Potentially pathogenic variants identified in patients with hypopituitarism by molecular inversion probe sequencing (MIPS), a new molecular approach for low cost gene panel sequencing

VISHNOPOLSKA SA; MARTI, MARCELO A.; CAMPER SA; PÉREZ MILLÁN, MARIA I.

Congreso; ENDO; 2019

Pituitary hormone deficiency or congenital hypopituitarism (CH) occurs in 1/3,000 ? 1/10,000 live births and is highly variable phenotypically. The condition is life-long and threatening if untreated, associated with significant morbidity and if undetected or inadequately treated, mortality. Diagnosis remains a challenge, particularly in the neonatal period. There is a need for improved diagnosis that would lead to reduced mortality and improved prognosis and treatment.We recently implemented a novel and cost-effective approach based on Molecular inversion probe sequencing (MIPS) to identify novel variants and candidate genes in sporadic trios and familial cases of combined pituitary deficiency (CPHD) in the absence or presence of any midline defects or optic nerve hypoplasia, and isolated growth hormone deficiency (IGHD). We captured 693 coding exons of 30 known genes and 37 candidate genes from studies in mice. We captured genomic DNA from 181 pediatric patients from Argentina with CPHD or IGHD (70% and 30 % of the cases, respectively) and 115 relatives and conducted next generation sequencing. We obtained a 600X average coverage per sample over targeted regions. We have identified a number of variant phenotypes associated with mutations in known genes (GH1, GLI2, LHX3, LHX4, PNPLA6, and HESX1) and in new candidate genes that require additional evidence about causality (BMP2, ACVRL1 and WDR11). PROP1 mutations are the most common known cause of CPHD, accounting for 11% of total cases worldwide. The rate varies greatly by ethnicity, and the rate was previously unknown for Argentina. We established that the rate of PROP1 mutations is very low in Argentina. We performed functional assays in cell culture to evaluate the effect of a novel GLI2 variant, pLeu761Phe. This patient presented a complex phenotype, including GH and antidiuretic hormone (ADH) deficiencies, hypoplasia of the anterior lobe, arachnoid cyst, right cerebellar hemisphere hypoplasia, myopia and astigmatism, neonatal insipidus diabetes, speech neurodevelopmental delay and mild left foot parrhesia. Preliminary results of luciferase assays showed that this variant, Gli2:p.Leu761Phe has a gain of function effect. We hypothesize that this mutation could affect the acetylation of the nearby 757 Lysine by modifying the recognition site of p300 acetylase. Acetylation of Gli2 at lysine 757 functions as a critical regulated step, controlling the activation status of Hedgehog pathway, one of the major networks that regulates the key events during embryonic development (1). Further work is needed to demonstrate this effect. In summary, using the first comprehensive screening panel for known genes and candidates for congenital hypopituitarism, we identified pathogenic, likely pathogenic or variants with uncertain significance but predicated to be damaging for at least 3 independent software in about 30% of the cases. Identification of disease-causing mutations in CH is complicated by phenotypic variation and incomplete penetrance. Identifying potential pathogenic variants will make it feasible to predict clinical outcomes from genetic data, which is necessary for patient diagnosis and prognosis, and for assessing the risk of future affected individuals.