Putative Pathogenic Variants Research Articles

Diagnostic Yield of Epilepsy Panel Testing in Patients With Seizure Onset Within the First Year of Life.

Purpose: We aimed to evaluate the diagnostic yield of epilepsy gene panel testing in epilepsy patients whose seizures began within the first year after birth. We included 112 patients with seizure onset before 12 months and no known etiology.Methods: Deep targeted sequencing with a custom-designed capture probe was performed to ensure the detection of germline or mosaic sequence variants and copy number variations (CNVs).Results: We identified pathogenic or likely pathogenic variants in 53 patients (47.3%, 53/112), including five with pathogenic CNVs. Two putative pathogenic mosaic variants in SCN8A and KCNQ2 were also detected and validated. Those with neonatal onset (61.5%, 16/26) or early infantile onset (50.0%, 29/58) showed higher diagnostic rates than those with late infantile onset (28.5%, 8/28). The diagnostic rate was similar between patients with a specific syndrome (51.9%, 27/52) and those with no recognizable syndrome (43.3%, 26/60).Conclusion: Epilepsy gene panel testing identified a genetic cause in nearly half of the infantile onset epilepsy patients. Since the phenotypic spectrum is expanding and characterizing it at seizure onset is difficult, this group should be prioritized for epilepsy gene panel testing.

Mutation analysis of LRP10 in Japanese patients with familial Parkinson's disease, progressive supranuclear palsy, and frontotemporal dementia

Mutations of the gene encoding low-density lipoprotein receptor–related protein 10 (LRP10) were recently detected in patients (heterogeneous races) with autosomal dominant inheritance of familial Parkinson's disease. The patients with Parkinson's disease, Parkinson's disease with dementia, and dementia with Lewy bodies whose brain pathology indicated deposit of alpha-synuclein along with the co-occurrence of tau pathology and amyloid-beta plaques presented LRP10 mutations. LRP10 is localized in the vesicular structures and trans-Golgi network; its alteration leads to alpha-synuclein aggregation. Thus, we conducted the genetic screening of LRP10 among 187 patients with familial Parkinson's disease and 19 patients with atypical parkinsonian disorders, including frontotemporal dementia, progressive supranuclear palsy, and corticobasal syndrome. There were no putative pathogenic variants among patients with Parkinson's disease. We detected one rare variant, p.D198N, in a patient with frontotemporal dementia, without a cosegregation study. Overall, our findings showed that LRP10 variants are not causative for disease in our cohort.

Encephalopathies with KCNC1 variants: genotype-phenotype-functional correlations.

ObjectiveTo analyze clinical phenotypes associated with KCNC1 variants other than the Progressive Myoclonus Epilepsy‐causing variant p.Arg320His, determine the electrophysiological functional impact of identified variants and explore genotype‐phenotype‐physiological correlations.MethodsTen cases with putative pathogenic variants in KCNC1 were studied. Variants had been identified via whole‐exome sequencing or gene panel testing. Clinical phenotypic data were analyzed. To determine functional impact of variants detected in the Kv3.1 channel encoded by KCNC1, Xenopus laevis oocyte expression system and automated two‐electrode voltage clamping were used.ResultsSix unrelated patients had a Developmental and Epileptic Encephalopathy and a recurrent de novo variant p.Ala421Val (c.1262C > T). Functional analysis of p.Ala421Val revealed loss of function through a significant reduction in whole‐cell current, but no dominant‐negative effect. Three patients had a contrasting phenotype of Developmental Encephalopathy without seizures and different KCNC1 variants, all of which caused loss of function with reduced whole‐cell currents. Evaluation of the variant p.Ala513Val (c.1538C > T) in the tenth case, suggested it was a variant of uncertain significance.InterpretationThese are the first reported cases of Developmental and Epileptic Encephalopathy due to KCNC1 mutation. The spectrum of phenotypes associated with KCNC1 is now broadened to include not only a Progressive Myoclonus Epilepsy, but an infantile onset Developmental and Epileptic Encephalopathy, as well as Developmental Encephalopathy without seizures. Loss of function is a key feature, but definitive electrophysiological separation of these phenotypes has not yet emerged.

Mutation and association analyses of dementia-causal genes in Han Chinese patients with early-onset and familial Alzheimer's disease

Alzheimer's disease (AD) is the most common cause of dementia in the elderly. It shares clinical and pathological features with other types of dementia, such as vascular dementia (VaD), Lewy body dementia (LBD), and frontotemporal dementia (FTD). We have hypothesized that there might be an overlapping molecular mechanism and genetic basis to the different types of dementia. In this study, we analyzed the mutation pattern of dementia-causal genes in 169 Han Chinese patients with familial and early-onset AD by using whole exome sequencing or targeted resequencing. We identified 9 potentially pathogenic mutations in the AD-causal genes APP, PSEN1, PSEN2, and 6 mutations in a group of non-AD dementia-causal genes including the FTD-causal gene GRN and the VaD-causal gene NOTCH3. A common splice-site variant rs514492 in the FTD-causal gene VCP showed a positive association with AD risk (P = 0.0003, OR = 1.618), whereas the rare missense variant rs33949390 (p. R 1628P) in the LBD-causal gene LRRK2 showed a protective effect on AD risk (P = 0.0004, OR = 0.170). The presence of putative pathogenic mutations and risk variants in these causal genes for different types of dementia in clinically diagnosed familial and early-onset AD patients suggests a need to screen for mutations of the dementia-causal genes in cases of AD to avoid misdiagnosis. These mutations also support the idea that there are overlapping pathomechanisms between AD and other forms of dementia.

Identification of putative pathogenic single nucleotide variants (SNVs) in genes associated with heart disease in 290 cases of stillbirth.

The incidence of stillbirth in Sweden has essentially remained constant since the 1980’s, and despite thorough investigation, many cases remain unexplained. It has been suggested that a proportion of stillbirth cases is caused by heart disease, mainly channelopathies. The aim of this study was to analyze DNA from 290 stillbirth cases without chromosomal abnormalities for pathogenic single nucleotide variants (SNVs) in 70 genes associated with cardiac channelopathies and cardiomyopathies. The HaloPlex Target Enrichment System (Agilent Technologies) was utilized to prepare sequencing libraries which were sequenced on the Illumina NextSeq platform. We found that 12.1% of the 290 investigated stillbirth cases had one (n = 31) or two (n = 4) variants with evidence supporting pathogenicity, i.e. loss-of-function variants (nonsense, frameshift, splice site substitutions), evidence from functional studies, or previous identification of the variants in affected individuals. Regarding identified putative pathogenic variants in genes associated with channelopathies, the prevalence was significantly higher in the stillbirth cohort (n = 23, 7.93%) than the corresponding prevalence of the same variants in the non-Finnish European population of the Exome Aggregation Consortium (2.70%, p<0.001) and SweGen, (2.30%, p<0.001). Our results give further support to the hypothesis that cardiac channelopathies might contribute to stillbirth. Screening for pathogenic SNVs in genes associated with heart disease might be a valuable complement for stillbirth cases where today’s conventional investigation does not reveal the underlying cause of fetal demise.

Clinical and genetic heterogeneity in Melkersson-Rosenthal Syndrome

Melkersson Rosenthal syndromes (MRS) is a rare autosomal dominantly inherited neurocutaneous syndrome characterised by a triad of facial (seventh cranial) nerve palsy, recurrent orofacial swelling and fissuring of the tongue. A recent report implicated a heterozygous missense variant in SLC27A1 (FATP1) as the cause of this condition in members of an affected Chinese family. We undertook Sanger sequencing of this gene in 14 affected unrelated individuals affected by MRS. We did not detect any putative pathogenic variants. Our data indicates that there is both clinical and genetic heterogeneity in this condition and that the causative gene remains to be identified for the majority of cases.

Distribution of KRAS, DDR2, and TP53 gene mutations in lung cancer: An analysis of Iranian patients.

PurposeLung cancer is the deadliest known cancer in the world, with the highest number of mutations in proto-oncogenes and tumor suppressor genes. Therefore, this study was conducted to determine the status of hotspot regions in DDR2 and KRAS genes for the first time, as well as in TP53 gene, in lung cancer patients within the Iranian population.Experimental designThe mutations in exon 2 of KRAS, exon 18 of DDR2, and exons 5–6 of TP53 genes were screened in lung cancer samples, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) using PCR and sequencing techniques.ResultsAnalysis of the KRAS gene showed only a G12C variation in one large cell carcinoma (LCC) patient, whereas variants were not found in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) cases. The Q808H variation in the DDR2 gene was detected in one SCC sample, while no variant was seen in the ADC and LCC subtypes. Variations in the TP53 gene were seen in all NSCLC subtypes, including six ADC (13.63%), seven SCC (15.9%) and two LCC (4.54%). Forty-eight variants were found in the TP53 gene. Of these, 15 variants were found in coding regions V147A, V157F, Q167Q, D186G, H193R, T211T, F212L and P222P, 33 variants in intronic regions rs1625895 (HGVS: c.672+62A>G), rs766856111 (HGVS: c.672+6G>A) and two new variants (c.560-12A>G and c.672+86T>C).ConclusionsIn conclusion, KRAS, DDR2, and TP53 variants were detected in 2%, 2.17% and 79.54% of all cases, respectively. The frequency of DDR2 mutation is nearly close to other studies, while KRAS and TP53 mutation frequencies are lower and higher than other populations, respectively. Three new putative pathogenic variants, for the first time, have been detected in Iranian patients with lung cancer, including Q808H in DDR2, F212L, and D186G in coding regions of TP53. In addition, we observed five novel benign variants, including Q167Q, P222P and T211T in coding sequence, and c.560-12A>G and c.672+86T>C, in intronic region of TP53. Mutations of KRAS and DDR2 were found in LCC and SCC subtypes, respectively, whereas mutations of TP53 were seen in SCC and ADC subtypes with higher frequencies and LCC subtype with lower frequency. Therefore, Iranian lung cancer patients can benefit from mutational analysis before starting the conventional treatment. A better understanding of the biology of these genes and their mutations will be critical for developing future targeted therapies.

Clinical and genetic characteristics of Chinese patients with familial or sporadic pediatric cataract

BackgroundPediatric cataract is a clinically and genetically heterogeneous disease which is a significant cause of lifelong visual impairment and treatable blindness. Our study aims to investigate the genotype spectrum in a group of Chinese patients with pediatric cataract.MethodsWe enrolled 39 families with pediatric cataract from October 2015 to April 2016. DNA samples of the probands were analyzed by target next-generation sequencing. Variants were validated using Sanger sequencing in the probands and available family members.ResultsIn our cohort of 39 cases with different types of pediatric cataract, 23 cases were found to harbor putative pathogenic variants in 15 genes: CRYAA, CRYBA1, CRYBA4, CRYBB1, CRYGC, CRYGD, MIP, GCNT2, IARS2, NHS, BCOR, BFSP2, FYCO1, MAF, and PAX6. The mutation detection rates in the familial and sporadic cases were 75 and 47.8%, respectively. Of the 23 causative variants, over half were novel.ConclusionsThis is a rare report of systematic mutation screening analysis of pediatric cataract in a comparably large cohort of Chinese patients. Our observations enrich the mutation spectrum of pediatric cataract. Next-generation sequencing provides significant diagnostic information for pediatric cataract cases, especially when considering sporadic and subtle syndromal cases.

Genetic basis of arrhythmogenic cardiomyopathy.

To date 16 genes have been associated with arrhythmogenic cardiomyopathy (ACM). Mutations in these genes can lead to a broad spectrum of phenotypic expression ranging from disease affecting predominantly the right or left ventricle, to biventricular subtypes. Understanding the genetic causes of ACM is important in diagnosis and management of the disorder. This review summarizes recent advances in molecular genetics and discusses the application of next-generation sequencing technology in genetic testing in ACM. Use of next-generation sequencing methods has resulted in the identification of novel causative variants and genes for ACM. The involvement of filamin C in ACM demonstrates the genetic overlap between ACM and other types of cardiomyopathy. Putative pathogenic variants have been detected in cadherin 2 gene, a protein involved in cell adhesion. Large genomic rearrangements in desmosome genes have been systematically investigated in a cohort of ACM patients. Recent studies have identified novel causes of ACM providing new insights into the genetic spectrum of the disease and highlighting an overlapping phenotype between ACM and dilated cardiomyopathy. Next-generation sequencing is a useful tool for research and genetic diagnostic screening but interpretation of identified sequence variants requires caution and should be performed in specialized centres.

Yield of the RYR2 Genetic Test in Suspected Catecholaminergic Polymorphic Ventricular Tachycardia and Implications for Test Interpretation.

Pathogenic RYR2 variants account for ≈60% of clinically definite cases of catecholaminergic polymorphic ventricular tachycardia. However, the rate of rare benign RYR2 variants identified in the general population remains a challenge for genetic test interpretation. Therefore, we examined the results of the RYR2 genetic test among patients referred for commercial genetic testing and examined factors impacting variant interpretability. Frequency and location comparisons were made for RYR2 variants identified among 1355 total patients of varying clinical certainty and 60 706 Exome Aggregation Consortium controls. The impact of the clinical phenotype on the yield of RYR2 variants was examined. Six in silico tools were assessed using patient- and control-derived variants. A total of 18.2% (218/1200) of patients referred for commercial testing hosted rare RYR2 variants, statistically less than the 59% (46/78) yield among clinically definite cases, resulting in a much higher potential genetic false discovery rate among referrals considering the 3.2% background rate of rare, benign RYR2 variants. Exclusion of clearly putative pathogenic variants further complicates the interpretation of the next novel RYR2 variant. Exonic/topologic analyses revealed overrepresentation of patient variants in exons covering only one third of the protein. In silico tools largely failed to show evidence toward enhancement of variant interpretation. Current expert recommendations have resulted in increased use of RYR2 genetic testing in patients with questionable clinical phenotypes. Using the largest to date catecholaminergic polymorphic ventricular tachycardia patient versus control comparison, this study highlights important variables in the interpretation of variants to overcome the 3.2% background rate that confounds RYR2 variant interpretation.

Integrative whole exome, transcriptome, and clinical profiling of biliary tract cancers (BTCs).

279 Background: BTC is clinically and genomically heterogeneous and next generation sequencing may identify disease subsets with distinct prognostic and therapeutic implications. Methods: Patients (pts) with BTC underwent whole exome and transcriptome sequencing via the Michigan Oncology Sequencing (MI-ONCOSEQ) platform between 09/2011 and 07/2017. Results: 53 pts (47.2% female) with median age 60 (range 17-72) years had 38 intrahepatic, 6 perihilar, and 4 extrahepatic distal cholangiocarcinoma (CCA) while 3 had gallbladder and 2 mixed CCA/hepatocellular carcinoma. Forty-one pts (77.3%) had advanced BTC at diagnosis and 40 (75.5%) received platinum doublet as first line therapy. The most frequent somatic mutations were TP53 (35.8%), BAP1 (18.9%), KRAS (17.0%), IDH1 (15.1%), PBRM1 (13.2%), ARID1A (11.3%), and SMAD4 (11.3%). Median overall survival (OS) in 8 pts with IDH1 mutation was 16.8 months (none received IDH1 inhibitor). Putative pathogenic germline variants were noted in 6 (11.3%) pts of which 4 were biallelic (MSH2, BRCA1, BRCA2 and MUTYH) and 2 monoallelic (ATM and FH). Germline mutation in FH has not been reported in BTC. Biallelic DNA damage repair pathway mutations were noted in 14 (26.4%; 11 somatic, 3 germline) pts and their median OS was 16.8 months. Of these 9 pts with advanced BTC received 1st line platinum therapy and had a median PFS of 9.3 months (4 PR, 3 SD). Nine (17%) pts had FGFR2 fusion (6 partners); median OS not yet reached (6 alive; 5 received FGFR inhibitor). Potentially targetable molecular alterations (IDH1, MSH2, BRCA1/2, PALB2, ATM, FGFR2, ERBB2) were identified in 27 (50.9%) pts. Copy number profile shows frequent loss of chr1p, chr3p, chr4, chr6q, chr8p, chr9, and gain of chr1q, chr7, and chr8q. Immune profiling &amp; pathway analysis of BTCs with clinical correlation is ongoing. Conclusions: Integrative sequencing of BTCs with clinical profiling will lead to more precise treatment of pts with BTCs.

Targeted next-generation sequencing analysis in couples at increased risk for autosomal recessive disorders

BackgroundMany of the genetic childhood disorders leading to death in the pre- or neonatal period or during early childhood follow autosomal recessive modes of inheritance and bear specific challenges for genetic counseling and prenatal diagnostics. Parents are carriers but clinically unaffected, and diseases are rare but have recurrence risks of 25% in the same family. Often, affected children (or fetuses) die before a genetic diagnosis can be established, post-mortem analysis and phenotypic descriptions are insufficient and DNA from affected fetuses or children is not available for later analysis. A genetic diagnosis showing biallelic causative mutations is, however, the requirement for targeted carrier testing in parents and prenatal and preimplantation genetic diagnosis in further pregnancies.MethodsWe undertook targeted next-generation sequencing (NGS) for carrier screening of autosomal recessive lethal disorders in 8 consanguineous and 5 non-consanguineous couples with one or more affected children. We searched for heterozygous variants (non-synonymous coding or splice variants) in parents’ DNA, using a set of 430 genes known to be causative for rare autosomal recessive diseases with poor prognosis, and then filtering for variants present in genes overlapping in both partners. Putative pathogenic variants were tested for cosegregation in affected fetuses or children where material was available.ResultsThe diagnosis for the premature death in children was established in 5 of the 13 couples. Out of the 8 couples in which no causative diagnosis could be established 4 consented to undergo further analysis, in two of those a potentially causative variant in a novel candidate gene was identified.ConclusionsFor the families in whom causative variants could be identified, these may now be used for prenatal and preimplantation genetic diagnostics. Our data show that NGS based gene panel sequencing of selected genes involved in lethal autosomal recessive disorders is an effective tool for carrier screening in parents and for the identification of recessive gene defects and offers the possibility of prenatal and preimplantation genetic diagnosis in further pregnancies in families that have experienced deaths in early childhood and /or multiple abortions.

Mutation Screening of the CHCHD2 Gene for Alzheimer's Disease and Frontotemporal Dementia in Chinese Mainland Population.

As an important multifunctional protein involved in regulation of mitochondrial metabolism, CHCHD2 was identified as a causative gene for Parkinson's disease (PD), yet the relationship between CHCHD2 and neurodegenerative dementia is not well understood. We directly sequenced the entire coding region of CHCHD2 gene in 150 AD patients, 84 FTD patients, and 417 controls. Four rare putative pathogenic variants of CHCHD2, including rs142444896 (c.5C>T, p.P2L), rs752705344 (c.15C>G, p.S5R), rs145190179 (c.94G>A, p.A32T), and rs182992574 (c.255T>A, p.S85R) were identified from a cohort composed of 150 AD and 84 FTD patients. These results suggest that CH CHD2 gene play an important role in other neurodegenerative disorders from our dementia study in China.

Genetic and clinical evidence of mitochondrial dysfunction in autism spectrum disorder and intellectual disability.

Clinical conditions commonly associated with mitochondrial disorders (CAMDs) are often present in autism spectrum disorders (ASD) and intellectual disability (ID). Therefore, the mitochondrial dysfunction hypothesis has been proposed as a transversal mechanism that may function in both disorders. Here, we investigated the presence of conditions associated with mitochondrial disorders and mitochondrial DNA (mtDNA) alterations in 122 subjects who presented ASD with ID (ASD group), 115 subjects who presented ID but not ASD (ID group) and 112 healthy controls (HC group). We assessed in the three study groups the presence of the clinical conditions through a questionnaire and the mtDNA content of two mitochondrial genes, MT-ND1 and MT-ND4, by qPCR. The mtDNA sequences of 98 ASD and 95 ID subjects were obtained by mtDNA-targeted next generation sequencing and analysed through the MToolBox pipeline to identify mtDNA mutations. Subjects with ASD and ID showed higher frequencies of constipation, edema, seizures, vision alterations, strabismus and sphincter incontinence than HCs subjects. ASD and ID subjects showed significantly lower mtDNA content than HCs in both MT-ND1 and MT-ND4 genes. In addition, we identified 49 putative pathogenic variants with a heteroplasmy level higher than 60%: 8 missense, 29 rRNA and 12 tRNA variants. A total of 28.6% of ASD and 30.5% of ID subjects carried at least one putative pathogenic mtDNA mutation. The high frequency of CAMDs, the low mtDNA content and the presence of putative pathogenic mtDNA mutations observed in both ASD and ID subjects are evidence of mitochondrial dysfunction in ASD and ID.

Postmortem Genetic Testing for Cardiac Ion Channelopathies in Stillbirths.

Although stillbirth is a significant health problem worldwide, the definitive cause of death remains elusive in many cases, despite detailed autopsy. In this study of partly explained and unexplained stillbirths, we used next-generation sequencing to examine an extended panel of 35 candidate genes known to be associated with ion channel disorders and sudden cardiac death. We examined tissue from 242 stillbirths (≥22 weeks), including those where no definite cause of death could be confirmed after a full autopsy. We obtained high-quality DNA from 70 cases, which were then sequenced for a custom panel of 35 genes, 12 for inherited long- and short-QT syndrome genes (LQT1-LQT12 and SQT1-3), and 23 additional candidate genes derived from genome-wide association studies. We examined the functional significance of a selected variant by patch-clamp electrophysiological recording. No predicted damaging variants were identified in KCNQ1 (LQT1) or KCNH2 (LQT2). A rare putative pathogenic variant was found in KCNJ2(LQT7) in 1 case, and several novel variants of uncertain significance were observed. The KCNJ2 variant (p. R40Q), when assessed by whole-cell patch clamp, affected the function of the channel. There was no significant evidence of enrichment of rare predicted damaging variants within any of the candidate genes. Although a causative link is unclear, 1 putative pathogenic and variants of uncertain significance variant resulting in cardiac channelopathies was identified in some cases of otherwise unexplained stillbirth, and these variants may have a role in fetal demise. URL: https://www.clinicaltrials.gov. Unique identifier: NCT01120886.

Mitochondrial DNA depletion, mitochondrial mutations and high TFAM expression in hepatocellular carcinoma.

We investigated the role of mitochondrial genetic alterations in hepatocellular carcinoma by directly comparing the mitochondrial genomes of 86 matched pairs of HCC and non-tumor liver samples. Substitutions in 637 mtDNA sites were detected, comprising 89.80% transitions and 6.60% transversions. Forty-six somatic variants, including 15 novel mutations, were identified in 40.70% of tumor tissues. Of those, 21 were located in the non-coding region and 25 in the protein-coding region. Twenty-two somatic nonsynonymous changes were identified as putative pathogenic variants, including 4 truncating mutations produced by three frameshifts (MT-ATP6 8628 insC; MT-ND5 13475 T-del, and MT-CYB 14984 insA) and 1 nonsense mutation in MT-CO3 9253 G>A. Among the somatic variants, only m.13676 A>G (MT-ND5), found in only 1 tumor, was heteroplasmic. Both inherited and somatic variants were predominately located in the D-loop region and the MT-ND5 gene. Tumor/non-tumor paired analysis showed that 69% of HCC samples contained significantly reduced mtDNA, compared with 49.0% of non-tumor counterparts. In 81.40% of HCC samples, mitochondrial transcription factor A (TFAM) was enriched in tumor cells but not in adjacent non-tumor cells. Neither mtDNA depletion nor TFAM overexpression correlated with the degree of cell differentiation, though TFAM expression correlated with tumor size.

Whole-Exome Sequencing Identifies Novel Variants for Tooth Agenesis

Tooth agenesis is a common craniofacial abnormality in humans and represents failure to develop 1 or more permanent teeth. Tooth agenesis is complex, and variations in about a dozen genes have been reported as contributing to the etiology. Here, we combined whole-exome sequencing, array-based genotyping, and linkage analysis to identify putative pathogenic variants in candidate disease genes for tooth agenesis in 10 multiplex Turkish families. Novel homozygous and heterozygous variants in LRP6, DKK1, LAMA3, and COL17A1 genes, as well as known variants in WNT10A, were identified as likely pathogenic in isolated tooth agenesis. Novel variants in KREMEN1 were identified as likely pathogenic in 2 families with suspected syndromic tooth agenesis. Variants in more than 1 gene were identified segregating with tooth agenesis in 2 families, suggesting oligogenic inheritance. Structural modeling of missense variants suggests deleterious effects to the encoded proteins. Functional analysis of an indel variant (c.3607+3_6del) in LRP6 suggested that the predicted resulting mRNA is subject to nonsense-mediated decay. Our results support a major role for WNT pathways genes in the etiology of tooth agenesis while revealing new candidate genes. Moreover, oligogenic cosegregation was suggestive for complex inheritance and potentially complex gene product interactions during development, contributing to improved understanding of the genetic etiology of familial tooth agenesis.

Contribution of exome sequencing for genetic diagnostic in arrhythmogenic right ventricular cardiomyopathy/dysplasia.

BackgroundArrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia (ARVC/D) is an inherited cardiomyopathy mainly caused by heterozygous desmosomal gene mutations, the major gene being PKP2. The genetic cause remains unknown in ~50% of probands with routine desmosomal gene screening. The aim of this study was to assess the diagnostic accuracy of whole exome sequencing (WES) in ARVC/D with negative genetic testing.MethodsWES was performed in 22 patients, all without a mutation identified in desmosomal genes. Putative pathogenic variants were screened in 96 candidate genes associated with other cardiomyopathies/channelopathies. The sequencing coverage depth of PKP2, DSP, DSG2, DSC2, JUP and TMEM43 exons was compared to the mean coverage distribution to detect large insertions/deletions. All suspected deletions were verified by real-time qPCR, Multiplex-Ligation-dependent-Probe-Amplification (MLPA) and cGH-Array. MLPA was performed in 50 additional gene-negative probands.ResultsCoverage-depth analysis from the 22 WES data identified two large heterozygous PKP2 deletions: one from exon 1 to 14 and one restricted to exon 4, confirmed by qPCR and MLPA. MLPA identified 2 additional PKP2 deletions (exon 1–7 and exon 1–14) in 50 additional probands confirming a significant frequency of large PKP2 deletions (5.7%) in gene-negative ARVC/D. Putative pathogenic heterozygous variants in EYA4, RBM20, PSEN1, and COX15 were identified in 4 unrelated probands.ConclusionA rather high frequency (5.7%) of large PKP2 deletions, undetectable by Sanger sequencing, was detected as the cause of ARVC/D. Coverage-depth analysis through next-generation sequencing appears accurate to detect large deletions at the same time than conventional putative mutations in desmosomal and cardiomyopathy-associated genes.

Integrative clinical genomics of metastatic cancer.

SUMMARYMetastasis is the primary cause of cancer-related deaths. While The Cancer Genome Atlas (TCGA) has sequenced primary tumor types obtained from surgical resections, much less comprehensive molecular analysis is available from clinically acquired metastatic cancers. Here, we perform whole exome and transcriptome sequencing of 500 adult patients with metastatic solid tumors of diverse lineage and biopsy site. The most prevalent genes somatically altered in metastatic cancer included TP53, CDKN2A, PTEN, PIK3CA, and RB1. Putative pathogenic germline variants were present in 12.2% of cases of which 75% were related to defects in DNA repair. RNA sequencing complemented DNA sequencing for the identification of gene fusions, pathway activation, and immune profiling. Integrative sequence analysis provides a clinically relevant, multi-dimensional view of the complex molecular landscape and microenvironment of metastatic cancers.

Abstract 4871: Whole-exome sequencing identifies a high frequency of germline deleterious variants in cancer predisposition genes in individuals with osteosarcoma

Abstract In children and adolescents, osteosarcoma (OS) is the most common malignant bone tumor. OS occurs in certain cancer predisposition syndromes at a higher than expected frequency. However, outside of these rare syndromes, OS is significantly more common and its genetic etiology remains poorly understood. We conducted an evaluation of rare exonic variants in 545 unselected OS cases compared with 1061 cancer-free controls using whole-exome sequencing of blood or buccal cell DNA to estimate the prevalence and burden of rare deleterious germline variants. We assessed potentially pathogenic rare variants in 126 established cancer predisposing genes (CPG) and known somatically mutated genes. We also surveyed the exome for genes with a higher burden of rare variants in 342 EUR cases with 994 EUR controls sequenced at the same time. Rare genetic variants, defined by a MAF &amp;lt;0.01 in 1000G, ESP or ExAC, that passed quality control filters were classified based on well-annotated databases (IARC-TP53, Telomerase Database, ARUP, BIC, Insight and COSMIC-hotspots), variant databases (e.g. Clinvar, HGMD and LOVD), and in silico predictions (Sift, Polyphen, Mutation assessor, MutationTaster, FATHMM and LTR). Rare variants matching known pathogenic variants, or inexact matches at the same residue, in selected well-annotated databases were labeled as putative pathogenic variants. High impact variants (frameshift, stop gain/loss, or known splice sites) not matching a previously identified pathogenic variant in the selected databases were categorized as probably pathogenic. Predictions based solely on in silico predictions were categorized as possibly pathogenic, uncertain or likely benign. A total of 14.5% of cases had a predicted pathogenic or high impact variant in an autosomal dominant CPG. A significantly higher pathogenic rare variant burden was present in EUR cases compared with EUR controls (Pburden=3.7x10-05). TP53 had the highest prevalence of pathogenic mutations (5% of EUR cases; Pburden=1.05x10-09). CDKN2A, MEN1, MLH1, MUTYH, PALB2 and VHL had a significantly higher rare variant burden in the EUR cases. 7.5% of cases had a predicted pathogenic or probably pathogenic variant in an autosomal recessive CPG (Pburden=5.4x10-03). Additionally, two males had a pathogenic variant in the X-linked genes, DKC1 and GPC3. In total, 21% of cases had a predicted pathogenic or high impact variant in a CPG (Pburden=2.4x10-07). Exome-wide analysis identified two novel genes with significantly higher rare variant burdens in the cases compared to controls. In conclusion, several CPGs and two novel genes, not previously associated with OS, had an enrichment of rare variants in OS and warrant further follow-up. Our results indicate that a clinically significant fraction of OS cases may harbor one or more mutations worthy of consideration for further investigation and genetic counseling. Citation Format: Roelof Koster, Bin Zhu, Meredith Yeager, Michael Dean, Matthew Gianferante, Lei Song, Joshua Sampson, NCI DCEG Cancer Genomics Research Laboratory, Julie Gastier-Foster, Richard Gorlick, Silvia Regina Caminada de Toledo, Antonio Petrilli, Ana Patiño-Garcia, Fernando Lecanda, Massimo Serra, Claudia Hattinger, Piero Picci, Katia Scotlandi, Adrienne Flanagan, Roberto Tirabosco, Maria Amary, Nilgün Kurucu, Inci Ergurhan Ilhan, Neriman Sari, Mandy Ballinger, David Thomas, Donald Barkauskas, Belynda Hicks, Margaret Tucker, Neil Caporaso, Robert Hoover, Stephen Chanock, Sharon Savage, Lisa Mirabello. Whole-exome sequencing identifies a high frequency of germline deleterious variants in cancer predisposition genes in individuals with osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4871. doi:10.1158/1538-7445.AM2017-4871