Multigene Panel Analysis Research Articles

Multi-gene panel analysis in BRCA1/2-negative patients suspected of hereditary breast and ovarian cancer syndrome: Real-world data from a single institution.

Although BRCA1/2 is most frequently associated with hereditary breast and ovarian cancer (HBOC), many other related genes have been implicated. Therefore, we investigated the prevalence of non-BRCA1/2 genes associated with hereditary cancer predisposition in BRCA1/2-negative patients from the Department of Genetic Medicine and Services with breast and ovarian cancer using a multi-gene panel (MGP) analysis. We conducted a retrospective MGP analysis (National Cancer Center Onco-Panel for Familial Cancer; NOP_FC) in BRCA1/2-negative patients with breast, ovarian, and overlapping breast/ovarian cancers who visited our genetic counseling between April 2004 and October 2022. NOP_FC was performed in 128 of the 390 BRCA test-negative cases (117 breast cancer, 9 ovarian cancer, and 2 overlapping breast/ovarian cancer cases). Among the BRCA1/2-negative patients, nine (7.7%) with breast cancer and one (11%) with ovarian cancer had pathogenic variants (PVs) in non-BRCA1/2 genes associated with breast and ovarian cancers, respectively. Five patients had PVs in RAD51D, two in PALB2, one in BARD1, one in ATM, and one in RAD51C. Additional MGP testing of germline genes associated with hereditary cancer predisposition syndrome in BRCA1/2-negative breast and ovarian cancer patients revealed PVs in non-BRCA1/2 breast cancer- and ovarian cancer-related genes in 7.7% of breast cancer and 11% of ovarian cancer. Therefore, additional testing may provide useful information for subsequent risk-reducing surgery and surveillance in BRCA1/2-negative patients.

Broadening the scope of multigene panel analysis for adult epilepsy patients.

Epilepsy is a suitable target for gene panel sequencing because a considerable portion of epilepsy is now explained by genetic components, especially in syndromic cases. However, previous gene panel studies on epilepsy have mostly focused on pediatric patients. We enrolled adult epilepsy patients meeting any of the following criteria: family history of epilepsy, seizure onset age ≤ 19 years, neuronal migration disorder, and seizure freedom not achieved by dual anti-seizure medications. We sequenced the exonic regions of 211 epilepsy genes in these patients. To confirm the pathogenicity of a novel MTOR truncating variant, we electroporated vectors with different MTOR variants into developing mouse brains. A total of 92 probands and 4 affected relatives were tested, and the proportion of intellectual disability (ID) and/or developmental disability (DD) was 21.7%. As a result, twelve probands (13.0%) had pathogenic or likely pathogenic variants in the following genes or regions: DEPDC5, 15q12-q13 duplication (n = 2), SLC6A1, SYNGAP1, EEF1A2, LGI1, MTOR, KCNQ2, MEF2C, and TSC1 (n = 1). We confirmed the functional impact of a novel truncating mutation in the MTOR gene (c.7570C > T, p.Gln2524Ter) that disrupted neuronal migration in a mouse model. The diagnostic yield was higher in patients with ID/DD or childhood-onset seizures. We also identified additional candidate variants in 20 patients that could be reassessed by further studies. Our findings underscore the clinical utility of gene panel sequencing in adult epilepsy patients suspected of having genetic etiology, especially those with ID/DD or early-onset seizures. Gene panel sequencing could not only lead to genetic diagnosis in a substantial portion of adult epilepsy patients but also inform more precise therapeutic decisions based on their genetic background. This study demonstrated the effectiveness of gene panel sequencing in adults with epilepsy, revealing pathogenic or likely pathogenic variants in 13.0% of patients. Higher diagnostic yields were observed in those with neurodevelopmental disorders or childhood-onset seizures. Additionally, we have shown that expanding genetic studies into adult patients would uncover new types of pathogenic variants for epilepsy, contributing to the advancement of precision medicine for individuals with epilepsy. In conclusion, our results highlight the practical value of employing gene panel sequencing in adult epilepsy patients, particularly when genetic etiology is clinically suspected.

Genetic Testing for Supravalvar Aortic Stenosis: What to Do When It Is Not Williams Syndrome.

We aimed to describe the frequency and yield of genetic testing in supravalvar aortic stenosis (SVAS) following negative evaluation for Williams-Beurensyndrome (WS). This retrospective cohort study included patients with SVAS at our institution who had a negative evaluation for WS from May 1991 to September 2021. SVAS was defined as (1) peak supravalvar velocity of ≥2 meters/second, (2) sinotubular junction or ascending aortic Z score <-2.0, or (3) sinotubular junction Z score <-1.5 with family history of SVAS. Patients with complex congenital heart disease, aortic valve disease as the primary condition, or only postoperative SVAS were excluded. Genetic testing and diagnoses were reported. Of 162 patients who were WS negative meeting inclusion criteria, 61 had genetic testing results available (38%). Chromosomal microarray had been performed in 44 of 61 and was nondiagnostic for non-WS causes of SVAS. Sequencing of 1 or more genes was performed in 47 of 61. Of these, 39 of 47 underwent ELN sequencing, 20 of 39 (51%) of whom had a diagnostic variant. Other diagnoses made by gene sequencing were Noonan syndrome (3 PTPN11, 1 RIT1), Alagille syndrome (3 JAG1), neurofibromatosis (1 NF1), and homozygous familial hypercholesterolemia (1 LDLR1). Overall, sequencing was diagnostic in 29 of 47 (62%). When WS is excluded, gene sequencing for SVAS is high yield, with the highest yield for the ELN gene. Therefore, we recommend gene sequencing using a multigene panel or exome analysis. Hypercholesterolemia can also be considered in individuals bearing the stigmata of this disease.

Mutation analysis of the TERT gene in ovarian cancer patients of the Turkish population by next generation sequencing method.

Ovarian cancer (OC) ranks seventh among malignant tumors worldwide. As one of the most common gynecological malignancies, ovarian cancer has the second-highest mortality rate, after cervical and uterine cancer. Next-Generation Sequencing (NGS) technology has enhanced multi-gene panel analysis and its clinical utility for identifying cancer-causing gene mutations. This study aimed to determine the presence of significant and nonsense mutations in telomerase reverse transcriptase (TERT), alpha-thalassemia/mental retardation, X-linked (ATRX), O-6-methylguanine-DNA methyltransferase (MGMT), and isocitrate dehydrogenase 1 and 2 (IDH1/IDH2) genes using the Next-Generation Sequencing (NGS) method. A cohort of 33 patients diagnosed with ovarian cancer was included in this investigation, and peripheral blood samples were collected from all participants. Significant and nonsense mutations in TERT, ATRX, MGMT, IDH1, and IDH2 genes were detected using the Next-Generation Sequencing method. Bioinformatics analysis was conducted using the QIAGEN Clinical Insight system. Twenty-four patients exhibited seven different TERT mutations, occurring in both exonic and intronic regions. One patient displayed a c.699-3delC deletion in the intronic region of the IDH1 gene, and the c.532G > A (p.V178I) mutation observed in three patients was assessed as potentially harmful. Additionally, novel mutations c.881A > G and c.995A > G were observed in the ATRX gene. The heterozygous novel mutation identified in the ATRX gene was confirmed through Sanger sequencing. These mutations were not previously associated with ovarian cancer and are considered novel candidate markers for ovarian cancer susceptibility. Confirmation of these results through larger cohort studies or functional investigations will contribute to a better understanding of the molecular mechanisms underlying ovarian cancer.

Only one beer can be mortal: a case report of two sisters with cardiac arrest due to a homozygous mutation in PPA2 gene

We report the long way to the correct diagnosis in two teenage sisters who developed a cardiac arrest after consuming minimal amounts of alcohol. The older girl dramatically survived two cardiac arrests at the age of 14 and 15 years. She underwent an extensive examination that revealed isolated cardiac abnormalities including fibrosis, dilated cardiomyopathy and inflammation. The younger girl also had a cardiac arrest at the age of 15 and died suddenly after consuming 1–2 beers, 3 years after her sister´s first incident. Autopsy of the heart revealed acute myocarditis without structural alterations. Multigene panel analysis (not including PPA2) showed SCN5A and CACNA1D variants in both sisters and their healthy mother. Six years later duo exome allowed the diagnosis of an autosomal recessive PPA2-related mitochondriopathy. We discuss the molecular results and clinical picture of our patients compared to other PPA2-related cases. We highlight the diagnostic contribution of multigene panels and exome analysis. The genetic diagnosis is important for medical care and for everyday life, specifically because alcohol intake can result in cardiac arrest and should be strictly avoided. Conclusion: Duo exome sequencing clarified the diagnosis of PPA2-related mitochondriopathy in two sisters with isolated cardiac features and sudden cardiac arrest triggered by minimal amounts of alcohol.What is Known:• Multigene-Panel or exome analysis is a valuable tool to identify genetic causes of hereditary cardiac arrhythmias.• Variants of unknown significance can lead to misinterpretation. PPA2-related mitochondriopathy is a very rare autosomal recessive condition that is normally fatal in infancy.What is New:• Duo exome analysis in two teeenage sisters with cardiac arrest revealed a homozygous mild PPA2 mutation as the underlying pathology restricted to the heart muscle

Exome Sequencing and Multigene Panel Testing in 1,411 Patients With Adult-Onset Neurologic Disorders.

Owing to their extensive clinical and molecular heterogeneity, hereditary neurologic diseases in adults are difficult to diagnose. The current knowledge about the diagnostic yield and clinical utility of exome sequencing (ES) for neurologic diseases in adults is limited. This observational study assesses the diagnostic value of ES and multigene panel analysis in adult-onset neurologic disorders. From January 2019 through April 2022, ES-based multigene panel testing was conducted in 1,411 patients with molecularly unexplained neurologic phenotypes at the Ghent University Hospital. Gene panels were developed for ataxia and spasticity, leukoencephalopathy, movement disorders, paroxysmal episodic disorders, neurodegeneration with brain iron accumulation, progressive myoclonic epilepsy, and amyotrophic lateral sclerosis. Single nucleotide variants, small indels, and copy number variants were analyzed. Across all panels, our analysis covered a total of 725 genes associated with Mendelian inheritance. A molecular diagnosis was established in 10% of the cases (144 of 1,411) representing 71 different monogenic disorders. The diagnostic yield depended significantly on the presenting phenotype with the highest yield seen in patients with ataxia or spastic paraparesis (19%). Most of the established diagnoses comprised disorders with an autosomal dominant inheritance (62%), and the most frequently mutated genes were NOTCH3 (13 patients), SPG7 (11 patients), and RFC1 (8 patients). 34% of the disease-causing variants were novel, including a unique likely pathogenic variant in APP (Ghent mutation, p.[Asn698Asp]) in a family presenting with stroke and severe cerebral white matter disease. 7% of the pathogenic variants comprised copy number variants detected in the ES data and confirmed by an independent technique. ES and multigene panel testing is a powerful and efficient tool to diagnose patients with unexplained, adult-onset neurologic disorders.

NEFL-Related Charcot-Marie Tooth Disease due to P440L Mutation in Two Italian Families: Expanding the Phenotype and Defining Modulating Factors

Introduction: Mutations in the neurofilament polypeptide light chain (NEFL) gene account for <1% of all forms of Charcot-Marie-Tooth (CMT) diseases and present with different phenotypes, including demyelinating, axonal and intermediate neuropathies, and with diverse pattern of transmission, with dominant and recessive inheritance being described. Methods: Here, we present clinical and molecular data in two new unrelated Italian families, affected with CMT. Results: We studied fifteen subjects (11 women, 4 men), age range 23–62 year. Onset of symptoms was mainly in childhood, with running/walking difficulties; some patients were pauci-asymptomatic; almost all shared variably distributed features of absent/reduced deep tendon reflexes, impaired gait, reduced sensation, and distal weakness in the legs. Skeletal deformities were seldom documented and were of mild degree. Additional features included sensorineural hearing loss in 3 patients, underactive bladder in 2 patients, and cardiac conduction abnormalities, requiring pacemaker implantation, in one child. Central nervous system (CNS) impairment was not documented in any subject. Neurophysiological investigation disclosed feature suggestive of demyelinating sensory-motor polyneuropathy in one family and resembling an intermediate form in the other. Multigene panel analysis of all known CMT genes revealed two heterozygous variants in NEFL: p.E488K and p.P440L. While the latter change segregated with the phenotype, the p.E488K variant appeared to act as a modifier factor being associated with axonal nerve damage. Conclusions: CMT related to P440L mutation in NEFL is associated with a mild, childhood-onset phenotype, showing prevalently sensory distal limbs involving and with motor impairment predominantly involving anterolateral leg muscles, in the absence of CNS involvement. Additional findings, never reported so far in patients with NEFL mutation, are cardiological and urinary dysfunctions. Our study expands the array of clinical features associated with NEFL-related CMT.

Clinical usefulness of NGS multi-gene panel testing in hereditary cancer analysis.

Introduction: A considerable number of families with pedigrees suggestive of a Mendelian form of Breast Cancer (BC), Ovarian Cancer (OC), or Pancreatic Cancer (PC) do not show detectable BRCA1/2 mutations after genetic testing. The use of multi-gene hereditary cancer panels increases the possibility to identify individuals with cancer predisposing gene variants. Our study was aimed to evaluate the increase in the detection rate of pathogenic mutations in BC, OC, and PC patients when using a multi-gene panel. Methods: 546 patients affected by BC (423), PC (64), or OC (59) entered the study from January 2020 to December 2021. For BC patients, inclusion criteria were i) positive cancer family background, ii) early onset, and iii) triple negative BC. PC patients were enrolled when affected by metastatic cancer, while OC patients were all submitted to genetic testing without selection. The patients were tested using a Next-Generation Sequencing (NGS) panel containing 25 genes in addition to BRCA1/2. Results: Forty-four out of 546 patients (8%) carried germline pathogenic/likely pathogenic variants (PV/LPV) on BRCA1/2 genes, and 46 (8%) presented PV or LPV in other susceptibility genes. Discussion: Our findings demonstrate the utility of expanded panel testing in patients with suspected hereditary cancer syndromes, since this approach increased the mutation detection rate of 15% in PC, 8% in BC and 5% in OC cases. In absence of multi-gene panel analysis, a considerable percentage of mutations would have been lost.

Targeted gene panel analysis of Japanese patients with maturity-onset diabetes of the young-like diabetes mellitus: Roles of inactivating variants in the ABCC8 and insulin resistance genes.

To investigate the genetic background of Japanese patients with suspected maturity-onset diabetes of the young (MODY). On 340 proband patients referred from across Japan, genomic variants were analyzed using a targeted multigene panel analysis combined with the multiplex ligation probe amplification (MLPA) analysis, mitochondrial m.3243A > G analysis and methylation-specific polymerase chain reaction of the imprinted 6q24 locus. Pathogenic/likely pathogenic variants were listed according to the 2015 American College of Medical Genetics and Genomics and the Association for Molecular Pathology criteria. Additionally, variants with a population frequency <0.001 and Combined Annotation Dependent Depletion score >20 (CS >20) were listed as rare variants of uncertain significance-CS >20. A total of 157 pathogenic/likely pathogenic variants and 44 rare variants of uncertain significance-CS >20 were identified. In the pathogenic/likely pathogenic variants, alterations in the GCK gene were the most common (82, 52.2%) followed by HNF1A (29,18.5%), HNF4A (13, 8.3%) and HNF1B (13, 8.3%). One patient was a 29.5% mosaic with a truncating INSR variant. In the rare variants of uncertain significance-CS >20, 20 (45.5%) were in the genes coding for the adenosine triphosphate-sensitive potassium channel, KCNJ11 or ABCC8, and four were in the genes of the insulin-signaling pathway, INSR and PIK3R1. Four variants in ABCC8 were previously reported in patients with congenital hyperinsulinism, suggesting the inactivating nature of these variants, and at least two of our patients had a history of congenital hyperinsulinism evolving into diabetes. In two patients with INSR or PIK3R1 variants, insulin resistance was evident at diagnosis. Causative genomic variants could be identified in at least 46.2% of clinically suspected MODY patients. ABCC8-MODY with inactivating variants could represent a distinct category of MODY. Genes of insulin resistance should be included in the sequencing panel for MODY.

Cancer risks associated with heterozygous ATM loss of function and missense pathogenic variants based on multigene panel analysis.

Cancer risks conferred by germline, heterozygous, ATM pathogenic/likely pathogenic variants (PSVs) are yet to be consistently determined. The current study assessed these risks by analysis of a large dataset of ATM heterozygote loss of function (LOF) and missense PSV carriers tested with a multigene panel (MGP). De-identified data of all individuals who underwent ATM sequencing as part of MGP between October 2015 and February 2020 were reviewed. In cancer cases, rates for the six most prevalent variants and for all LOF and missense PSV combined were compared with rates of the same PSV in ethnically matched, healthy population controls. Statistical analysis included Chi-square tests and odds ratios calculations. For female breast cancer cases, LOF )1794/219,269) and missense (301/219,269) ATM PSVs were seen at higher rates compared to gnomAD non-cancer controls (n = 157/56,001 and n = 27/61,208; p < 0.00001, respectively). Notably, the rate of the c.103C > T variant was higher in controls than in breast cancer cases [p = 0.001; OR 0.31 (95% CI 0.1-0.6)]. For all cancer cases combined, compared with non-cancer population controls, LOF (n = 143) and missense (n = 15) PSVs reported in both datasets were significantly more prevalent in cancer cases [ORLOF 1.7 (95% 1.5-1.9) ORmissense 3.0 (95% CI 2.3-4); p = 0.0001]. Both LOF and missense heterozygous ATM PSVs are more frequently detected in cases of several cancer types (breast, ovarian, prostate, lung, pancreatic) compared with healthy population controls. However, not all ATM PSVs confer an increased cancer risk (e.g., breast).

Adenomatous Polyposis Phenotype in BMPR1A and SMAD4 Variant Carriers.

Variants in SMAD4 or BMPR1A cause juvenile polyposis syndrome, a rare autosomal dominant condition characterized by multiple gastrointestinal hamartomatous polyps. A phenotype of attenuated adenomatous polyposis without hamartomatous polyps is rare. We describe a retrospective cohort of individuals with SMAD4 or BMPR1A heterozygous germline variants, having ≥10 cumulative colorectal adenomas and/or colorectal cancer without hamartomatous polyps. All individuals had multigene panel and duplication/deletion analysis to exclude other genetic syndromes. The study cohort included 8 individuals. The pathogenic potential of the variants was analyzed. Variants detected included 4 missense variants, 1 nonsense variant, 1 splice site variant, and 2 genomic deletions. Features of pathogenicity were present in most variants, and cosegregation of the variant with polyposis or colorectal cancer was obtained in 7 of the 8 families. Three of 8 individuals had colorectal cancer (age less than 50 years) in addition to the polyposis phenotype. Two individuals had extraintestinal neoplasms (pancreas and ampulla of Vater). The clinical phenotype of SMAD4 and BMPR1A variants may infrequently extend beyond the classical juvenile polyposis syndrome phenotype. Applying multigene panel analysis of hereditary cancer-related genes in individuals with unexplained polyposis can provide syndrome-based clinical surveillance for carriers and their family members.

Abstract P2-09-10: Different CNVs account for 10.4% of pathogenic variants in 1418 patients referred for hereditary breast cancer testing

Abstract Background: Breast cancer is the most frequently diagnosed cancer in women and about 10% of breast cancer cases are hereditary. BRCA1 and BRCA2 are the genes most frequently associated with Hereditary Breast Cancer, although there are numerous other genes, such as PALB2, CHEK2 and ATM, that require to be considered as well. Germline Copy Number Variation (CNV) is one mutation type that is an important contributor to hereditary breast cancer. Nowadays, next-generation sequencing (NGS) technologies has contributed to multi-gene panel analysis used in clinical practice. Methods: In total, 1418 individuals were tested for breast cancer predisposition, using a solution-based capture approach. Targeted NGS was performed with a panel of 36 genes. The capture-based approach allowed for computational analysis of CNVs from NGS data. Results: We investigate the performance of the CNV module of the commercial software suite SeqPilot (JSI Medical Systems) and the non-commercial tool panelcn.MOPS. Both algorithms are specifically developed for CNV analysis of sequencing data reporting 99-100% sensitivity and up to 100% specificity for the prediction of CNVs up to the level of a single gene exon. All CNVs detected with these algorithms were then verified experimentally using the MLPA technique as an orthogonal assay. At least one pathogenic/likely pathogenic variant was identified in 289 samples (20.4%). CNVs accounted for 10.4% (30/289), referring to the deletion of one or more exons of a gene. Interestingly, 50% of deletions were single exon and approximately 36% of CNVs were detected in genes other than BRCA1/2. In specific, of the 30 CNVs detected, 60% occurred in BRCA1, 3.3% in BRCA2, 20% in CHEK2, 6.7% in FANCA, 6.7% in PMS2, and 3.3% in ATM. The majority of CNVs in BRCA1 were deletions of exons 19, 22, and 22-23 whereas deletions of exons 9-10 were the most common deletions in CHEK2. Detailed information of all CNVs detected is provided in Table 1. Conclusions: Our results suggest that CNV analysis should not be restricted to BRCA1/2 due to the significant proportion of CNVs (36%) in additional breast cancer predisposition genes. Furthermore, in silico CNV detection tools provide a cost-effective and feasible methodology for the identification of CNVs from NGS experiments. This outlines the clinical utility of comprehensive genetic testing that includes full sequencing and CNV analysis in hereditary breast cancer facilitating personalized management decisions for patients. Table 1.Pathogenic Copy Number Variations (CNVs) identified in this studyGeneHGVS nomenclatureOther nomenclature# detectedATMNM_000051:c.(-30+1_-29-1)_(331+1_332-1)deldeletion of exons 2-41BRCA1NM_007294:c.(5467+1_5468-1)-(*1_?)deldeletion of exon 237BRCA1NM_007294:c.(5406+1_5407-1)_(*1_?)deldeletion of exons 23-245BRCA1NM_007294:c.(5193+1_5194-1)-(5277+1_5278-1)deldeletion of exon 196BRCA2NM_000059:c.(6841+1_6842-1)_(7007+1_7008-1)deldeletion of exons 12-131CHEK2NM_007194:c.(908+1_909-1)_(1095+1_1096-1)deldeletion of exons 9-104CHEK2NM_007194:c.(792+1_793-1)_(846+1_847-1)deletion of exon 72FANCANM_000135:c.(1626+1_1627-1)_ (2852+1_2853-1)deldeletion of exons 18-291FANCANM_000135:c.(893+1_894-1)_(1359+1_1360-1)deldeletion of exons 11-141PMS2NM_000535: c.(903+1_904-1)_(988+1_989-1)deldeletion of exon 91PMS2NM_000535:c.(705+1_706-1)_(2006+1_2007-1)deldeletion of exons 7-111 Citation Format: Nikolaos Tsoulos, Konstantinos Agiannitopoulos, Georgia Pepe, Eirini Papadopoulou, Georgios N Tsaousis, Despina Apostolopoulou, Angeliki Meintani, Vassileios Venizelos, Christos Markopoulos, Rodoniki Iosifidou, Sofia Karageorgopoulou, Christos Christodoulou, Ioannis Natsiopoulos, Konstantinos Papazisis, Maria Vasilaki-Antonatou, Eleftherios Kabletsas, Amanta Psyrri, Stylianos Giassas, Dimitrios Ziogas, Efthalia Lalla, Anna Koumarianou, Christos Papadimitriou, Vahit Ozmen, Sualp Tansan, Kerim Kaban, Tahsin Ozatlı, Dan Tudor Eniu, Angelica Chiorean, Alexandru Blidaru, George Nasioulas. Different CNVs account for 10.4% of pathogenic variants in 1418 patients referred for hereditary breast cancer testing [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-09-10.

The Contribution of Deleterious Rare Alleles in ENPP1 and Osteomalacia Causative Genes to Atypical Femoral Fracture.

Atypical femoral fractures (AFFs) are very rare atraumatic or mild trauma fractures in the subtrochanteric region or femoral shaft. Some unique genetic variants in Asian populations might confer susceptibility to AFF, since the incidence of AFFs is higher in Asian populations. Because rare variants have been found to be causative in some diseases and the roles of osteomalacia causative genes have not been reported, we investigated rare variants in genes causing abnormal mineralization. Exome sequencing was performed to detect variants in gene coding and boundary regions, and the frequencies of deleterious rare alleles were compared between Japanese patients with AFF (n = 42) and controls of the 4.7KJPN panel of Tohoku Medical Megabank by whole genome sequencing (n = 4773). The frequency of the deleterious rare allele of ENPP1 was significantly increased in AFF (P = .0012, corrected P [Pc] = .0155, OR 4.73, 95% CI 2.15-10.40). In multigene panel analysis, the frequencies of deleterious rare alleles of candidate genes were increased in AFF (P = .0025, OR 2.72, 95% CI 1.49-4.93). Principal component analysis of bone metabolism markers identified a subgroup of patients with AFF with higher frequencies of deleterious rare alleles in ENPP1 (P = 4.69 × 10-5, Pc = .0006, OR 8.47, 95% CI 3.76-19.09) and the candidate genes (P = 1.08 × 10-5, OR 5.21, 95% CI 2.76-9.86). AFF is associated with genes including ENPP1 that cause abnormal mineralization, suggesting that osteomalacia is an underlying condition predisposing to AFF and that higher incident rates of AFFs in Asian populations might be explained by the genetic risk factors including ENPP1.

Singleton exome sequencing of 90 fetuses with ultrasound anomalies revealing novel disease-causing variants and genotype\u2013phenotype correlations

Exome sequencing has been increasingly implemented in prenatal genetic testing for fetuses with morphological abnormalities but normal rapid aneuploidy detection and microarray analysis. We present a retrospective study of 90 fetuses with different abnormal ultrasound findings, in which we employed the singleton exome sequencing (sES; 75 fetuses) or to a lesser extent (15 fetuses) a multigene panel analysis of 6713 genes as a primary tool for the detection of monogenic diseases. The detection rate of pathogenic or likely pathogenic variants in this study was 34.4%. The highest diagnostic rate of 56% was in fetuses with multiple anomalies, followed by cases with skeletal or renal abnormalities (diagnostic rate of 50%, respectively). We report 20 novel disease-causing variants in different known disease-associated genes and new genotype–phenotype associations for the genes KMT2D, MN1, CDK10, and EXOC3L2. Based on our data, we postulate that sES of fetal index cases with a concurrent sampling of parental probes for targeted testing of the origin of detected fetal variants could be a suitable tool to obtain reliable and rapid prenatal results, particularly in situations where a trio analysis is not possible.

Neurological Characteristics of Pediatric Glycogen Storage Disease.

Glycogen storage diseases (GSD) encompass a group of rare inherited diseases due dysfunction of glycogen metabolism. Hypoglycemia is the most common primary manifestation of GSD, and disturbances in glucose metabolism can cause neurological damage. The aims of this study were to first investigate the metabolic, genetic, and neurological profiles of children with GSD, and to test the hypothesis whether GSD type I would have greater neurological impact than GSD type IX. A cross-sectional study was conducted with 12 children diagnosed with GSD [Types: Ia (n=5); 1, Ib (n=1); 4, IXa (n=5); and 1, IXb (n=1)]. Genetic testing was conducted for the following genes using multigene panel analysis. The biochemical data and magnetic resonance imaging of the brain presented by the patients were evaluated. The criteria of adequate metabolic control were adopted based on the European Study on Glycogen Storage Disease type I consensus. Pathogenic mutations were identified using multigene panel analyses. The mutations and clinical chronology were related to the disease course and neuroimaging findings. Adequate metabolic control was achieved in 67% of patients (GSD I, 43%; GSD IX, 100%). Fourteen different mutations were detected, and only two co-occurring mutations were observed across families (G6PC c.247C>T and c.1039C>T). Six previously unreported variants were identified (5 PHKA2; 1 PHKB). The proportion of GSD IX was higher in our cohort compared to other studies. Brain imaging abnormalities were more frequent among patients with GSD I, early-symptom onset, longer hospitalization, and inadequate metabolic control. The frequency of mutations was similar to that observed among the North American and European populations. None of the mutations observed in PHKA2 have been described previously. Therefore, current study reports six GSD variants previously unknown, and neurological consequences of GSD I. The principal neurological impact of GSD appeared to be related to inadequate metabolic control, especially hypoglycemia.

BARD1 Pathogenic Variants are Associated with Triple-Negative Breast Cancer in a Spanish Hereditary Breast and Ovarian Cancer Cohort.

Only a small fraction of hereditary breast and/or ovarian cancer (HBOC) cases are caused by germline variants in the high-penetrance breast cancer 1 and 2 genes (BRCA1 and BRCA2). BRCA1-associated ring domain 1 (BARD1), nuclear partner of BRCA1, has been suggested as a potential HBOC risk gene, although its prevalence and penetrance are variable according to populations and type of tumor. We aimed to investigate the prevalence of BARD1 truncating variants in a cohort of patients with clinical suspicion of HBOC. A comprehensive BARD1 screening by multigene panel analysis was performed in 4015 unrelated patients according to our regional guidelines for genetic testing in hereditary cancer. In addition, 51,202 Genome Aggregation Database (gnomAD) non-Finnish, non-cancer European individuals were used as a control population. In our patient cohort, we identified 19 patients with heterozygous BARD1 truncating variants (0.47%), whereas the frequency observed in the gnomAD controls was 0.12%. We found a statistically significant association of truncating BARD1 variants with overall risk (odds ratio (OR) = 3.78; CI = 2.10–6.48; p = 1.16 × 10−5). This association remained significant in the hereditary breast cancer (HBC) group (OR = 4.18; CI = 2.10–7.70; p = 5.45 × 10−5). Furthermore, deleterious BARD1 variants were enriched among triple-negative BC patients (OR = 5.40; CI = 1.77–18.15; p = 0.001) compared to other BC subtypes. Our results support the role of BARD1 as a moderate penetrance BC predisposing gene and highlight a stronger association with triple-negative tumors.

ERCC3, a new ovarian cancer susceptibility gene?

Hereditary breast and ovarian cancer syndrome (HBOC) is an inherited disorder with an increased risk of breast cancer (BC) and ovarian cancers(OC). Mutations in BRCA1-BRCA2 explains less than a half of cases. In the last decade several genes with different penetrance have been associated with an increased risk of BC or OC. A recurrent heterozygous ERCC3 truncating mutation increases the risk for breast cancer in patients with Ashkenazi Jewish ancestry. Our study aimed to investigate the role of ERCC3 truncating variants in a cohort of patients with suspicion of HBOC. ERCC3 screening by multigene-panel analysis in 1311 unrelated patients after our regional consensus for genetic testing in hereditary cancer was done. In addition, 453 Spanish cancer-free individuals and 51,343 GnomAD non-Finnish, non-cancer European individuals were used as control populations. We identified 13 patients with heterozygous ERCC3 truncating variants (0.99%). Five of them also carried a mutation in a high- /moderate-penetrance HBOC gene (BRCA1, BRCA2, CHEK2, and TP53) being Multilocus Inherited Neoplasia Alleles syndrome (MINAS) patients. The frequency in 453 Spanish controls was of 0.22%; similar to that observed in 51,343 non-Finnish European GnomAD population (0.24%). We found an almost statistically significant association of truncating ERCC3 variants with BC (odds ratio [OR]=2.25, confidence interval [CI]=0.6-5.93,P=0.11), and we observed for the first time a significant association with OC (OR=4.74, CI=1-14.34,P=0.028), that holds even after removing MINAS cases. To our knowledge, this is the largest HBOC series comprehensively analysed for ERCC3 mutations, and the first study identifying ERCC3 as a cancer risk for OC.

Comprehensive Constitutional Genetic and Epigenetic Characterization of Lynch-Like Individuals.

The causal mechanism for cancer predisposition in Lynch-like syndrome (LLS) remains unknown. Our aim was to elucidate the constitutional basis of mismatch repair (MMR) deficiency in LLS patients throughout a comprehensive (epi)genetic analysis. One hundred and fifteen LLS patients harboring MMR-deficient tumors and no germline MMR mutations were included. Mutational analysis of 26 colorectal cancer (CRC)-associated genes was performed. Pathogenicity of MMR variants was assessed by splicing and multifactorial likelihood analyses. Genome-wide methylome analysis was performed by the Infinium Human Methylation 450K Bead Chip. The multigene panel analysis revealed the presence of two MMR gene truncating mutations not previously found. Of a total of 15 additional MMR variants identified, five -present in 6 unrelated individuals- were reclassified as pathogenic. In addition, 13 predicted deleterious variants in other CRC-predisposing genes were found in 12 probands. Methylome analysis detected one constitutional MLH1 epimutation, but no additional differentially methylated regions were identified in LLS compared to LS patients or cancer-free individuals. In conclusion, the use of an ad-hoc designed gene panel combined with pathogenicity assessment of variants allowed the identification of deleterious MMR mutations as well as new LLS candidate causal genes. Constitutional epimutations in non-LS-associated genes are not responsible for LLS.

Abstract P5-03-09: Multigene panel analysis in breast and ovarian cancer

Abstract Background: Genetic testing is of prognostic and predictive importance in patients with ovarian cancer and breast cancer. The best explored mutations are those of the BRCA 1 and BRCA2 genes, and the result of BRCA testing impacts treatment and prophylactic options. Data on other breast-cancer-associated genes are accumulating. Methods: All patients who underwent genetic counseling and testing for BRCA 1 and BRCA 2 between 2007 and 2019 at the Medical University of Graz were reviewed. If BRCA testing was negative patients were offered panel testing for other breast and ovarian cancer associated genes including ATM, PETEN, RAD50, RAD51D, RAD51C, TP53, STK11, ATM, NBN, CDH, MSH6, MSH2, MLH1, CHEK2, PALB2, BRIP1, SMO, PETEN, SMO. Results: In total 1905 patients were counseled for genetic testing between 2007 to 2019. In 292 patients (mean age 53yrs) panel gene testing was performed. 114 (39%) of these 292 BRCA-negative patients (55 with breast cancer, 24 with ovarian cancer) had a pathogenic variant (n=32) or a mutation of unknown significance (n=82). The most common pathogenic variant occurred in the CHEK2 gene (n=15). Interpretation: In this series approximately one third of high-risk patients who tested negative for a BRCA mutation had a mutation of unknown significance and/or pathogenic mutation in gene panel testing. This indicates that genetic panel testing should be considered more frequently. Citation Format: Elisabeth Katharina Trapp, Michael Speicher, Jochen B Geigl, Vassiliki Kolovetsiou-Kreiner, Julia Reisinger, Josef Haas, Edgar Petru, Karl Tamussino, Gunda Pristauz. Multigene panel analysis in breast and ovarian cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-03-09.

NGS-based multi-gene panel analysis in early-onset colorectal cancer patients

Abstract Background Colorectal cancer (CRC) keeps an upward incidence and rejuvenation trend in Kazakhstan, as well as worldwide, which is the motive for revising traditional risk factors and searching for cause-effective relationships at the genome level. Genetic nature of early-onset CRC is still unclear. The aim of this study was to determine the spectrum of cancer-related gene mutations among early-onset CRC patients. Methods The study included 125 CRC patients aged between 17 and 50 from Kazakhstan. DNAs were extracted from blood using the GeneJET Purification Kit. Next-generation sequencing (NGS) was done on the MiSeq using the TruSightCancer Kit. NGS data were analyzed by the MiSeq Reporter and Variant Studio. SIFT and PolyPhen-2 were used to predict potential pathogenic effects of missense variants on protein structure and function. Results We were able to detect 24 pathogenic/likely pathogenic mutations in 20 (16%) cases, out of which 8 were novel. Out of all mutations, five were detected in the APC gene, three in FANCI, three in BRCA2, two in BRCA1, two in MLH1, and one mutation each, in MSH6, MUTYH, BLM, NBN, ATM, BMPR1A, CHEK2, AIP, and DICER1 genes. The analysis of mutation type has revealed 10 frameshift mutations, 5 missense mutations, 5 stop-gain mutations, 1 in-frame deletion, and 3 mutations involving uncorrected splicing. All mutations were in the heterozygous state. Most of the mutations were not available in the 1000G, ESP6500 and ExAC databases. 5% were available in the dbSNP database, 29.1% in the COSMIC, and 38.4% in the ClinVar and/or LOVD. Pathogenic mutations in high penetrance CRC genes were higher in patients with family history of cancer (FHC) (21.1%, P = 0.0002) and in patients with primary multiple tumors (20.0 %, P = 0.0004) compared with patients without/unknown FHC (3.1%). Conclusions Molecular genetic study of CRC in young patients using NGS allows to identify CRC cases with syndromic and sporadic nature, to stratify the level of risk for a particular patient and his/her relatives, to influence on the diagnostic and therapeutic approaches and clinical examinations. The findings from this study show the diagnostic value of the detected pathogenic mutations in key CRC genes from the Kazakhstan population as promising biomarkers for CRC diagnosis in young people. Legal entity responsible for the study Institute of General Genetics and Cytology. Funding Ministry of Healthcare of the Republic of Kazakhstan. Disclosure All authors have declared no conflicts of interest.