Clinical Whole Exome Sequencing Research Articles

Identification of mutations using whole exome sequencing in eight fetuses presenting with short femur

BackgroundFemur length is one of the important indicators for evaluating fetal growth and development. Short femur is a common prenatal ultrasound finding. This study aimed to investigate the genetic etiology of fetal short femur using trio-based whole exome sequencing (WES), so as to provide evidence for prenatal diagnosis and evaluate the application of trio-WES in prenatal diagnosis of fetal short femur. MethodsWe retrospectively analyzed the clinical phenotype and WES results of eight fetuses with short femur diagnosed by prenatal ultrasound. The results of WES were validated by Sanger sequencing. The pathogenicity of the mutations was evaluated. Minigene assay was performed to investigate the effects of intronic mutation on mRNA splicing. The pregnancy outcome was followed up. ResultsA total of seven mutations were detected in eight short femur fetuses. Among them, COL2A1 (p.Gly1107Glu), GNAS (p.Lys739Glu) and FGFR3 (c.1075 + 95C > G) were novel mutations that had not been reported. Minigene assay showed that c.1075 + 95C > G in FGFR3 partially retained a 90 bp sequence in intron 8. ConclusionsThe results of this study enriched the mutant spectrums of COL2A1, GNAS and FGFR3 genes, and demonstrated the value of trio-WES in prenatal diagnosis of fetuses with short femur.

Harmonizing tumor mutational burden analysis: Insights from a multicenter study using in silico reference data sets in clinical whole-exome sequencing (WES).

Tumor mutational burden (TMB) is a significant biomarker for predicting immune checkpoint inhibitor response, but the clinical performance of whole-exome sequencing (WES)-based TMB estimation has received less attention compared to panel-based methods. This study aimed to assess the reliability and comparability of WES-based TMB analysis among laboratories under routine testing conditions. A multicenter study was conducted involving 24 laboratories in China using in silico reference data sets. The accuracy and comparability of TMB estimation were evaluated using matched tumor-normal data sets. Factors such as accuracy of variant calls, limit of detection (LOD) of WES test, size of regions of interest (ROIs) used for TMB calculation, and TMB cutoff points were analyzed. The laboratories consistently underestimated the expected TMB scores in matched tumor-normal samples, with only 50% falling within the ±30% TMB interval. Samples with low TMB score (<2.5) received the consensus interpretation. Accuracy of variant calls, LOD of the WES test, ROI, and TMB cutoff points were important factors causing interlaboratory deviations. This study highlights real-world challenges in WES-based TMB analysis that need to be improved and optimized. This research will aid in the selection of more reasonable analytical procedures to minimize potential methodologic biases in estimating TMB in clinical exome sequencing tests. Harmonizing TMB estimation in clinical testing conditions is crucial for accurately evaluating patients' response to immunotherapy.

Abstract 6908: Integrating PDX GBM in vivo models with patient history and whole exome sequencing: Advancing relevance and precision in preclinical studies

Abstract Glioblastoma poses a significant challenge in oncology, necessitating improved translational models that faithfully replicate the intricate tumor microenvironment. Patient-derived xenograft (PDX) models, are key in glioblastoma multiforme (GBM) preclinical research. PDX models (orthotopic and subcutaneous) provide a more clinically relevant representation compared to CDX xenografts. Analyzing PDX tumors in their native environment allows for a nuanced understanding of GBM biology, tumor-stroma interactions, and treatment responses. This abstract explores the rationale and advantages of utilizing PDX GBM in vivo models coupled with patient diagnosis and treatment history, empowered by tumor whole exome sequencing (WES), to enhance the relevance of preclinical studies in evaluating GBM-specific treatments. TD2 has access to over 100 GBM PDX tumors models with fully annotated patient details and treatment history. Moreover, 68 of these PDXs also have WES data, that allows most appropriate model selection to ensure alignment with the therapeutic development strategy. Below is a detailed analysis of two GBM PDX models, GBM6 and GBM46, using patient history and WES data to define criteria for success in assessing novel preclinical therapeutic agents. GBM6, was obtained prior to treatment from a 65-year-old man with glioblastoma multiforme, that contains 642 mutations, including EGFR VIII amplification. GBM46, was obtained from a 55-year-old man with prior treatment, contained 646 mutations, including RAS pathway alterations and EGFR (MET and FLT3) amplification. When grown subcutaneously the anti-tumor activity of cetuximab against GBM6 and GBM46 resulted in 41% and 48% growth inhibition, respectively. Traditional treatments have also been evaluated in the orthotopic setting, with temozolomide (TMZ) showing a nominal improvement in lifespan (11%) as expected for the unmethylated MGMT found in the GBM6 model. By contrast, GBM46 from a patient with prior treatment with TMZ, OSI-774, radiation, and BCNU, exhibited a 58% increase in overall lifespan when treated with TMZ alone. Orthotopic PDX GBM models preserve many molecular characteristics, enabling the study of intrinsic GBM mutations for developing novel therapeutic strategies. While orthotopic models better capture GBM's dynamic nature, traditional xenograft systems remain valuable as rapid screening tools. Models with defined clinical treatment history and WES data allows for testing new agents in models that more closely resemble the clinical development path for a given new agent and aids in deciphering factors influencing therapeutic resistance. These models have the potential to reshape preclinical research paradigms, accelerating the translation of promising therapeutic agents increasing the likelihood of clinical benefit for patients with glioblastoma multiforme. Citation Format: Erin E. Trachet, Paul M. Gonzales, Stephen Gately. Integrating PDX GBM in vivo models with patient history and whole exome sequencing: Advancing relevance and precision in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6908.

Development of a coding SNP panel for tracking the origin of whole-exome sequencing samples.

Whole-exome sequencing (WES) is widely used to diagnose complex genetic diseases and rare conditions. The implementation of a robust and effective quality control system for sample identification and tracking throughout the WES process is essential. We established a multiplex panel that included 22 coding single-nucleotide polymorphism (cSNP) loci. The personal identification and paternity identification abilities of the panel were evaluated, and a preliminary validation of the practical feasibility of the panel was conducted in a clinical WES case. These results indicate that the cSNP panel could be a useful tool for sample tracking in WES.

Clinicopathological and molecular genetic characteristics of ELOC mutated renal cell carcinoma

Objective: To investigate the clinicopathological features, molecular genetic features, differential diagnosis and prognosis of ELOC mutated renal cell carcinoma. Methods: From January 2015 to June 2022, 11 cases of renal cell carcinoma with clear-cell morphology, expression of CAⅨ and CK7 and no 3p deletion were collected. Two cases of ELOC mutant renal cell carcinoma were diagnosed using whole exome sequencing (WES). The clinical features, morphology, immunophenotype, FISH and WES results were analyzed. The relevant literature was reviewed. Results: The two patients were both male, aged 29 and 51 years, respectively. They were both found to have a renal mass by physical examination. The maximum diameters of the tumors were 3.5 cm and 2.0 cm, respectively. At the low magnification, the tumors were well-defined. The tumor cells showed a pushing border and were separated by thick fibrous bands, forming nodules. The tumor cells were arranged in a variety of patterns, including tubular, papillary, solid nest or alveolar. At high magnification, the tumor cells were large, with well-defined cell borders and clear cytoplasm or fine eosinophilic granules. CAⅨ was diffusely box-like positive in both cases. Case 1 was partially and moderately positive for CK7, strongly positive for CD10, diffusely and moderately positive for P504S, and weakly positive for 34βE12. In case 2, CK7 and CD10 were both partially, moderately positive and P504s were diffusely positive, but 34βE12 was negative. FISH results showed that both cases had no 3p deletion. ELOC c.235T>A (p.Y79N) mutation was identified using WES in case 1, while ELOC c.236_237inv (p.Y79C) mutation was identified in case 2. Conclusions: As a new clinical entity, ELOC mutated renal cell carcinoma may be underdiagnosed due to its overlap with clear cell renal cell carcinoma in morphology and immunophenotype. The diagnosis of renal cell carcinoma with ELOC mutation should be confirmed by morphology, immunohistochemistry, FISH and gene mutation detection. However, more additional cases are needed to explain its biological behavior and prognosis.

Multicentric pilot study to standardize clinical whole exome sequencing (WES) for cancer patients

A growing number of druggable targets and national initiatives for precision oncology necessitate broad genomic profiling for many cancer patients. Whole exome sequencing (WES) offers unbiased analysis of the entire coding sequence, segmentation-based detection of copy number alterations (CNAs), and accurate determination of complex biomarkers including tumor mutational burden (TMB), homologous recombination repair deficiency (HRD), and microsatellite instability (MSI). To assess the inter-institution variability of clinical WES, we performed a comparative pilot study between German Centers of Personalized Medicine (ZPMs) from five participating institutions. Tumor and matched normal DNA from 30 patients were analyzed using custom sequencing protocols and bioinformatic pipelines. Calling of somatic variants was highly concordant with a positive percentage agreement (PPA) between 91 and 95% and a positive predictive value (PPV) between 82 and 95% compared with a three-institution consensus and full agreement for 16 of 17 druggable targets. Explanations for deviations included low VAF or coverage, differing annotations, and different filter protocols. CNAs showed overall agreement in 76% for the genomic sequence with high wet-lab variability. Complex biomarkers correlated strongly between institutions (HRD: 0.79–1, TMB: 0.97–0.99) and all institutions agreed on microsatellite instability. This study will contribute to the development of quality control frameworks for comprehensive genomic profiling and sheds light onto parameters that require stringent standardization.

Computational pharmacogenotype extraction from clinical next-generation sequencing.

Next-generation sequencing (NGS), including whole genome sequencing (WGS) and whole exome sequencing (WES), is increasingly being used for clinic care. While NGS data have the potential to be repurposed to support clinical pharmacogenomics (PGx), current computational approaches have not been widely validated using clinical data. In this study, we assessed the accuracy of the Aldy computational method to extract PGx genotypes from WGS and WES data for 14 and 13 major pharmacogenes, respectively. Germline DNA was isolated from whole blood samples collected for 264 patients seen at our institutional molecular solid tumor board. DNA was used for panel-based genotyping within our institutional Clinical Laboratory Improvement Amendments- (CLIA-) certified PGx laboratory. DNA was also sent to other CLIA-certified commercial laboratories for clinical WGS or WES. Aldy v3.3 and v4.4 were used to extract PGx genotypes from these NGS data, and results were compared to the panel-based genotyping reference standard that contained 45 star allele-defining variants within CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, CYP4F2, DPYD, G6PD, NUDT15, SLCO1B1, TPMT, and VKORC1. Mean WGS read depth was >30x for all variant regions except for G6PD (average read depth was 29 reads), and mean WES read depth was >30x for all variant regions. For 94 patients with WGS, Aldy v3.3 diplotype calls were concordant with those from the genotyping reference standard in 99.5% of cases when excluding diplotypes with additional major star alleles not tested by targeted genotyping, ambiguous phasing, and CYP2D6 hybrid alleles. Aldy v3.3 identified 15 additional clinically actionable star alleles not covered by genotyping within CYP2B6, CYP2C19, DPYD, SLCO1B1, and NUDT15. Within the WGS cohort, Aldy v4.4 diplotype calls were concordant with those from genotyping in 99.7% of cases. When excluding patients with CYP2D6 copy number variation, all Aldy v4.4 diplotype calls except for one CYP3A4 diplotype call were concordant with genotyping for 161 patients in the WES cohort. Aldy v3.3 and v4.4 called diplotypes for major pharmacogenes from clinical WES and WGS data with >99% accuracy. These findings support the use of Aldy to repurpose clinical NGS data to inform clinical PGx.

Expanding the Allelic Heterogeneity of ANO10-Associated Autosomal Recessive Cerebellar Ataxia.

The term autosomal recessive cerebellar ataxia (ARCA) encompasses a diverse group of heterogeneous degenerative disorders of the cerebellum. Spinocerebellar ataxia autosomal recessive 10 (SCAR10) is a distinct classification of cerebellar ataxia caused by variants in the ANO10 gene. Little is known about the molecular role of ANO10 or its role in disease. There is a wide phenotypic spectrum among patients, even among those with the same or similar genetic variants. This study aimed to characterize the molecular consequences of variants in ANO10 and determine their pathologic significance in patients diagnosed with SCAR10. We presented 4 patients from 4 families diagnosed with spinocerebellar ataxia with potential pathogenic variants in the ANO10 gene. Patients underwent either clinical whole-exome sequencing or screening of a panel of known neuromuscular disease genes. Effects on splicing were studied using reverse transcriptase PCR to analyze complementary DNA. Western blots were used to examine protein expression. One individual who presented clinically at a much earlier age than typical was homozygous for an ANO10 variant (c.1864A > G [p.Met622Val]) that produces 2 transcription products by altering an exonic enhancer site. Two patients, both of Lebanese descent, had a homozygous intronic splicing variant in ANO10 (c.1163-9A > G) that introduced a cryptic splice site acceptor, producing 2 alternative transcription products and no detectable wild-type protein. Both these variants have not yet been associated with SCAR10. The remaining patient was found to have compound heterozygous variants in ANO10 previously associated with SCAR10 (c.132dupA [p.Asp45Argfs*9] and c.1537T > C [p.Cys513Arg]). We presented rare pathogenic variants adding to the growing list of ANO10 variants associated with SCAR10. In addition, we described an individual with a much earlier age at onset than usually associated with ANO10 variants. This expands the phenotypic and allelic heterogeneity of ANO10-associated ARCA.

Reduced resource utilization with early use of next-generation sequencing in rare genetic diseases in an Asian cohort.

Children with genetic diseases endure a prolonged and costly "diagnostic odyssey." The use of whole exome sequencing (WES) and whole genome sequencing (WGS) has improved the diagnosis rate, ending the odyssey. However, the additionalcosts associated WES/WGS has impeded their adoption in Asian settings. We aim to estimate the expected change to the mean number of diagnostic tests used, and the associated costs from a decision to use WES early in the diagnostic pathways of pediatric phenotypes, as compared to Existing Practice. Retrospective data from a patient cohort recruited under the Singapore Undiagnosed Disease Program from a tertiary hospital in Singapore, for the period October 2004 to September 2020, was analyzed. Four phenotype-specific subgroups were used: multiple congenital anomalies (MCA) without developmental delay; global developmental delay (GDD); neuromuscular disorder (NMD) and primary immunodeficiency disorder (PID). Patients had undergone a traditional diagnostic pathway and received a diagnosis either through clinical exome or WES or WGS. A costs only analysis was performed, by tabulating the outcomes "test quantity" and "test costs" incurred by patients. The outcomes were compared with alternate diagnostic pathways which incorporates the early introduction of WES trio testing. To include uncertainty in cost outcomes, simulation studies were done on uncertain parameters. Cost outcomes are reported in Singapore dollars (S$). The 92 included patients had MCA (n=48), GDD (n=29), NMD (n=10), or PID (n=5). Patients were aged between 18 days and 26 years, 52.2% were males. The majority were of Chinese ethnicity (81.5%). If patients had access to WES directly, test quantity reduced by 97.38% for MCA, 96.98% for GDD, 96.56% for NMD, and 99.84% for PID. The expected cost savings per patient were $5940 for MCA (US$4433), $5342 for GDD (US$3986), $4622 for NMD (US$3449), and $58,497 for PID (US$43,654). Uncertainty assessment for MCA and GDD patients showed a respective likelihood of 86.9% and 97.4% for cost savings. Adoption of alternate diagnostic pathways with early WES in selected pediatric subgroups are likelt to reduce costs, when compared to Existing Practice. Benefits arising from earlier diagnosis, and the potential cost savings could mitigate the large initial cost of implementing WES in Asian settings.

Italian Precision Medicine in Pediatric Oncology: Moving beyond Actionable Alterations

Neuroblastoma (NB) is the most common extracranial solid tumor encountered in childhood. Although there has been significant improvement in the outcomes of patients with high-risk disease, the prognosis for patients with metastatic relapse or refractory disease is poor. Hence, the clinical integration of genome sequencing into standard clinical practice is necessary in order to develop personalized therapy for children with relapsed or refractory disease. The PeRsonalizEdMEdicine (PREME) project focuses on the design of innovative therapeutic strategies for patients suffering from relapsed NB. We performed whole exome sequencing (WES) of patient-matched tumor-normal samples to identify genetic variants amenable to precision medicine. Specifically, two patients were studied (First case: a three-year-old male with early relapsed NB; Second case: a 20-year-old male who relapsed 10 years after the first diagnosis of NB). Results were reviewed by a multi-disciplinary molecular tumor board (MTB) and clinical reports were issued to the ordering physician. WES revealed the mutation c.G320C in the CUL4A gene in case 1 and the mutation c.A484G in the PSMC2 gene in case 2. Both patients were treated according to these actionable alterations, with promising results. The effective treatment of NB is one of the main challenges in pediatric oncology. In the era of precision medicine, the need to design new therapeutic strategies for NB is fundamental. Our results demonstrate the feasibility of incorporating clinical WES into pediatric oncology practice.

Three Cases of Familial Moyamoya Disease with RASA1 Mutations—A Case Report

The prevalence of moyamoya disease (MMD) is relatively high in East Asia, whereas the susceptibility genes of MMD have not been identified. Here, we reported 3 patients diagnosed with MMD from 1 single family, including a 53-year-old mother (case 1) and her 32-year-old and 29-year-old daughters (cases 2 and 3). The younger daughter was diagnosed with cerebral hemorrhage. Computed tomographic angiogram showed the typical signs of MMD in 3 patients. Clinical whole-exome sequencing was performed in 3 daughters of case 1, and RASA1 mutations in chr5: 87,376,389 and NM_002890.2: c.2012-4C>T were determined to have the strongest correlation with MMD. RASA1 mutations were verified in case 1, husband of case 1 and the descendant of case 3 by using Sanger sequencing. According to the findings of literature review, this is the first study indicating the association between RASA1 mutations and MMD.

Genetic variants in rare diseases identified by WES analysis

Next-generation sequencing tests have become a part of the diagnostic process in most fields of medicine. Especially with whole-exome sequencing (WES) studies, the rate of diagnosis has increased in rare hereditary diseases. In this study, we aimed to present the results together with the clinical findings of 65 cases whose diseases are suspected to be of genetic origin. Between 2016 and 2019, patients who underwent WES testing in Bursa Yüksek İhtisas Training and Research Hospital Medical Genetics Unit were retrospectively screened and included in the study with their analysis results and clinical findings. In 27 of the 65 cases (41.5 %) included in the study, 30 significant variants were found in relation to their clinical findings. Twenty of these variants (66.7 %) have not been previously reported in literature. Rare diseases encountered in patients within a wide age range, from the fetus to 66 years of age, are presented along with their clinical findings and WES results. Thus, this study contributes to the mutation spectrum of hereditary diseases.

Comparison of actionable events detected in cancer genomes by whole-genome sequencing, in silico whole-exome and mutation panels

BackgroundNext-generation sequencing is used in cancer research to identify somatic and germline mutations, which can predict sensitivity or resistance to therapies, and may be a useful tool to reveal drug repurposing opportunities between tumour types. Multigene panels are used in clinical practice for detecting targetable mutations. However, the value of clinical whole-exome sequencing (WES) and whole-genome sequencing (WGS) for cancer care is less defined, specifically as the majority of variants found using these technologies are of uncertain significance.Patients and methodsWe used the Cancer Genome Interpreter and WGS in 726 tumours spanning 10 cancer types to identify drug repurposing opportunities. We compare the ability of WGS to detect actionable variants, tumour mutation burden (TMB) and microsatellite instability (MSI) by using in silico down-sampled data to mimic WES, a comprehensive sequencing panel and a hotspot mutation panel.ResultsWe reveal drug repurposing opportunities as numerous biomarkers are shared across many solid tumour types. Comprehensive panels identify the majority of approved actionable mutations, with WGS detecting more candidate actionable mutations for biomarkers currently in clinical trials. Moreover, estimated values for TMB and MSI vary when calculated from WGS, WES and panel data, and are dependent on whether all mutations or only non-synonymous mutations were used. Our results suggest that TMB and MSI thresholds should not only be tumour-dependent, but also be sequencing platform-dependent.ConclusionsThere is a large opportunity to repurpose cancer drugs, and these data suggest that comprehensive sequencing is an invaluable source of information to guide clinical decisions by facilitating precision medicine and may provide a wealth of information for future studies. Furthermore, the sequencing and analysis approach used to estimate TMB may have clinical implications if a hard threshold is used to indicate which patients may respond to immunotherapy.

A Novel Mutation of ORNT1 Detected in a Hyperornithinemia-Hyperammonemia-Homocitrullinuria Syndrome Child by Clinical Whole-Exome Sequencing.

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, an inborn error of metabolism, is an inherited syndrome caused by loss-of-function mutations in the SLC25A15, resulting in ornithine translocase1 (ORNT1) deficiency. Disrupted ornithine transportation in an affected individual usually manifests with the accumulation of intermediate metabolites, leading to neurological impairment, hepatitis, and/or protein intolerance at various ages of onset. In this paper, we report a compound heterozygous mutation in SLC25A15 from a 2-year-old girl who presented with neurological alterations and hepatic failure. Before developing neurological sequelae, she had signs of globally delayed development. The accumulation of toxic metabolites may explain these neurological consequences. After biochemical confirmation of HHH, whole-exome sequencing (WES) was performed, which identified mutations at codons 21 and 179 of SLC25A15 that are predicted to result in the loss of function of ORNT1. Each of the mutations was found to be inherited from one of her parents. After therapy, her toxic metabolites decreased significantly. In conclusion, HHH syndrome frequently manifests with nonspecific symptoms and unapparent biochemical profiles, which may lead to delayed diagnosis. Correction of the accumulating metabolites is necessary to prevent irreversible neurological impairment. Furthermore, performing a WES provides a shortcut for accurate diagnosis.

Common disease-associated gene variants in a Saudi Arabian population.

BACKGROUND: Screening programs for the most prevalent conditions occurring in a country is an evidence-based prevention strategy. The burden of autosomal recessive disease variations in Saudi Arabia is high because of the highly consanguineous population. The optimal solution for estimating the carrier frequency of the most prevalent diseases is carrier screening.OBJECTIVES: Identify the most influential recessive alleles associated with disease in the Saudi population.DESIGN: We used clinical whole-exome sequencing data from an in-house familial database to evaluate the most prevalent genetic variations associated with disease in a Saudi population.SETTINGS: King Abdullah International Medical Research Center (KAIMRC) and King Abdulaziz Medical City.METHODS: Whole exome sequencing data obtained from clinical studies of family members, a cohort of 1314 affected and unaffected individuals, were filtered using the in-house pipeline to extract the most prevalent variant in the dataset.MAIN OUTCOME MEASURES: Most prevalent genetic variations associated with disease in the Saudi population.SAMPLE SIZE: 1314 affected and unaffected individuals.RESULTS: We identified 37 autosomal recessive variants and two heterozygous X-linked variants in 35 genes associated with the most prevalent disorders, which included hematologic (32%), endocrine (21%), metabolic (11%) and immunological (10%) diseases.CONCLUSION: This study provides an update of the most frequently occurring alleles, which support future carrier screening programs.LIMITATIONS: Single center that might represent the different regions but may be biased. In addition, most of the families included in the database are part of the proband's genetic identification for specific phenotypes.CONFLICT OF INTEREST: None.

Ending diagnostic odyssey using clinical whole-exome sequencing (CWES)

Abstract Objectives Most rare diseases are genetic diseases. Due to the diversity of rare diseases and the high likelihood of patients with rare diseases to be undiagnosed or misdiagnosed, it is not unusual that these patients undergo a long diagnostic odyssey before they receive a definitive diagnosis. This situation presents a clear need to set up a dedicated clinical service to end the diagnostic odyssey of patients with rare diseases. Methods Therefore, in 2014, we started an Undiagnosed Diseases Program in Hong Kong with the aim of ending the diagnostic odyssey of patients and families with rare diseases by clinical whole-exome sequencing (CWES), who have not received a definitive diagnosis after extensive investigation. Results In this program, we have shown that genetic diseases diagnosed by CWES were different from that using traditional approaches indicating that CWES is an essential tool to diagnose rare diseases and ending diagnostic odysseys. In addition, we identified several novel genes responsible for monogenic diseases. These include the TOP2B gene for autism spectrum disorder, the DTYMK gene for severe cerebral atrophy, the KIF13A gene for a new mosaic ectodermal syndrome associated with hypomelanosis of Ito, and the CDC25B gene for a new syndrome of cardiomyopathy and endocrinopathy. Conclusions With the incorporation of CWES in an Undiagnosed Diseases Program, we have ended diagnostic odysseys of patients with rare diseases in Hong Kong in the past 7 years. In this program, we have shown that CWES is an essential tool to end diagnostic odysseys. With the declining cost of next-generation sequencers and reagents, CWES set-ups are now affordable for clinical laboratories. Indeed, owing to the increasing availability of CWES and treatment modalities for rare diseases, precedence can be given to both common and rare medical conditions.

Lessons learned from unsolicited findings in clinical exome sequencing of 16,482 individuals

Unsolicited findings (UFs) are uncovered unintentionally and predispose to a disease unrelated to the clinical question. The frequency and nature of UFs uncovered in clinical practice remain largely unexplored. We here evaluated UFs identified during a 5-year period in which 16,482 index patients received clinical whole-exome sequencing (WES). UFs were identified in 0.58% (95/16,482) of index patients, indicating that the overall frequency of UFs in clinical WES is low. Fewer UFs were identified using restricted disease-gene panels (0.03%) than when using whole-exome/Mendeliome analysis (1.03%). The UF was disclosed to 86 of 95 individuals, for reasons of medical actionability. Only 61% of these UFs reside in a gene that is listed on the “ACMG59”-list, representing a list of 59 genes for which the American College of Medical Genetics recommends UF disclosure. The remaining 39% were grouped into four categories: disorders similar to “ACMG59”-listed disorders (25%); disorders for which disease manifestation could be influenced (7%); UFs providing reproductive options (2%); and UFs with pharmacogenetic implications (5%). Hence, our experience shows that UFs predisposing to medically actionable disorders affect a broader range of genes than listed on the “ACMG59”, advocating that a pre-defined gene list is too restrictive, and that UFs may require ad hoc evaluation of medical actionability. While both the identification and disclosure of UFs depend on local policy, our lessons learned provide general essential insight into the nature and odds of UFs in clinical exome sequencing.

SYN1 Mutation Causes X-Linked Toothbrushing Epilepsy in a Chinese Family

Toothbrushing epilepsy is a rare form of reflex epilepsy (RE) with sporadic incidence. To characterize the genetic profile of reflex epilepsy patients with tooth brushing-induced seizures in a Chinese family. Solo clinical whole-exome sequencing (WES) of the proband, a 37-year-old Chinese man, was performed to characterize the genetic etiology of toothbrushing epilepsy. Mutations in the maternal X-linked synapsin 1 (SYN1) identified in the proband and his family members were confirmed by Sanger sequencing. The pathogenicity of these mutations was determined using in silico analysis. The proband had four episodes of toothbrushing-induced seizures. The semiology included nausea, twitching of the right side of the mouth and face, followed by a generalized tonic-clonic seizure (GTCS). The proband's elder maternal uncle had three toothbrushing-induced epileptic seizures at the age of 26. The proband's younger maternal uncle had no history of epileptic seizures but had a learning disability and aggressive tendencies. We identified a deleterious nonsense mutation, c.1807C>T (p.Q603Ter), in exon 12 of the SYN1 gene (NM_006950), which can result in a truncated SYN1 phosphoprotein with altered flexibility and hydropathicity. This novel mutation has not been reported in the 1000G, EVS, ExAC, gnomAD, or HGMD databases. We identified a novel X-linked SYN1 exon 12 mutant gene in a Chinese family with toothbrushing epilepsy. Our findings provide novel insights into the mechanism of this complex form of reflex epilepsy that could potentially be applied in disease diagnosis.

EP221 - Single center experience in clinical whole-exome sequencing

eP221 - Single center experience in clinical whole-exome sequencing

Mitochondrial DNA Analysis from Exome Sequencing Data Improves Diagnostic Yield in Neurological Diseases.

A rapidly expanding catalog of neurogenetic disorders has encouraged a diagnostic shift towards early clinical whole exome sequencing (WES). Adult primary mitochondrial diseases (PMDs) frequently exhibit neurological manifestations that overlap with other nervous system disorders. However, mitochondrial DNA (mtDNA) is not routinely analyzed in standard clinical WES bioinformatic pipelines. We reanalyzed 11,424 exomes, enriched with neurological diseases, for pathogenic mtDNA variants. Twenty‐four different mtDNA mutations were detected in 64 exomes, 11 of which were considered disease causing based on the associated clinical phenotypes. These findings highlight the diagnostic uplifts gained by analyzing mtDNA from WES data in neurological diseases. ANN NEUROL 2021;89:1240–1247