole exome sequencing.Conclusion. Whole exome sequencing is very important for finding the molecular cause of hereditary motor and sensory neuropathies. In most cases, additional methods should be used to clarify the pathogenicity of variants detected by whole exome sequencing. However, it is necessary to remember that the most common cause of the disease is a large duplication of the region 17p11.2.

Diagnostic Utility of Exome Sequencing for Kidney Disease

BackgroundExome sequencing is emerging as a first-line diagnostic method in some clinical disciplines, but its usefulness has yet to be examined for most constitutional disorders in adults, including chronic kidney disease, which affects more than 1 in 10 persons globally.MethodsWe conducted exome sequencing and diagnostic analysis in two cohorts totaling 3315 patients with chronic kidney disease. We assessed the diagnostic yield and, among the patients for whom detailed clinical data were available, the clinical implications of diagnostic and other medically relevant findings.ResultsIn all, 3037 patients (91.6%) were over 21 years of age, and 1179 (35.6%) were of self-identified non-European ancestry. We detected diagnostic variants in 307 of the 3315 patients (9.3%), encompassing 66 different monogenic disorders. Of the disorders detected, 39 (59%) were found in only a single patient. Diagnostic variants were detected across all clinically defined categories, including congenital or cystic renal disease (127 of 531 patients [23.9%]) and nephropathy of unknown origin (48 of 281 patients [17.1%]). Of the 2187 patients assessed, 34 (1.6%) had genetic findings for medically actionable disorders that, although unrelated to their nephropathy, would also lead to subspecialty referral and inform renal management.ConclusionsExome sequencing in a combined cohort of more than 3000 patients with chronic kidney disease yielded a genetic diagnosis in just under 10% of cases. (Funded by the National Institutes of Health and others.)

Diagnostic utility of exome sequencing in the evaluation of neuromuscular disorders.

ObjectiveTo evaluate the diagnostic yield and workflow of genome-scale sequencing in patients with neuromuscular disorders (NMDs).MethodsWe performed exome sequencing in 93 undiagnosed patients with various NMDs for whom a molecular diagnosis was not yet established. Variants on both targeted and broad diagnostic gene lists were identified. Prior diagnostic tests were extracted from the patient's medical record to evaluate the use of exome sequencing in the context of their prior diagnostic workup.ResultsThe overall diagnostic yield of exome sequencing in our cohort was 12.9%, with one or more pathogenic or likely pathogenic variants identified in a causative gene associated with the patient's disorder. Targeted gene lists had the same diagnostic yield as a broad NMD gene list in patients with clear neuropathy or myopathy phenotypes, but evaluation of a broader set of disease genes was needed for patients with complex NMD phenotypes. Most patients with NMD had undergone prior testing, but only 10/16 (63%) of these procedures, such as muscle biopsy, were informative in pointing to a final molecular diagnosis.ConclusionsGenome-scale sequencing or analysis of a panel of relevant genes used early in the evaluation of patients with NMDs can provide or clarify a diagnosis and minimize invasive testing in many cases.

Utility and limitations of exome sequencing as a genetic diagnostic tool for conditions associated with pediatric sudden cardiac arrest/sudden cardiac death.

BackgroundConditions associated with sudden cardiac arrest/death (SCA/D) in youth often have a genetic etiology. While SCA/D is uncommon, a pro-active family screening approach may identify these inherited structural and electrical abnormalities prior to symptomatic events and allow appropriate surveillance and treatment. This study investigated the diagnostic utility of exome sequencing (ES) by evaluating the capture and coverage of genes related to SCA/D.MethodsSamples from 102 individuals (13 with known molecular etiologies for SCA/D, 30 individuals without known molecular etiologies for SCA/D and 59 with other conditions) were analyzed following exome capture and sequencing at an average read depth of 100X. Reads were mapped to human genome GRCh37 using Novoalign, and post-processing and analysis was done using Picard and GATK. A total of 103 genes (2,190 exons) related to SCA/D were used as a primary filter. An additional 100 random variants within the targeted genes associated with SCA/D were also selected and evaluated for depth of sequencing and coverage. Although the primary objective was to evaluate the adequacy of depth of sequencing and coverage of targeted SCA/D genes and not for primary diagnosis, all patients who had SCA/D (known or unknown molecular etiologies) were evaluated with the project’s variant analysis pipeline to determine if the molecular etiologies could be successfully identified.ResultsThe majority of exons (97.6 %) were captured and fully covered on average at minimum of 20x sequencing depth. The proportion of unique genomic positions reported within poorly covered exons remained small (4 %). Exonic regions with less coverage reflect the need to enrich these areas to improve coverage. Despite limitations in coverage, we identified 100 % of cases with a prior known molecular etiology for SCA/D, and analysis of an additional 30 individuals with SCA/D but no known molecular etiology revealed a diagnostic answer in 5/30 (17 %). We also demonstrated 95 % of 100 randomly selected reported variants within our targeted genes would have been picked up on ES based on our coverage analysis.ConclusionsES is a helpful clinical diagnostic tool for SCA/D given its potential to successfully identify a molecular diagnosis, but clinicians should be aware of limitations of available platforms from technical and diagnostic perspectives.Electronic supplementary materialThe online version of this article (doi:10.1186/s40246-015-0038-y) contains supplementary material, which is available to authorized users.

A commentary on The diagnostic utility of exome sequencing in Joubert syndrome and related disorders

A commentary on The diagnostic utility of exome sequencing in Joubert syndrome and related disorders

The diagnostic utility of exome sequencing in Joubert syndrome and related disorders

Joubert syndrome (JS) and related disorders (JSRD) are autosomal recessive and X-linked disorders characterized by hypoplasia of the cerebellar vermis with a characteristic 'molar tooth sign' on brain imaging and accompanying neurological symptoms including episodic hyperpnoea, abnormal eye movements, ataxia and intellectual disability. JSRD are clinically and genetically heterogeneous, and, to date, a total of 17 causative genes are known. We applied whole-exome sequencing (WES) to five JSRD families and found mutations in all: either CEP290, TMEM67 or INPP5E was mutated. Compared with conventional Sanger sequencing, WES appears to be advantageous with regard to speed and cost, supporting its potential utility in molecular diagnosis.