Genetic diagnosis of congenital hypopituitarism by a target gene panel: novel pathogenic variants in GLI2, OTX2 and GHRHR

Aim: Congenital hypopituitarism has an incidence of 1:3500–10,000 births and is defined by the impaired production of pituitary hormones. Early diagnosis has an impact on management and genetic counselling. The clinical and genetic heterogeneity of hypopituitarism poses difficulties to select the order of genes to analyse. The objective of our study is to screen hypopituitarism genes (candidate and previously related genes) simultaneously using a target gene panel in patients with congenital hypopituitarism. Methods: Screening of 117 subjects with congenital hypopituitarism for pathogenic variants in 26 genes associated with congenital hypopituitarism by massively parallel sequencing using a customized target gene panel. Results: We found three novel pathogenic variants in OTX2 c.295C>T:p.Gln99*, GLI2 c.1681G>T:p.Glu561* and GHRHR c.820_821insC:p.Asp274Alafs*113, and the previously reported variants in GHRHR c.57+1G>A and PROP1 [c.301_302delAG];[c.109+1G>A]. Conclusions: Our results indicate that a custom-designed panel is an efficient method to screen simultaneously variants of biological and clinical relevance for congenital GH deficiency. A genetic diagnosis was possible in 5 out of 117 (4%) patients of our cohort. We identified three novel pathogenic variants in GHRHR, OTX2 and GLI2 expanding the spectrum of variants associated with congenital hypopituitarism

Massive-parallel sequencing by gene panel in the diagnosis of congenital hypopituitarism

Convencionou-se chamar de hipopituitarismo congênito (HC) a deficiência de um ou mais hormônios hipofisários. O HC pode se apresentar na forma de insuficiência isolada do hormônio de crescimento (DIGH) ou combinada com a deficiência de outros hormônios hipofisários - deficiência hipotálamo-hipofisária múltipla (DHHM) e apresenta uma prevalência de um a cada 3.500 a 10.000 nascimentos. O diagnóstico precoce apresenta impacto no tratamento clínico inicial e, posteriormente, no aconselhamento genético. A maioria dos estudos realizados até o momento, que identificou uma etiologia genética para a deficiência hormonal, utilizou a técnica de Sanger. Mais recentemente, o sequenciamento paralelo em larga escala (SPLE) vem sendo utilizado como uma forma de investigação genética mais rápida e a um custo menor, permitindo estender esse processo de sequenciamento a milhares de reações de forma simultânea e automatizada. Os objetivos deste estudo foram: 1) desenvolver um painel customizado de sequenciamento paralelo em larga escala (SPLE) para investigação da etiologia genética da deficiência do hormônio de crescimento congênito e 2) correlacionar os achados moleculares encontrados com o fenótipo. Para este estudo, foram selecionados: 117 pacientes acompanhados no serviço de endocrinologia do Hospital das Clínicas da Universidade de São Paulo (HC-FMUSP), Serviço de Endocrinologia do Hospital do Servidor Público Estadual de São Paulo, Instituto de Assistência Médica ao Servidor Público Estadual (HSPE-IAMSPE), Hospital de Transplantes Euryclides de Jesus Zerbini de São Paulo e Hospital de Niños Santísima Trinidad em Cordoba, Argentina. Métodos: os pacientes foram sequenciados por SPLE, utilizando um painel contendo 26 genes associados ao hipopituitarismo. Como resultados foram encontradas quatro novas variantes patogênicas nos genes OTX2 c.295C > T, GLI2 c.1681G > T, GHRHR c.820_821insC:p.Asp274Alafs*113 e PROP1 c.109+1G > A e variantes previamente classificadas como patogênicas pelos critérios da ACMG / AMP nos genes GHRHR c.57+1G > A e PROP1 c.301_302delAG em cinco pacientes. Os resultados indicaram que um painel projetado sob medida é um método útil para rastrear, simultaneamente, variantes de relevância biológica e clínica para a deficiência congênita de hormônio de crescimento (GH). Um diagnóstico genético foi possível em cinco de 117 (4%) pacientes desta coorte. Foram identificadas quatro novas variantes patogênicas nos genes GHRHR, GLI2, OTX2 e PROP1, expandindo o espectro de variantes associadas ao hipopituitarismo congênitoCongenital hypopituitarism can be present as isolated growth hormone deficiency (IGHD) or combined with the impaired production of other pituitary hormones (CPHD) and has an incidence of 1: 3,500 to 10,000 births. Genetic diagnosis has an impact on management and genetic counseling. Most studies to identify the genetic etiology of the hormonal deficiency used Sanger sequencing. Massive-parallel sequencing has been used as a method of faster genetic investigation with reduced costs, allowing extending this sequencing process to thousands of reactions in a simultaneous and automated manner. The objectives of our study are: 1) developing a customized panel for massively-parallel sequencing (MPS) to investigate the genetic etiology of congenital hypopituitarism, and 2) correlate the molecular findings with the phenotype. In this study, 117 subjects were studied from the endocrinology units of Hospital das Clínicas da Universidade de São Paulo (HC-FMUSP), Hospital do Servidor Público Estadual de São Paulo, Instituto de Assistência Médica ao Servidor Público Estadual (HSPE-IAMSPE), Hospital de Transplantes Euryclides de Jesus Zerbini de São Paulo and Hospital de Niños Santísima Trinidad de Cordoba, Argentina. Methods: patients were screened by a target panel containing 26 genes associated to hypopituitarism. As result, four novel pathogenic variants were found in OTX2 c.295C > T, GLI2 c.1681G > T, GHRHR c.820_821insC and PROP1 c.109+1G > A and the previously reported variants in PROP1 c.301_302delAG and GHRHR c.57+1G > A in five patients. In conclusion, our results indicate that a custom designed panel is an efficient method to screen simultaneously variants of biological and clinical relevance for congenital GH deficiency. A genetic diagnosis was possible in 5 out of 117 (4%) patients of our cohort expanding the spectrum of variants associated with congenital hypopituitaris

The phenotypic spectrum associated with OTX2 mutations in humans

The transcription factor OTX2 is implicated in ocular, craniofacial, and pituitary development

High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 variants underlying pituitary hormone deficiency

International audiencePituitary hormone deficiency occurs in ∼1:4,000 live births. Approximately 3% of the cases are due to mutations in the alpha isoform of POU1F1, a pituitary-specific transcriptional activator. We found four separate heterozygous missense variants in unrelated individuals with hypopituitarism that were predicted to affect a minor isoform, POU1F1 beta, which can act as a transcriptional repressor. These variants retain repressor activity, but they shift splicing to favor the expression of the beta isoform, resulting in dominant-negative loss of function. Using a high-throughput splicing reporter assay, we tested 1,070 single-nucleotide variants in POU1F1. We identified 96 splice-disruptive variants, including 14 synonymous variants. In separate cohorts, we found two additional synonymous variants nominated by this screen that co-segregate with hypopituitarism. This study underlines the importance of evaluating the impact of variants on splicing and provides a catalog for interpretation of variants of unknown significance in POU1F1