Genetic diagnostic yield of rare endocrine diseases through Next-Generation Sequencing: our-7-year experience based on targeted gene panels

Currently, NGS-based genetic testing has become an indispensable component of the comprehensive diagnostic workup in rare endocrine diseases. For this reason, our clinical genetics laboratory has designed a custom NGS panel including all the known candidate and susceptibility genes for Congenital Hypogonadotropic Hypogonadism (CHH) and Pheochromocytoma/Paraganglioma (Pheo/PGL/HNPGL), respectively. In addition, the VHL mutational screening has been developed to diagnose the von Hippel-Lindau syndrome (VHL) in suspected cases. This study aimed to evaluate the diagnostic yield basedon genetic testing in the above-mentioned rare endocrine diseases. After the NGS sequencing, a comprehensive bioinformatic analysis of each variant was performed in order to evaluate their pathogenicity and genotype-phenotype correlations were examined. The first part of this thesis focused on CHH, which is a congenital disorder covering a widespectrum of signs/symptoms, from classical forms with absent puberty, including Kallmannsyndrome and other syndromic conditions, to milder forms with Adult-Onset Hypogonadism (AOH). To date more than 40 candidate genes have been identified in approximately 40-50% of cases with a genetic diagnosis of CHH. We have enrolled 26 affected patients, of whom 12 showed the classic forms of disease whereas 14 were AOH. The total genetic diagnostic yield of our NGS panel, including 34 candidate genes with strong/definitive clinical evidence, was 23%, with the highest diagnostic rate in classic forms (42%) and the lowest one in the AOH (7%). The majority of mutated genes are well knowncausative genes, with the exception of DMXL2, for which only a few familial cases have beendescribed so far. Interestingly, the unique case of AOH with a clear genetic diagnosis carriedan affected DMXL2 allele, which is responsible for a mild reproductive phenotype, according to the literature. Our finding contributes to elucidate the role of DMXL2 gene in the aetiology of CHH. The second part of this thesis focused on rare endocrine tumours. Among them, Pheo/PGL/HNPGL arise from neural crest cells and affect adrenal gland (Pheo) or extra-adrenal sites (PGL/HNPGL). More than 20 susceptibility genes have been reported to predispose to Pheo/PGL/HNPGL, with a diagnostic yield of approximately 30%. On the other hand, the VHL syndrome is a monogenic disorder arising from germline mutations in the VHL gene and involving multiple organs. All these conditions follow an autosomal mode of inheritance, with an incomplete and age-dependent penetrance related to the specific gene defect. We have enrolled 95 patients affected by Pheo/PGL/HNPGL and 8 suspected VHL cases. Concerning the former, our NGS panel containing 15 susceptibility genes revealed a genetic diagnostic yield of 20%, with the highest diagnostic rate in PGLs (33%) and the lowest one in Pheos (14%). The majority of mutated genes belongs to the SDHx family, in line with previous data. Interestingly, one Pheo patient carrying two germline defects in the SDHD and VHL genes received a diagnosis of VHL syndrome, thanks to the gene panel results. Regarding patients with suspected VHL, we diagnosed a “true” VHL syndrome in 25% of cases. This study highlights the power of genetic testing as a diagnostic tool with relevant implications in genetic counselling and clinical management of the mutation carriers. Based on our results, CHH patients should be well-characterized prior to genetic testing, as the presence of a disease-causing genotype is almost exclusively found in classic forms. Hence, the genotype of AOH patients seems to be different from those with CHH. In case of rare endocrine tumours, we confirmed that genetic testing is relevant for correct and early diagnosis, leading to a better prognosis and to an appropriate treatment, of both the patient and her/his family members through an active surveillance.