ClinVar Database Research Articles

Identification of novel single-nucleotide variants altering RNA splicing of PKD1 and PKD2.

The development of sequencing techniques identified numerous genetic variants, and accurate evaluation of the clinical significance of these variants facilitates the diagnosis of Mendelian diseases. In the present study, 549 rare single- nucleotide variants of uncertain significance were extracted from the ADPKD and ClinVar databases. MaxEntScan scoresplice is an in silico splicing prediction tool that was used to analyze rare PKD1 and PKD2 variants of unknown significance. An in vitro minigene splicing assay was used to verify 37 splicing-altering candidates that were located within seven residues of the splice donor sequence excluding canonical GT dinucleotides or within 21 residues of the acceptor sequence excluding canonical AG dinucleotides of PKD1 and PKD2. We demonstrated that eight PKD1 variants alter RNA splicing and were predicted to be pathogenic.

P-PN036. Loss of function mutation in HINT1 gene presenting with neuromyotonia and hereditary neuropathy

Introduction. Histidine Triad Nucleotide binding protein 1 (HINT1) also known as Protein kinase C inhibitor 1 (PRKCNH-1); Protein kinase C interacting protein 1 (PKCI-1), mapped to chromosome 5q31, is a purine phosphoramidase homodimer and is ubiquitously expressed. HINT1 is a tumour suprresor gene and is involved in several apoptotic pathways. Biallelic variants in the HINT1 gene are known to cause Neuromyotonia with Axonal Neuropathy (NMAN). Zimon et al. have identified 8 HINT1 variants in 33 families of NMAN. Results. A 13-year-old male child, born of consanguineous parentage; presented for the first time in 2018 with of bilateral foot drop of 2 years duration. This was followed one and half year later by bilateral forearm and hand muscle weakness in the form of finger and wrist drop with ulner deviation of the wrist. Hyperpigmentation of the knuckles was noted on the examination. Investigations were negative for screening of inborn errors of metabolism by Tandem mass spectrometry. His serum vitamin B12 level was 692 pg/ml (180-914 pg/ml). ENMG demonstrated neurogenic myotonia. NGS of the proband reported a pathogenic start-loss variation c.3G>C_NM005340.7 (p.Met1Ile) in the HINT1 gene aligned to human reference genome (GRCh37/hg19). The variant is not reported in the ClinVar and the Human gemone mutation (HGMD) databases. A variant at c.2T>C has been reported as likely pathogenic for NMAN in the ClinVar database. The variant is reported as damaging by SIFT, possibly damaging by PolyPhen2, and disease causing by MutationTaster2. CADD score is 24. The variant is not reported in gnomAD, 1000 genome and ExAC population frequency databases. Conclusion. Rare genetic causes of CMT are important contributions to the full allelic spectrum of the disease. Here we report an ultra-rare NMAN case of Indian origin; mediated by a novel, pathogenic, biallelic start-loss variant in the HINT1 gene.

IVS-II-16 (G>C) (HBB: c.315+16G>C) or IVS-II-666 (C>T) (HBB: c.316-185C>T) Mutations Trigger an Hb S (HBB: c.20A>T)/β+-Thalassemia Phenotype in an Hb S Trait Patient

Sickle cell trait is a medical condition caused by the presence of both mutant Hb S (HBB: c.20A>T) and normal Hb A alleles. Although sickle cell trait is typically considered to be asymptomatic and benign, genetic modifiers and mutations can lead to severe clinical complications. In this study, the possible pathogenicity of the IVS-II-16 (G>C) (HBB: c.315+16G>C) and IVS-II-666 (C>T) (HBB: c.316-185C>T) mutations, which are considered to be neutral polymorphisms, and the association between the Hb S mutation are presented. To the best of our knowledge, these polymorphisms have not been previously reported in any sickle cell trait patient, and no relevant studies have been conducted. We recently studied a 40-year-old woman (proband), diagnosed to be an Hb S/β-thalassemia (β-thal) carrier. β-Globin mutations were analyzed using a DNA sequencer based on the Sanger method. The HbVar and ClinVar databases show IVS-II-16 and IVS-II-666 to be intronic mutations. However, statements in these data banks contradict our findings. In the present study, a transfusion-dependent Hb S patient, behaving as an Hb S/β-thal case due to these mutations, was reported. These mutations have not been previously reported in an Hb S patient. Although the IVS-II-16 and IVS-II-666 mutations were previously reported as benign, they converted the Hb S phenotype to transfusion-dependent Hb S/β-thal when combined with Hb S. In this regard, IVS-II-16 and IVS-II-666 mutations may not be innocent, as previously thought.

Abstract 2175: Identification of Somatic BRCA1/2 mutation profile in tissue-based and liquid biopsy-based next generation sequencing in Egyptian patients with breast cancer

Abstract Aim: Identification of BRCA1/2 mutations in Breast Cancer (BC) patients may help in providing genetic information to customize the therapeutic management of patients. Assessing liquid biopsies for BRCA1/2 somatic mutations could overcome the limitation of information gained from tissue biopsy. Consequently, this study aimed to identify the frequencies of pathogenic BRCA1 & BRCA2 somatic mutations in both liquid and tissue biopsies in Egyptian BC patients. Material and methods: The study was conducted on 45 BC patients. Liquid and tissue biopsies were collected from each patient and they were profiled for BRCA1/2 mutations using next generation sequencing (NGS). Qiaseq UMI-based targeted panel was used for performing libraries. Ion proton platform was used for sequencing. The detected genetic variants at depth of coverage 500x were annotated against Cosmic, dbSNP, and Exac all databases. Polyphen-2, Sift prediction tools and Clinvar database were used to detect the biological function of the detected variants using IVA software. Results: Variants analysis revealed that Among 45 patients, 37 (82.2%) patients were carrying one or more damaging BRCA1/2 somatic mutations. A total number of BRCA1/2 somatic variants detected was 24 and they were classified to 13 pathogenic, 7 likely pathogenic and 4 benign variants. Tissue and plasma samples of BC patients were found to harbor one detectable pathogenic somatic mutation in BRCA1 gene; c.1961delA with frequencies of 75.5% vs 17.7%, respectively. While, both samples had 8 detectable pathogenic BRCA2 somatic variants, the top 3 most frequent mutations among them were: c.5351delA (82.2% vs 40%), c.2957delA (75.56% vs 42.2%) and c.9253delA (68.89% vs 24.4%). One deleterious variant was detected merely in tissue sample; c.8940delA (55.5%). Whereas, 3 damaging variants were detected merely in plasma samples: c.8053delA (22.2%), c.6762delT (20%), c.4631delA (6.6%).Likely pathogenic BRCA2 somatic variants were also identified in both samples; c.7177delA (71.1% vs 53.3%) and c.10248delA (37.7% vs 11.1%). Moreover, 5 likely pathogenic variants were merely found in plasma samples. The most frequently detected variant among them was c.1114A>C (42.2%). Our data showed that exon 10 in BRCA1 and exon 11 in BRCA2 are the most affected exons as they harbored the most frequent detected pathogenic variants. Conclusion: Our preliminary results showed the most frequent pathogenic and likely pathogenic BRCA1/2 somatic mutations in liquid and tissue biopsies in Egyptian BC patients. This data may help in developing personalized targeted therapies that efficiently target the BRCA1/2 mutations. Citation Format: Mai M. Lotfy, Amira Salah El-Din Youssef, Auhood Nassar, Mahmoud Kamel, Zeinab K. Hassan, Amany Abd-Elhameed Abou-Bakr, Ahmed Farahat, Mohamed M. Gomaa, Abdel Rahman N. Zekri. Identification of Somatic BRCA1/2 mutation profile in tissue-based and liquid biopsy-based next generation sequencing in Egyptian patients with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2175.

Abstract 192: MutAnt: Mutation annotation machine learning algorithm for pathogenicity evaluation of single nonsynonymous nucleotide substitutions in cancer cells

Abstract With the wealth of next-generation sequencing (NGS) produced over the last decade, distinguishing between pathogenic and neutral single nucleotide missense mutations is essential to understanding disease pathogenesis and cancer treatment selection and optimization. Current experimental methods, such as overexpression assays, siRNA, knock-out models, and studies in cultured cells or model organisms, can help uncover the phenotype of a set of mutations in genes but are not optimal for the identification of pathogenic mutations in thousands of potential candidates. In this study, we present a new mutation annotation tool, MutAnt, for the prediction of mutation pathogenicity that achieves superior performance by integrating state-of-the-art machine learning techniques with feature selection and hyperparameter tuning. To develop MutAnt, a supervised machine learning algorithm was trained to classify pathogenic and neutral single nonsynonymous nucleotide substitutions. The training dataset was based on ClinVar v20190102, the CancerGenomeInterpreter database of validated pathogenic mutations, and benign mutations from the database of Cancer Passenger Mutations (dbCPM). The Boruta algorithm with Shapley values was used for feature selection, and the Bayesian optimization was applied for the model hyperparameter tuning. Diverse validation methods were utilized in the development of MutAnt, such as cross-validation; the Database of Curated Mutations (DoCM); holdout on new mutations documented in the latest released version of ClinVar database, which contains validated disease-associated single nucleotide polymorphisms (SNPs) not previously annotated in existing algorithms; mutations with high allele frequency; test dataset III from VariBench; and correlation with the mean function score in a saturation genome editing experiment for the BRCA gene. MutAnt exhibited a stronger performance than other methods on all of the tested validation strategies, which was demonstrated by its accuracy (high f1-score, 0.95 to 0.99) and sensitivity-specificity value (ROC-AUC value, 0.97 to 0.99) in classifying mutations in each validation dataset. Furthermore, the present approach allowed for the prediction of novel mutations that are not covered by extensive population studies with higher accuracy than other methods. This was demonstrated by its prediction of the clinically relevant BRCA variants in the saturation genome editing experiment. In conclusion, MutAnt outperformed all compared mutation annotation algorithms in this study for the classification of clinically relevant benign and pathogenic mutations in the ClinVar dataset. Importantly, this approach allows prediction of novel mutations, subsequently allowing for the selection of targeted therapies for a broader set of patients. Citation Format: Aleksandr Sarachakov, Viktor Svekolkin, Zoia Antysheva, Jessica H. Brown, Alexander Bagaev, Nathan Fowler. MutAnt: Mutation annotation machine learning algorithm for pathogenicity evaluation of single nonsynonymous nucleotide substitutions in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 192.

Whole-exome sequencing reveals a novel homozygous mutation in the COQ8B gene associated with nephrotic syndrome

Nephrotic syndrome arising from monogenic mutations differs substantially from acquired ones in their clinical prognosis, progression, and disease management. Several pathogenic mutations in the COQ8B gene are known to cause nephrotic syndrome. Here, we used the whole-exome sequencing (WES) technology to decipher the genetic cause of nephrotic syndrome (CKD stage-V) in a large affected consanguineous family. Our study exposed a novel missense homozygous mutation NC_000019.9:g.41209497C > T; NM_024876.4:c.748G > A; NP_079152.3:p.(Asp250Asn) in the 9th exon of the COQ8B gene, co-segregated well with the disease phenotype. Our study provides the first insight into this homozygous condition, which has not been previously reported in 1000Genome, ClinVar, ExAC, and genomAD databases. In addition to the pathogenic COQ8B variant, the WES data also revealed some novel and recurrent mutations in the GLA, NUP107, COQ2, COQ6, COQ7 and COQ9 genes. The novel variants observed in this study have been submitted to the ClinVar database and are publicly available online with the accessions: SCV001451361.1, SCV001451725.1 and SCV001451724.1. Based on the patient's clinical history and genomic data with in silico validation, we conclude that pathogenic mutation in the COQ8B gene was causing kidney failure in an autosomal recessive manner. We recommend WES technology for genetic testing in such a consanguineous family to not only prevent the future generation, but early detection can help in disease management and therapeutic interventions.

Potential Familial Predisposition to Hematologic Neoplasm Detected Incidentally in Adult Patients Presenting with Myeloid Neoplasms

Introduction: Germline mutations in CEBPA, ETV6, RUNX1, and GATA2 have been reported to be associated with predisposition to hematologic malignancies. Next Generation Sequencing (NGS), used to identify somatic mutations to aid in targeted therapies, may incidentally uncover inherited germline mutations. Recognizing familial predisposition is important as it may impact management and surveillance. We assessed the prevalence of potentially clinically significant germline mutations detected incidentally by NGS in samples from patients investigated for myeloid disorders.Methods: NGS was performed to identify somatic mutations in 54 genes, on 6100 consecutive bone marrow aspirates or peripheral blood specimens between January 2014 and mid-2017. The average depth of sequencing was 10,000x. DNA was extracted using the QIAamp DNA Mini Kit and subjected to the TruSight Myeloid panel (Illumina, San Diego, CA). Most (92%) of the common dbSNP variants (>10% allele frequency in dbSNP) were found between 45-55% variant allele frequency (VAF) or 98-100% VAF for hetero- and homozygous states. This suggests that common SNPs are mostly found between those frequencies; hence, alleles with these frequencies were defined as candidate germline variants. However, to further enrich for germline variants, we only analyzed those cases with at least one other somatic variant with VAF of 5-40%; the rational being that it is unlikely that a sample would have two true independent somatic mutation events in different subclones. Following this enrichment process, 2951 specimens were eligible for further analysis for potential germline mutations. The ClinVar and dbSNP databases were used to assess allele frequencies and clinical significance.Results: In 2951 of 6100 specimens, we reported at least one somatic mutation with a variant allele frequency in the range of 5-40%. A total of 5322 variants were identified in this subset of specimens. Among the candidate germline mutations in 4 genes implicated in myeloid neoplasm predisposition, we identified 93 unique putative loss of function variants that met our germline criteria. Nine of these mutations were identified in more than 3 cases and more than half of these were represented in ClinVar as germline loss of function variants. One of them, RUNX1 : p.Arg201Gln, identified in 5 patients, is described as a germline pathogenic variant. In contrast, the other 84 variants identified in the 4 genes were rare (observed in 1 or 2 cases) and few (7/84) had been characterized in dbSNP. Most loss of function germline variants are unique and rare with few hotspots, which is consistent with our data.Analysis of germline mutations in genes, which have not yet been implicated in predisposition to myeloid neoplasm, revealed 4858 candidate mutations in 23 genes: 15.4% (750/4858) were unique, 13.6% (102/750) were more common (> 3 cases), while most (86.4%; 648/750) were found in only 1 or 2 cases. Some of the more common variants were identified in ClinVar as pathogenic germline variants, including IDH2 (p.Arg140Gln; MIM: 613657), identified in 84 cases, and a TP53 loss of function mutation (p.Pro72Arg) identified in 424 cases.Conclusion: Germline mutations, GATA2, RUNX1, CEBPA, and ETV6, known to predispose to myeloid malignancies, may be identified incidentally during testing for somatic mutations in myeloid malignancies.In addition, if the NGS panel includes genes other than those known to predispose towards myeloid malignancies, pathogenic variants of wide ranging clinical implication may be uncovered, as observed in the cases with mutation in IDH2 or TP53 . Although the specific mutation observed in IDH2 has been identified most often as a somatic mutation, it has been reported to be inherited in one instance. Inheriting this allele can confer D-2-hydroxyglutaric aciduria-2 (D2HGA2). The TP53 mutation observed is known to be involved in other hereditary cancers (e.g. Li-Fraumeni syndrome) and could affect the recommended therapy of antineoplastic agents.Rare incidentally identified potential germline mutations remain a challenge as their clinical significance may be unknown. These mutations require additional investigation to determine if they could contribute to clinical management. DisclosuresFunari:NeoGenomics: Employment. Thangavelu:NeoGenomics: Employment. Ma:NeoGenomics: Employment. De Dios:NeoGenomics: Employment. Agersborg:NeoGenomics: Employment. Blocker:NeoGenomics: Employment. Albitar:NeoGenomics: Employment.

Clinical heterogeneity and molecular genetic causes in a cohort of patients with disorders/differences of sex development

Background. Disorders of sex development (DSD) are a group of rare congenital conditions. Clinical management of patients with DSD is often difficult and requires multidisciplinary approach.
 Aim. Analysis of the frequency of establishing genetic causes in various forms of DSD by using an original targeted sequencing panel with subsequent establishment of associations of the identified genetic variants with the nature of clinical manifestations.
 Materials and methods. Conducted a clinical examination, karyotype analysis followed by the next generation sequencing (NGS) using MiSeq (Illumina) with the twenty-eight patients with different forms of 46, XY DSD were included. We designed HaloPlex (Agilent) gene panel that included coding regions of 80 candidate genes associated with DSD. All variants identified by NGS were confirmed by Sanger sequencing. We performed bioinformatics analysis using OMIM, 1000 genomes, ESP6500, Genome Aggregation Database projects. To assess the clinical significance of the identified variants we used ClinVar database and American College of Medical Genetics and Genomics criteria.
 Results. Out of 28 patients pathogenic, likely pathogenic, variants with unknown significance were identified in 11 patients (39%). In combination with clinical phenotype these variants were determined as causative for DSD. Nine patients (82%) had likely causative variants in one gene (of monogenic origin), while 18% had variants in two genes simultaneously (of oligogenic origin). 43% of the identified gene variants have not been previously reported. The variants in NR5A1 were associated with gonadal dysgenesis in two patients; the variants in MAP3K1 were also found in another two patients with gonadal dysgenesis, variants in AR in three patients with CAIS, variant in MAMLD1 was associated with proximal form of hypospadias, variant in CYP17A1 was associated with testosterone biosynthetic defect. Among the two patients with variants of oligogenic origin, one had variants in MAP3K1 and MAMLD1 genes and was clinically characterized by hypospadias; the second had variants in AR and SEMA3A and was diagnosed with PAIS. There were also two patients with variants in NR5A1 of familial inheritance.
 Conclusion. NGS-based targeted sequencing is a promising technique to improve the differential diagnosis, genetic counseling and management strategies for patients with DSD. Complex clinical examination followed by molecular genetic analysis improves the diagnosis, genetic counseling, and management strategies for patients with DSD including the assignment of sex of rearing.

MO045THE APPLICATION OF A NGS KIDNEY PANEL REVEALED KEY CHALLENGES OF PKD1-2 ANALYSIS: INTERPRETATION OF MISSENSE VARIANTS, SIGNIFICANCE OF VARIANTS IN DUPLICATED REGIONS AND HIGH ALLELIC HETEROGENEITY

Abstract Background and Aims Genetic testing has changed the clinical management of inherited kidney diseases patients, improving prognosis, surveillance and therapy. On the other hand, it has put geneticists and clinicians in front of new challenges, as the heterogeneity of these disorders and the high number of variants, with no clear genotype-phenotype correlation. Method 108 patients underwent genetic analysis through a kidney focused NGS panel, named Nephroplex, containing 119 genetic loci associated with inherited kidney disorders. The study aimed to addressed the genetic landscape of cystic individuals and to analyze PKD1 and PKD2 variants in non-cystic individuals. Results Following diagnostic criteria, patients were divided as cystic kidney diseases (n=36) and non-cystic kidney diseases (n=72). Among the group of cystic patients, a causative mutation was detected in 51% of cases. We found thirty-seven PKD1 and PKD2 variants in 26 out of 35 individuals. In particular, 12 variants were shown to be damaging and nine of that were reported in public database, as CLINVAR and Mayo Clinic databases. Among pathogenic variants, twelve were truncating and the remaining were missense variants. Of note, 7 out of 12 damaging PKD1 mutations were located in duplicated regions. Moreover, in three cystic patients, we found a (i) a frameshift hemizygote OFD1 mutation (ii) compound heterozygote PKHD1 variants and (iii) a frameshift MUC1 variant, framing the diagnosis of oro-facio-digital type 1, autosomal recessive polycystic kidney disease and autosomal dominant tubulointerstitial disease, respectively. Interestingly, we detected 28 PKD1-2 rare variants in 21 out of 75 adult non cystic patients (28%). The most were observed in PKD1 genes (82% vs 18% in PKD2). Eighteen of 28 variants were described in the literature as likely benign or as mutations of uncertain significance, while we found 10 novel variants. In silico analysis revealed as pathogenic a frameshift mutation located in exon 15. Of note, the great part of these variations reside into the duplicated PKD1 regions. Conclusion Our data showed that genetic analysis of ADPKD retains unique challenges, given the high degree of homology of PKD1 with his pseudogenes and the high allelic heterogeneity in non-cystic individuals.

In silico analysis of BRCA1 and BRCA2 missense variants and the relevance in molecular genetic testing

Over the years since the genetic testing of BRCA1 and BRCA2 has been conducted for research and later introduced into clinical practice, a high number of missense variants have been reported in the literature and deposited in public databases. Polymorphism Phenotyping v2 (PolyPhen-2) and Sorting Intolerant from Tolerant (SIFT) are two widely applied bioinformatics tools used to assess the functional impacts of missense variants. A total of 2605 BRCA1 and 4763 BRCA2 variants from the ClinVar database were analysed with PolyPhen2 and SIFT. When SIFT was evaluated alongside PolyPhen-2 HumDiv and HumVar, it had shown top performance in terms of negative predictive value (NPV) (100%) and sensitivity (100%) for ClinVar classified benign and pathogenic BRCA1 variants. Both SIFT and PolyPhen-2 HumDiv achieved 100% NPV and 100% sensitivity in prediction of pathogenicity of the BRCA2 variants. Agreement was achieved in prediction outcomes from the three tested approaches in 55.04% and 68.97% of the variants of unknown significance (VUS) for BRCA1 and BRCA2, respectively. The performances of PolyPhen-2 and SIFT in predicting functional impacts varied across the two genes. Due to lack of high concordance in prediction outcomes among the two tested algorithms, their usefulness in classifying the pathogenicity of VUS identified through molecular testing of BRCA1 and BRCA2 is hence limited in the clinical setting.

Whole-Exome Sequencing Reveals a Rare Variant of OTOF Gene Causing Congenital Non-syndromic Hearing Loss Among Large Muslim Families Favoring Consanguinity.

Non-syndromic hearing loss (NSHL) is one of the most frequent auditory deficits in humans characterized by high clinical and genetic heterogeneity. Very few studies have reported the relationship between OTOF (Locus: DFNB9) and hereditary hearing loss in India. We aimed to decipher the genetic cause of prelingual NSHL in a large affected Muslim consanguineous families using whole-exome sequencing (WES). The study was performed following the guidelines and regulations of the Indian Council of Medical Research (ICMR), New Delhi. The population was identified from Jammu and Kashmir, the Northernmost part of India. Near about 100 individuals were born deaf-mute in the village of 3,000 inhabitants. A total of 103 individuals (with 52 cases and 51 controls) agreed to participate in this study. Our study revealed a rare non-sense homozygous mutation NC_000002.11:g.2:26702224G>A; NM_001287489.2:c.2122C>T; NP_001274418.1:p.(Arg708∗) in the 18th exon of the OTOF gene. Our study provides the first insight into this homozygous condition, which has not been previously reported in ExAC, 1,000 Genome and genomAD databases. Furthermore, the variant was confirmed in the population cohort (n = 103) using Sanger sequencing. In addition to the pathogenic OTOF variant, the WES data also revealed novel and recurrent mutations in CDH23, GJB2, MYO15A, OTOG, and SLC26A4 genes. The rare pathogenic and the novel variants observed in this study have been submitted to the ClinVar database and are publicly available online with the accessions SCV001448680.1, SCV001448682.1 and SCV001448681.1. We conclude that OTOF-related NSHL hearing loss is prevalent in the region due to successive inbreeding in its generations. We recommend premarital genetic testing and genetic counseling strategies to minimize and control the disease risk in future generations.

The landscape of germline mutations and the relationship with tumor mutation burden in Chinese patients with lung cancer.

10522 Background: Lung cancer is one of the most common types of cancer, ranking the first in the incidence and mortality of malignant tumors in the world and China. Although studies have been reported that genetic susceptibility to lung cancer is associated with certain germline mutations, the relationship between lung cancer risk and inherited genetic factors remains relatively elusive. However, the effect of germline mutation on TMB in lung cancer has not been explored. Herein, DNA genomic profiling was performed through NGS with a 539-gene panel to explore the germline mutations and the relationship with TMB in Chinese patients with lung cancer. Methods: We retrospectively analyzed the germline mutations through a comprehensive 539-gene profiling of 3541 Chinese patients with lung cancer. 539-gene panel contained germline mutations in 90 hereditary tumor-associated genes. We screened out the pathogenic and likely pathogenic germline mutations according to the standards and guidelines for the interpretation of sequence variants of The American College of Medical Genetics and Genomics (ACMG), and picked out there is no records in Clinvar database and no literature report. TMB of tissue or blood ctDNA in 3541 patients were further analyzed in with pathogenic mutations (P group), with likely pathogenic mutations (LP group), and no germline mutations group (Non-P group). The difference in TMB was analyzed via the Wilcoxon sign test. Results: In 3541 patients with lung cancer, 177 (4.999%) patients were identified harboring pathogenic or likely pathogenic germline mutations, in which 78 P group and 99 LP group, the rest 3364 were Non-P group. The highest prevalence of germline mutation was found in BRCA2 (0.565%), ATM (0.339%), MUTYH (0.282%), and BRCA1 (0.254%). In 177 patients with pathogenic or likely pathogenic germline mutations, 67 mutations were recorded as UNK (unknow) in Clinvar database and no literature report. The media TMB of tissue in P group, LP group and Non-P group were 5.149, 5.535 and 5.547 respectively. The media TMB of blood ctDNA in P group, LP group and Non-P group were 4.257, 3.945 and 4.483 respectively. There was no statistical difference in TMB between P and Non-P groups (tissue p = 0.98; ctDNA p = 0.5). Conclusions: In our study, we firstly identified 67 novel germline mutations and studied on the relationship between germline mutations and TMB in lung cancer, which expanded the understanding of germline mutations.

Predicting Pathogenicity of CDH1 Gene Variants in Patients with Early-onset Diffuse Gastric Cancer from Western Mexico.

Early-onset diffuse gastric cancer (EODGC) occurs at or before 50 years of age. Pathogenic mutations and germline deletions in the CDH1 gene (E-cadherin) are well-documented genetic factors associated with the causes of EODGC. The objective of the study was to study CDH1 germline variants and their potential functional impact in patients with EODGC in a Mexican population. We studied seven EODGC patients from a biomedical research center in western Mexico. Variants were identified by Sanger sequencing and multiplex ligation-dependent probe amplification. The DeepSEA and SNPClinic v.1.0 software and the Ensembl (1000 Genomes Project, 1kGP) and ClinVar databases were used to predict functional single-nucleotide polymorphisms (SNPs). The genetic admixture of the Mexican patients was corroborated by 22 short tandem repeat loci genotyping and structure analysis. We found 12 germline CDH1 variants in all EODGC patients, and all of them are considered as polymorphisms: rs34561447, rs5030625, rs16260, rs1330727101, rs28372783, rs942269593, rs3743674, rs1801552, rs34939176, rs33964119, rs3556654, and rs1801026. The prediction of regulatory SNPs in the promoter suggests a role for a retrovirus in EODGC that induces the transcription of interferon-related genes through toll-like receptor-interferon response factor 3 signaling, as three SNPs in the CDH1 promoter alter three binding sites for this transcription factor. In addition, SNPs rs28372783 and rs1801026 could alter upstream stimulatory factors 1 (USF1)/USF2-mediated telomerase-dependent lymphocyte activation in EODGC. Other interesting result is a CTCF-dependent shorter CDH1 isoform lacking exon 14, probably due to exon-skipping mediated by rs33964119. Classical pathogenic germline mutations in the CDH1 gene were not found in these 7 EODGC patients. However, the in silico approaches revealed the possible involvement of a retrovirus and a shorter E-cadherin isoform in EODGC. Nevertheless, further in vitro and in vivo assays are needed to confirm these predictions.

A bioinformatics approach to investigating the structural and functional consequences of SNPs in TMPRSS2 for COVID‐19 infection

SARS‐CoV‐2 is a highly infectious virus that is responsible for the COVID‐19 global pandemic that swept the world in 2020. Disease outcomes range from asymptomatic to fatal. The virus initiates entry into host cells by the binding of its spike protein to the ACE2 receptor. Entry is finalized by the activation of spike glycoprotein by proteases including transmembrane protease, serine 2 (TMPRSS2) and FURIN which cleave the spike protein of the virus. Single nucleotide polymorphisms (SNPs) in TMPRSS2 may lead to functional changes which could underlie differences in disease severity. TMPRSS2 is also known to activate different respiratory illnesses including coronaviruses and influenza A (Shen et al., 2020). Previous studies have shown that knockout TMPRSS2 mice appeared healthy, experienced a decrease in viral spread within the respiratory system, and had a less severe immune response when infected with SARS‐CoV and MERS‐CoV (Baughn et al., 2020). Thus, we asked whether genetic variations in TMPRSS2 in humans lead to differences in infection rates or severity of disease symptoms of SARS‐CoV‐2. We examined the NCBI dbSNP database to identify SNPs in the TMPRSS2 gene. As of 10 December 2020, we found there were 11,023 intron variants, 393 missense variants, 186 synonymous variants, 3 in‐frame insertion variants, 2 in‐frame deletion variants, and 1 initiator codon variant reported. To narrow these down to 23 SNPs of interest, we first searched the ClinVar database to identify SNPs with general clinical significance, followed by searching the literature to determine SNPs specifically related to SARS‐CoV‐2 severity. One missense variant, rs12329760, results in an amino acid substitution, V160M, which has been predicted to alter TMPRSS2 function. A subset of these SNPs show differences in frequency in world populations, and we wondered if these SNPs had structural and functional consequences for the protein. A crystal structure of TMPRSS2 is not currently available. To visualize the structural consequences of amino acid substitutions, we performed homology modeling on TMPRSS2 (UniProt O15393) using the structure prediction software HHPred, RaptorX, and SwissModel based on the ~30% similarity to hepsin. The predicted structures of TMPRSS2 with various amino acid substitutions were then docked to the SARS‐CoV‐2 spike protein using I‐TASSER and Haddock 2.4 to observe differences in binding interactions and therefore determine which sequence changes are predicted to alter binding interactions, potentially contributing to the wide variation of symptoms caused by COVID‐19.Baughn, L. B., Sharma, N., Elhaik, E., Sekulic, A., Bryce, A. H., & Fonseca, R. (2020). Targeting TMPRSS2 in SARS‐CoV‐2 Infection. Mayo Clinic proceedings, 95(9), 1989–1999. https://doi.org/10.1016/j.mayocp.2020.06.018Shen, L.W.; Mao, H.J.;, Wu, Y.L.; Tanaka,Y.; Zhang,W. (2017) TMPRSS2: A potential target for treatment of influenza virus and coronavirus infections, Biochimie, 142, 1‐10. https://doi.org/10.1016/j.biochi.2017.07.016

Identification of a novel LAMA2 c.2217G\u2009>\u2009A, p.(Trp739*) mutation in a Moroccan patient with congenital muscular dystrophy: a case report

BackgroundMerosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a rare autosomal recessive genetic condition caused by deleterious mutations in the LAMA2 gene encoding the laminin-α2 chain. It is the most frequent subtype of congenital muscular dystrophies (CMDs) characterized by total laminin-α2 deficiency with muscle weakness at birth or in the first six months of life. To the best of our knowledge, this study reports the first molecular diagnosis and genetic defect of this heterogeneous form of CMD performed in a Moroccan medical genetic center using next-generation sequencing (NGS). It allows us to expand the mutational spectrum of the LAMA2 gene.Case presentationWe report the case of a female Moroccan child with clinical and paraclinical features in favor of a CMD. She has global congenital hypotonia with generalized muscle weakness, psychomotor retardation, increased serum creatine kinase, and normal brain scan at the age of six months. Targeted NGS leads to the identification of a novel homozygous nonsense mutation c.2217G > A, p.(Trp739*) in the exon 16 of LAMA2. Sanger sequencing confirmed this mutation in the affected patient and showed that her parents are heterozygous carriers.ConclusionsA modern genetic analysis by NGS improves the genetic diagnosis pathway for adequate genetic counseling of affected families more precisely. An accession number from the National Center for Biotechnology Information (NCBI) ClinVar database was retrieved for this novel LAMA2 mutation.

Analysis of BRCA1/2 variants of unknown significance in the prospective Korean Hereditary Breast Cancer study

Genetic testing for BRCA1 and BRCA2 is crucial in diagnosing hereditary breast and ovarian cancer syndromes and has increased with the development of multigene panel tests. However, results classified as variants of uncertain significance (VUS) present challenges to clinicians in attempting to choose an appropriate management plans. We reviewed a total of 676 breast cancer patients included in the Korean Hereditary Breast Cancer (KOHBRA) study with a VUS on BRCA mutation tests between November 2007 and April 2013. These results were compared to the ClinVar database. We calculated the incidence and odds ratios for these variants using the Korean Reference Genome Database. A total of 58 and 91 distinct VUS in BRCA1 and BRCA2 were identified in the KOHBRA study (comprising 278 and 453 patients, respectively). A total of 27 variants in the KOHBRA study were not registered in the Single Nucleotide Polymorphism database. Among BRCA1 VUSs, 20 were reclassified as benign or likely benign, four were reclassified as pathogenic or likely pathogenic, and eight remained as VUSs according to the ClinVar database. Of the BRCA2 VUSs, 25 were reclassified as benign or likely benign, two were reclassified as pathogenic or likely pathogenic, and 33 remained as VUS according to the ClinVar database. There were 12 variants with conflicting interpretations of pathogenicity for BRCA1 and 18 for BRCA2. Among them, p.Leu1780Pro showed a particularly high odds ratio. Six pathogenic variants and one conflicting variant identified using ClinVar could be reclassified as pathogenic variants in this study. Using updated ClinVar information and calculating odds ratios can be helpful when reclassifying VUSs in BRCA1/2.

Detection of activating mutations in liquid biopsy of Egyptian breast cancer patients using targeted next-generation sequencing: a pilot study

BackgroundBreast cancer (BC) is the 2nd most prevalent malignancy worldwide and is the most prevalent cancer among Egyptian women. The number of newly described cancer-associated genes has grown exponentially since the emergence of next-generation sequencing (NGS) technology. We aim to identify activating mutations in liquid biopsy of Egyptian breast cancer patients using targeted NGS technology. We also demonstrate the microsatellite instability (MSI) status using BAT25, BAT26, and NR27 markers which are tested on the Bioanalyzer 2100 system.ResultsTwenty-one variants were detected in 15 genes: 7 Substitution-Missense, 12 Substitution-coding silent, and 2 Substitution-intronic. Regarding ClinVar database, out of 21 variants there were 14 benign variants, 3 variants with conflicting interpretations of pathogenicity, 3 variants not reported, and 1 drug response variant. TP53 p.(Pro72Arg) missense mutations were found in 75% of patients. PIK3CA p.(Ile391Met), KDR p.(Gln472His) missense mutations were detected in 25% of patients each. Two patients revealed APC gene missense mutation with p.(Ile1307Lys) and p.(Glu1317Gln) variants. Only one patient showed ATM p.(Phe858Leu) gene mutation and one showed FGFR3 p.(Ala719Thr) variant. Regarding microsatellite instability (MSI) status, 2/8 (25%) patients were MSS, 3/8 (37.5%) patients were MSI-L, and 3/8 (37.5%) patients were MSI-HI.ConclusionIt is essential to use and validate minimally invasive liquid biopsy for activating mutations detection by next-generation sequencing especially in patients with inoperable disease or bone metastasis. This work should be extended with larger patient series with comparison of genetic mutations in liquid-based versus tissue-based biopsy and longer follow up period.

Predicting Ovarian/Breast Cancer Pathogenic Risks of Human BRCA1 Gene Variants of Unknown Significance.

High-throughput sequencing is gaining popularity in clinical diagnoses, but more and more novel gene variants with unknown clinical significance are being found, giving difficulties to interpretations of people's genetic data, precise disease diagnoses, and the making of therapeutic strategies and decisions. In order to solve these issues, it is of critical importance to figure out ways to analyze and interpret such variants. In this work, BRCA1 gene variants with unknown clinical significance were identified from clinical sequencing data, and then, we developed machine learning models so as to predict the pathogenicity for variants with unknown clinical significance. Through performance benchmarking, we found that the optimized random forest model scored 0.85 in area under receiver operating characteristic curve, which outperformed other models. Finally, we applied the best random forest model to predict the pathogenicity of 6321 BRCA1 variants from both sequencing data and ClinVar database. As a result, we obtained the predictive pathogenic risks of BRCA1 variants of unknown significance.

Variants associated with urea cycle disorders in Japanese patients: Nationwide study and literature review.

Urea cycle disorders (UCDs) are inherited metabolic diseases that lead to hyperammonemia with variable clinical manifestations. Using data from a nationwide study, we investigated the onset time, gene variants, clinical manifestations, and treatment of patients with UCDs in Japan. Of the 229 patients with UCDs diagnosed and/or treated between January 2000 and March 2018, identified gene variants and clinical information were available for 102 patients, including 62 patients with ornithine transcarbamylase (OTC) deficiency, 18 patients with carbamoyl phosphate synthetase 1 (CPS1) deficiency, 16 patients with argininosuccinate synthetase (ASS) deficiency, and 6 patients with argininosuccinate lyase (ASL) deficiency. A total of 13, 10, 4, and 5 variants in the OTC, CPS1, ASS, and ASL genes were respectively identified as novel variants, which were neither registered in ClinVar databases nor previously reported. The onset time and severity in patients with UCD could be predicted based on the identified gene variants in each patient from this nationwide study and previous studies. This genetic information may help in predicting the long-term outcome and determining specific treatment strategies such as liver transplantation in patients with UCDs.

Actionable secondary findings in arrhythmogenic right ventricle cardiomyopathy genes: impact and challenge of genetic counseling.

Comprehensive genetic analysis yields in a higher diagnostic rate but also in a higher number of secondary findings (SF). American College of Medical Genetics and Genomics (ACMG) published a list of 59 actionable genes for which disease causing sequence variants are recommended to be reported as SF including 27 genes linked to inherited cardiovascular disease (CVD) such as arrhythmia syndromes, cardiomyopathies and vascular and connective tissue disorders. One of the selected conditions represented in the actionable gene list is the arrhythmogenic right ventricle cardiomyopathy (ARVC), an inherited heart muscle disease with a particularly high risk of sudden cardiac death (SCD). Since clinical symptoms are frequently absent before SCD, a genetic finding is a promising option for early diagnosis and possible intervention. However, the variant interpretation and the decision to return a SF is still challenging. To determine the frequency of medically actionable SF linked to CVD we analyzed data of 6,605 individuals who underwent high throughput sequencing for noncardiac diagnostic requests. In particular, we critically assessed and classified the variants in the ARVC genes: DSC2, DSG2, DSP, PKP2 and TMEM43 and compared our findings with the population-based genome Aggregation Database (gnomAD) and ARVC-afflicted individuals listed in ClinVar and ARVC database. 1% (69/6,605) of tested individuals carried pathogenic SF in one of the 27 genes linked to CVD, of them 13 individuals (0.2%) carried a pathogenic SF in a ARVC gene. Overall, 582 rare variants were identified in all five ARVC genes, 96% of the variants were missense variants and 4% putative LoF variants (pLoF): frameshift, start/stop-gain/loss, splice-site. Finally, we selected 13 of the 24 pLoF variants as pathogenic SF by careful data interpretation. Since SF in actionable ARVC genes can allow early detection and prevention of disease and SCD, detected variant must undergo rigorous clinical and laboratory evaluation before it can be described as pathogenic and returned to patients. Returning a SF to a patient should be interdisciplinary, it needs genetic counselling and clinicians experienced in inherited heart disease.