Clinical and molecular characterization of DSD patients: Impact of Next Generation Sequencing in diagnosis

317 p.Disorders of sex development (DSD) encompass a high heterogeneous range of conditionsin which the optimal clinical management of the individuals comprise clinical description, biochemicaltesting and genetic analysis. With the arrival of next-generation sequencing (NGS) new genes andpathways have been implicated in the pathogenesis of the disease. However, only 50% of the 46,XY DSDpatients will receive a definitive diagnosis. The aim of this work is the clinical and molecularcharacterization of a DSD cohort. Patients and methods: Blood samples or DNA from a total of 125independent patients with a DSD referred from several Spanish centres and one Swiss hospital werestudied. Clinical characterization was done after examination of the data sheet provided by the clinicians.The molecular study was performed either by single-gene testing or NGS using a targeted gene panel of48 DSD-related genes. Parents were also analysed when possible. Genetic variants were analysed usingin silico prediction programmes and were classified according to its potential pathogenicity. Furtherfunctional studies of the genetic changes identified in three genes were also performed. Results: Overall,we made a genetic diagnosis in 4.4% of the individuals. In 46,XY DSD, the diagnostic rate increased upto 46.5% while in 46.XX DSD it was 29.2%. Novel changes classified as pathogenic or likely pathogenicwere mostly located in the NR5A1 and LHCGR genes. Functional impact of variants in NR5A1 andLHCGR showed a significant reduced transactivation activity while the variant of unknown significancein GATA4 showed similar activity compared to wild type. Conclusions: The heterogeneous phenotypesencompassed in this condition difficult the clinical diagnosis and highlight the need of a genetic study.The molecular analysis of DSD-related genes identified a causative genetic variant in the 40.2% of thepatients analysed in this cohort. Targeted gene sequencing panel is useful for the detection of singlenucleotidevariants, however further improvements are needed to detect copy number variations.Functional studies are a valuable tool to confirm the effect of a variant in the pathogenesis of the disease

Clinical and molecular characterization of DSD patients: Impact of Next Generation Sequencing in diagnosis

317 p.Disorders of sex development (DSD) encompass a high heterogeneous range of conditionsin which the optimal clinical management of the individuals comprise clinical description, biochemicaltesting and genetic analysis. With the arrival of next-generation sequencing (NGS) new genes andpathways have been implicated in the pathogenesis of the disease. However, only 50% of the 46,XY DSDpatients will receive a definitive diagnosis. The aim of this work is the clinical and molecularcharacterization of a DSD cohort. Patients and methods: Blood samples or DNA from a total of 125independent patients with a DSD referred from several Spanish centres and one Swiss hospital werestudied. Clinical characterization was done after examination of the data sheet provided by the clinicians.The molecular study was performed either by single-gene testing or NGS using a targeted gene panel of48 DSD-related genes. Parents were also analysed when possible. Genetic variants were analysed usingin silico prediction programmes and were classified according to its potential pathogenicity. Furtherfunctional studies of the genetic changes identified in three genes were also performed. Results: Overall,we made a genetic diagnosis in 4.4% of the individuals. In 46,XY DSD, the diagnostic rate increased upto 46.5% while in 46.XX DSD it was 29.2%. Novel changes classified as pathogenic or likely pathogenicwere mostly located in the NR5A1 and LHCGR genes. Functional impact of variants in NR5A1 andLHCGR showed a significant reduced transactivation activity while the variant of unknown significancein GATA4 showed similar activity compared to wild type. Conclusions: The heterogeneous phenotypesencompassed in this condition difficult the clinical diagnosis and highlight the need of a genetic study.The molecular analysis of DSD-related genes identified a causative genetic variant in the 40.2% of thepatients analysed in this cohort. Targeted gene sequencing panel is useful for the detection of singlenucleotidevariants, however further improvements are needed to detect copy number variations.Functional studies are a valuable tool to confirm the effect of a variant in the pathogenesis of the disease

Novel Variant in the CNNM2 Gene Associated with Dominant Hypomagnesemia

The maintenance of magnesium (Mg2+) homeostasis is essential for human life. The Cystathionine-beta-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) have been described to be involved in maintaining Mg2+ homeostasis. Among these CNNMs, CNNM2 is expressed in the basolateral membrane of the kidney tubules where it is involved in Mg2+ reabsorption. A total of four patients, two of them with a suspected disorder of calcium metabolism, and two patients with a clinical diagnosis of primary tubulopathy were screened for mutations by Next-Generation Sequencing (NGS). We found one novel likely pathogenic variant in the heterozygous state (c.2384C>A; p.(Ser795*)) in theCNNM2gene in a family with a suspected disorder of calcium metabolism. In this family, hypomagnesemia was indirectly discovered. Moreover, we observed three novel variants of uncertain significance in heterozygous state in the other three patients (c.557G>C; p.(Ser186Thr), c.778A>T; p.(Ile260Phe), and c.1003G>A; p.(Asp335Asn)). Our study shows the utility of Next-Generation Sequencing in unravelling the genetic origin of rare diseases. In clinical practice, serum Mg2+ should be determined in calcium and PTH-related disorders.This study was supported by three grants from the Department of Health (2017111014, 2018111097 and 2019111052) and one grant from the Department of Education (IT1281-19) of the Basque Government. This work is generated within the Endocrine European Reference Network (Project ID number of Endo-ERN: 739527). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Five Patients with Disorders of Calcium Metabolism Presented with GCM2 Gene Variants

The GCM2 gene encodes a transcription factor predominantly expressed in parathyroid cells that is known to be critical for development, proliferation and maintenance of the parathyroid cells. A cohort of 127 Spanish patients with a disorder of calcium metabolism were screened for mutations by Next-Generation Sequencing (NGS). A targeted panel for disorders of calcium and phosphorus metabolism was designed to include 65 genes associated with these disorders. We observed two variants of uncertain significance (p.(Ser487Phe) and p.Asn315Asp), one likely pathogenic (p.Val382Met) and one benign variant (p.Ala393_Gln395dup) in the GCM2 gene in the heterozygous state in five families (two index cases had hypocalcemia and hypoparathyroidism, respectively, and three index cases had primary hyperparathyroidism). Our study shows the utility of NGS in unravelling the genetic origin of some disorders of the calcium and phosphorus metabolism, and confirms the GCM2 gene as an important element for the maintenance of calcium homeostasis. Importantly, a novel variant in the GCM2 gene (p.(Ser487Phe)) has been found in a patient with hypocalcemia.This study was supported by three grants from the Department of Health (2017111014, 2018111097 and 2019111052) and one grant from the Department of Education (IT1281-19) of the Basque Government. This work is generated within the Endocrine European Reference Network (Project ID number of Endo-ERN: 739527). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Rare Germline DICER1 Variants in Pediatric Patients With Cushing's Disease: What Is Their Role?

Context: The DICER1 syndrome is a multiple neoplasia disorder caused by germline mutations in the DICER1 gene. In DICER1 patients, aggressive congenital pituitary tumors lead to neonatal Cushing's disease (CD). The role of DICER1 in other corticotropinomas, however, remains unknown. Objective: To perform a comprehensive screening for DICER1 variants in a large cohort of CD patients, and to analyze their possible contribution to the phenotype. Design, setting, patients, and interventions: We included 192CD cases: ten young-onset (age <30 years at diagnosis) patients were studied using a next generation sequencing panel, and 182 patients (170 pediatric and 12 adults) were screened via whole-exome sequencing. In seven cases, tumor samples were analyzed by Sanger sequencing. Results: Rare germline DICER1 variants were found in seven pediatric patients with no other known disease-associated germline defects or somatic DICER1 second hits. By immunohistochemistry, DICER1 showed nuclear localization in 5/6 patients. Variant transmission from one of the parents was confirmed in 5/7 cases. One patient had a multinodular goiter; another had a family history of melanoma; no other patients had a history of neoplasms. Conclusions: Our findings suggest that DICER1 gene variants may contribute to the pathogenesis of non-syndromic corticotropinomas. Clarifying whether DICER1 loss-of-function is disease-causative or a mere disease-modifier in this setting, requires further studies.This work was supported by the Intramural Research Programs of Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) and National Institute for Neurological Diseases and Stroke, National Institutes of Health, a grant from the Basque Department of Education (IT795-13), a grant from the Basque Department of Health (GV2018111082), the Merck Serono Research award from Fundacion Salud 2000 (15-EP-004) and the Jose Igea 2018 grant, sponsored by Pfizer, from Fundacion Sociedad Espanola de Endocrinologia Pediatrica (SEEP)