Clinical Evaluation of a Custom Gene Panel as a Tool for Precision Male Infertility Diagnosis by Next-Generation Sequencing

Background: Up to 15% of couples are infertile and male factor infertility accounts for approximately 50% of these cases. Male infertility is a multifactorial pathological condition. The genetic of male infertility is very complex and at least 2000 genes are involved in its etiology. Genetic testing by next-generation sequencing (NGS) technologies can be relevant for its diagnostic value in male infertile patients. Therefore, the aim of this study was to implement the diagnostic offer with the use of an NGS panel for the identification of genetic variants. Methods: We developed an NGS gene panel that we used in 22 male infertile patients. The panel consisted of 110 genes exploring the genetic causes of male infertility; namely spermatogenesis failure due to single-gene mutations, central hypogonadism, androgen insensitivity syndrome, congenital hypopituitarism, and primary ciliary dyskinesia. Results: NGS and a subsequent sequencing of the positive pathogenic or likely pathogenic variants, 5 patients (23%) were found to have a molecular defect. In particular, pathogenic variants were identified in TEX11, CCDC39, CHD7, and NR5A1 genes. Moreover, 14 variants of unknown significance and 7 novel variants were found that require further functional studies and family segregation. Conclusion: This extended NGS-based diagnostic approach may represent a useful tool for the diagnosis of male infertility. The development of a custom-made gene panel by NGS seems capable of reducing the proportion of male idiopathic infertility.

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Male Infertility Diagnosis: Improvement of Genetic Analysis Performance by the Introduction of Pre-Diagnostic Genes in a Next-Generation Sequencing Custom-Made Panel

Frontiers in Endocrinology ◽

10.3389/fendo.2020.605237 ◽

2021 ◽

Vol 11 ◽

Author(s):

Vincenza Precone ◽

Rossella Cannarella ◽

Stefano Paolacci ◽

Gian Maria Busetto ◽

Tommaso Beccari ◽

...

Keyword(s):

Next Generation Sequencing ◽

Male Infertility ◽

Next Generation ◽

Male Population ◽

Pathogenic Variants ◽

Custom Made ◽

Number Of Genes ◽

General Male Population ◽

Generation Sequencing ◽

Spermatogenic Failure

BackgroundInfertility affects about 7% of the general male population. The underlying cause of male infertility is undefined in about 50% of cases (idiopathic infertility). The number of genes involved in human spermatogenesis is over two thousand. Therefore, it is essential to analyze a large number of genes that may be involved in male infertility. This study aimed to test idiopathic male infertile patients negative for a validated panel of “diagnostic” genes, for a wide panel of genes that we have defined as “pre-diagnostic.”MethodsWe developed a next-generation sequencing (NGS) gene panel including 65 pre-diagnostic genes that were used in 12 patients who were negative to a diagnostic genetic test for male infertility disorders, including primary spermatogenic failure and central hypogonadism, consisting of 110 genes.ResultsAfter NGS sequencing, variants in pre-diagnostic genes were identified in 10/12 patients who were negative to a diagnostic test for primary spermatogenic failure (n = 9) or central hypogonadism (n = 1) due to mutations of single genes. Two pathogenic variants of DNAH5 and CFTR genes and three uncertain significance variants of DNAI1, DNAH11, and CCDC40 genes were found. Moreover, three variants with high impact were found in AMELY, CATSPER 2, and ADCY10 genes.ConclusionThis study suggests that searching for pre-diagnostic genes may be of relevance to find the cause of infertility in patients with apparently idiopathic primary spermatogenic failure due to mutations of single genes and central hypogonadism.

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SAT0531 NEXT GENERATION SEQUENCING AND AUTOINFLAMMATORY SYNDROMES: CLINICAL AND GENETICAL CORRELATION ON AN ADULT POPULATION FROM A REFERRAL THIRD-CARE CENTRE

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.5067 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 1223-1223

Author(s):

J. R. Marques Soares ◽

M. Antolin Mate ◽

E. Garcia Arumi ◽

E. Tizzano Ferrari ◽

S. Bujan Rivas

Keyword(s):

Next Generation Sequencing ◽

Genetic Data ◽

Clinical Suspicion ◽

Gene Panel ◽

Adult Patients ◽

Next Generation ◽

Related Gene ◽

Unknown Significance ◽

Level Hospital ◽

Generation Sequencing

Background:Systemic autoinflammatory diseases (sAID) are a group of conditions with recurrent episodes of inflammation in absence of infection or autoimmune response. Its physiopathology mainly lies on mono/poligenic mutations involving genes related to the innate immune system response. Next Generation Sequencing (NGS) platformss have been a big step forward on sAID diagnosis, although a clinical and genetic correlation is still needed.Objectives:To review the sAID related gene panel variants identified using NGS sAID gene panel on a cohort of adult patients screened for sAID from a referral third-level hospital.To correlate genetic and clinical findings for sAID related variants identified in order to the clinical suspicion diagnosis of sAID.Methods:A retrospective review of a cohort of adult (≥ 16 yo) patients with available NGS sAID related gene panel (MiSeq Illumina sequencing platform including intron and exon variants from up to 17 sAID genes, with coverage depth > x100) among 2014 and 2019 was performed.Demographic, clinical and genetic data were collected in a database.Genetic variants were classified according to the American College of Medical Genetics/Association for Molecular Pathology classification as benign/likely benign/variable of unknown significance (VUS)/likely pathogenic/pathogenic. In case of polymorphisms or lack of genetic data, the variants were named as unclassified.A description of the cohort and an analysis of the correlation assessment between clinical data and genetic findings were performed.Results:246 out of 299 (82%) patients with NGS sAID gene panel had clinical data available. 170/246 (69%) were adult patients. The medium age was 48 yo, and the M/F ratio was 2.46. 87/170 (51%) adult patients presented 122 variants involving sAID genes (60/87 patients with a single variant). All the variants out of 7 seven were heterozygous variants.Variants were classified according to ACMG/AMP as follow: pathogenic/probably pathogenic: 22/122 (18%), unknown significance: 74/122 (60.6%), benign/probably benign: 6/122 (4.91%). 20/122 (16.4%) were unclassified variants or polymorphisms.The most frequent variants identified involved MEFV (54/122), NOD2/CARD15 (18/122) and TNFRSF1A (17/122 including 12 p.Arg121Gln variants) genes.37/122 (30%) variants correlated with the clinical picture in 33 patients, allowing to confirm the suspected diagnosis. Among the 122 variants, 7 not previously communicated variants were identified.No somatic variants were found.Conclusion:NGS sAID related gene panel is a useful tool for sAID diagnosis. In this cohort of 170 adult patients from a referral third-level hospital, genetic tests identified sAID related variants in almost half of them.20% of patients who underwent genetic NGS sAID related gene panel studies were finally diagnosed with sAID.The identification of a genetic variant (even pathogenic / likely pathogenic variant) is not diagnostic for sAID if there is not a suggestive clinical picture.Despite genetic findings, a careful evaluation of clinical – genetic correlation is needed to confirm the suspicion diagnosis, especially for low penetrance variants like TNFRSF1A p. Arg121Gln.References:Diagnostic utility of a targeted next-generation sequencing gene panel in the clinical suspicion of systemic autoinflammatory diseases: a multi-center study. Karacan I, Balamir A, Uğurlu S, et al. . Rheumatol Int. 2019 May;39(5):911-919. doi: 10.1007/s00296-019-04252-5. Epub 2019 Feb 19.Disclosure of Interests:None declared

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Next generation sequencing-based gene panel tests for the management of solid tumors

Cancer Science ◽

10.1111/cas.13837 ◽

2018 ◽

Vol 110 (1) ◽

pp. 6-15 ◽

Cited By ~ 20

Author(s):

Masayuki Nagahashi ◽

Yoshifumi Shimada ◽

Hiroshi Ichikawa ◽

Hitoshi Kameyama ◽

Kazuaki Takabe ◽

...

Keyword(s):

Next Generation Sequencing ◽

Solid Tumors ◽

Gene Panel ◽

Next Generation ◽

Generation Sequencing

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Next-Generation Sequencing Approach to Non–Small Cell Lung Carcinoma Yields More Actionable Alterations

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2017-0046-oa ◽

2017 ◽

Vol 142 (3) ◽

pp. 353-357 ◽

Cited By ~ 12

Author(s):

Mitra Mehrad ◽

Somak Roy ◽

Humberto Trejo Bittar ◽

Sanja Dacic

Keyword(s):

Next Generation Sequencing ◽

Lung Adenocarcinoma ◽

Small Cell ◽

Gene Panel ◽

Next Generation ◽

Small Cell Lung ◽

Genomic Alterations ◽

Generation Sequencing ◽

Gene Alterations ◽

Cancer Panel

Context.— Different testing algorithms and platforms for EGFR mutations and ALK rearrangements in advanced-stage lung adenocarcinoma exist. The multistep approach with single-gene assays has been challenged by more efficient next-generation sequencing (NGS) of a large number of gene alterations. The main criticism of the NGS approach is the detection of genomic alterations of uncertain significance. Objective.— To determine the best testing algorithm for patients with lung cancer in our clinical practice. Design.— Two testing approaches for metastatic lung adenocarcinoma were offered between 2012–2015. One approach was reflex testing for an 8-gene panel composed of DNA Sanger sequencing for EGFR, KRAS, PIK3CA, and BRAF and fluorescence in situ hybridization for ALK, ROS1, MET, and RET. At the oncologist's request, a subset of tumors tested by the 8-gene panel was subjected to a 50-gene Ion AmpliSeq Cancer Panel. Results.— Of 1200 non–small cell lung carcinomas (NSCLCs), 57 including 46 adenocarcinomas and NSCLCs, not otherwise specified; 7 squamous cell carcinomas (SCCs); and 4 large cell neuroendocrine carcinomas (LCNECs) were subjected to Ion AmpliSeq Cancer Panel. Ion AmpliSeq Cancer Panel detected 9 potentially actionable variants in 29 adenocarcinomas that were wild type by the 8-gene panel testing (9 of 29, 31.0%) in the following genes: ERBB2 (3 of 29, 10.3%), STK11 (2 of 29, 6.8%), PTEN (2 of 29, 6.8%), FBXW7 (1 of 29, 3.4%), and BRAF G469A (1 of 29, 3.4%). Four SCCs and 2 LCNECs showed investigational genomic alterations. Conclusions.— The NGS approach would result in the identification of a significant number of actionable gene alterations, increasing the therapeutic options for patients with advanced NSCLCs.

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Targeted next generation sequencing with an extended gene panel does not impact variant detection in mitochondrial diseases

BMC Medical Genetics ◽

10.1186/s12881-018-0568-y ◽

2018 ◽

Vol 19 (1) ◽

Cited By ~ 10

Author(s):

Morgane Plutino ◽

Annabelle Chaussenot ◽

Cécile Rouzier ◽

Samira Ait-El-Mkadem ◽

Konstantina Fragaki ◽

...

Keyword(s):

Next Generation Sequencing ◽

Mitochondrial Diseases ◽

Gene Panel ◽

Next Generation ◽

Targeted Next Generation Sequencing ◽

Variant Detection ◽

Generation Sequencing

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Targeted Next-Generation Sequencing of Cancer-Related Genes in a Norwegian Patient Cohort With Head and Neck Squamous Cell Carcinoma Reveals Novel Actionable Mutations and Correlations With Pathological Parameters

Frontiers in Oncology ◽

10.3389/fonc.2021.734134 ◽

2021 ◽

Vol 11 ◽

Author(s):

Harsh N. Dongre ◽

Hilde Haave ◽

Siren Fromreide ◽

Fredrik A. Erland ◽

Svein Erik Emblem Moe ◽

...

Keyword(s):

Next Generation Sequencing ◽

Head And Neck ◽

Squamous Cell ◽

Ffpe Tissue ◽

Next Generation ◽

Targeted Next Generation Sequencing ◽

Mutational Burden ◽

Custom Made ◽

Targeted Ngs ◽

Generation Sequencing

BackgroundTargeted next-generation sequencing (NGS) is increasingly applied in clinical oncology to advance personalized treatment. Despite success in many other tumour types, use of targeted NGS panels for assisting diagnosis and treatment of head and neck squamous cell carcinomas (HNSCC) is still limited.AimThe focus of this study was to establish a robust NGS panel targeting most frequent cancer mutations in long-term preserved formalin-fixed paraffin-embedded (FFPE) tissue samples of HNSCC from routine diagnostics.Materials and MethodsTumour DNA obtained from archival FFPE tissue blocks of HNSCC patients treated at Haukeland University Hospital between 2003-2016 (n=111) was subjected to mutational analysis using a custom made AmpliSeq Library PLUS panel targeting 31 genes (Illumina). Associations between mutational burden and clinical and pathological parameters were investigated. Mutation and corresponding clinicopathological data from HNSCC were extracted for selected genes from the Cancer Genome Atlas (TCGA) and used for Chi-square and Kaplan-Meier analysis.ResultsThe threshold for sufficient number of reads was attained in 104 (93.7%) cases. Although the specific number of PCR amplified reads detected decreased, the number of NGS-annotated mutations did not significantly change with increased tissue preservation time. In HPV-negative carcinomas, mutations were detected mainly in TP53 (73.3%), FAT1 (26.7%) and FLG (16.7%) whereas in HPV-positive, the common mutations were in FLG (24.3%) FAT1 (17%) and FGFR3 (14.6%) genes. Other less common pathogenic mutations, including well reported SNPs were reproducibly identified. Presence of at least one cancer-specific mutations was found to be positively associated with an extensive desmoplastic stroma (p=0.019), and an aggressive type of invasive front (p=0.035), and negatively associated with the degree of differentiation (p=0.041). Analysis of TCGA data corroborated the association between cancer-specific mutations and tumour differentiation and survival analysis showed that tumours with at least one mutation had shorter disease-free and overall survival (p=0.005).ConclusionsA custom made targeted NGS panel could reliably detect several specific mutations in archival samples of HNSCCs preserved up to 17 years. Using this method novel associations between mutational burden and clinical and pathological parameters were detected and actionable mutations in HPV-positive HNSCC were discovered.

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