MON-472 Potentially Pathogenic Variants Identified in Patients with Hypopituitarism by Molecular Inversion Probe Sequencing (MIPS), a New Molecular Approach for Low Cost Gene Panel Sequencing

Congenital hypopituitarism occurs in 1/3,000 - 1/10,000 live births and is highly variable phenotypically. The condition is life-long and life-threatening if untreated. Diagnosis remains a challenge, particularly in the neonatal period. There is a need for better diagnosis that would lead to 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 with CPHD or IGHD and 115 relatives and conducted next generation sequencing. We obtained a 600X average coverage per sample over targeted regions. We scored rare variants as pathogenic, likely pathogenic, or variants with uncertain significance that are predicted to be damaging using at least 3 independent software programs. We identified such variants in known genes (GH1, GLI2, LHX3, LHX4, PNPLA6, and HESX1) and in new candidate genes that require additional evidence demonstrating causality (BMP2, ACVRL1 and WDR11). GLI2 heterozygous loss of function mutations have been reported in over 50 patients with CPHD and variable associated features. We identified a GLI2 p.Leu761Phe variant in a patient with 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 paresis. We performed functional assays in cell culture and preliminary results suggested that GLI2 p.Leu761Phe has a gain of function effect on transcription. We hypothesize that this mutation affects the acetylation of the nearby p.Lys757 by modifying the recognition site of p300 acetylase. Acetylation of GLI2 at this lysine is a critical regulated step, controlling the activation status of the Hedgehog pathway, which is a major regulator of embryonic development. 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 variants that are likely disease-causing in about 30% of the cases. Identifying 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.