Autor(es): Pereira-Caetano, Iris ; Mendonça, Joana ; Mirante, Alice ; Cardoso, Rita ; Rodrigues, Márcia ; Oliveira, João Paulo ; Grangeia, Ana ; Barbosa, David ; Vieira, Luís ; Gonçalves, João
Data: 2023
Identificador Persistente: http://hdl.handle.net/10400.18/9154
Origem: Repositório Científico do Instituto Nacional de Saúde
Assunto(s): Dsorders of Sexual Development; DSD; SRY; Sexual; Sex Determination; Molecular Diagnosis; Doenças Genéticas
Introduction: Next generation sequencing (NGS) is increasingly used in the molecular diagnosis of rare diseases, such as disorders of sexual development (DSD), allowing the analysis of a greater number of genes in a single assay, providing faster results with reduced costs. DSD patients may present high phenotypic overlap (genital ambiguity, sex reversal, delayed/absent puberty, infertility) posing challenges to clinical diagnosis. NGS technology using multigene panels has higher hypothesis to identify the genetic cause and novel genetic variants in a large number of cases. Methodology: 33 genomic DNA samples from DSD patients were sequenced on MiSeq using the Ampliseq technology (Illumina). A customized gene panel covering 40 genes was used to prepare the libraries of the target sequences. The 40 genes were subdivided into 5 subpanels: primary sex determination, sex differentiation, hypogonadotropic hypogonadism/infertility, steroidogenesis and premature ovarian insufficiency. Variant classification, according to ACMG-AMP, was based on bioinformatics tools (ex. VEP, HSF, VarSome, Alamut) and databases (gnomAD, HGMD, ClinVar, dbSNP). Pathogenic, Likely pathogenic and VUS variants were confirmed by Sanger sequencing. Results: 16 of the 33 patients were previously studied in our laboratory with negative results for the main genes associated with DSD (SRY, AR, ANOS1, GNRHR, CYP21A2). We identified 9 causative variants (8P/1LP) in 7 patients (21.2%) in AR, HSD17B3, LHCGR, FGFR1 and NR5A1. One patient was a compound heterozygous for HSD17B3 and another simultaneously homozygous for LHCGR and hemizygous for AR. The remaining 5 were homozigous, heterozigous or hemizygous for HSD17B3, LHCGR, NR5A1, FGFR1 or AR. One VUS, FGFR1:c.566G>A p.Arg189His, requires familial studies to revaluate its pathogenicity. Discussion: Multigene NGS studies allows to increase the rate of variant detection, mainly in genes not included in a first molecular approach. It also contributes to establishing or confirming the clinical diagnosis, assisting in decisions regarding the treatment and reproductive management of patients and families, as well as in genetic counselling.
