Novel Pathogenic Variants in LHX3, LHX4 and GLI2 Identified in Pediatric Patients with Congenital Hypopituitarism: from Variant Calling to Variant Testing

Abstract Congenital hypopituitarism (CH), septo-optic dysplasia (SOD), and holoprosencephaly (HPE) constitute an important group of structural birth defects that cause significant morbidity and life-long consequences for quality of life and care. The genetic causes are highly overlapping. As such, these disorders can be considered as a spectrum of related disorders. Improved insight into genetic causes would be valuable for patients, families, and medical geneticists. Very few systematic genetic screens have been carried out for patients with CH. We implemented genetic screening using single-molecule molecular inversion probes sequencing to identify causative mutations in a set of 67 genes previously reported in CH patients and the spectrum encompassing SOD and HPE. We captured genomic DNA from 170 Argentinean pediatric patients with CH, and 54% of the patients in this cohort have craniofacial, ophthalmologic, and/or central nervous system defects. We found candidate pathogenic, likely pathogenic and variants uncertain significance (VUS) in 23% of the cases. In order to evaluate the functional consequences of VUS in LHX3, LHX4, and GLI2, we performed in-vitro functional assays to study the activity of the mutated proteins. To test LHX3/4 variants we co-transfected HEK293T cells with wild type (WT) or mutated LHX3/4 variant plasmids and luciferase reporter genes driven by the ɑGSU promoter or GH1 promoter and assayed for luciferase activity. For GLI2 functional analysis we used the cell line NIH/3T3-CG, stably transfected to express GFP under the presence of GLI2 activated form. Endogenous Gli2 was knocked out by CRISPR-Cas9 and clones were selected for absence of GFP expression upon activation of the sonic hedgehog pathway. We tested the ability of transfected WT or mutated GLI2 expression plasmids to restore GFP fluorescence. We concluded that variants LHX3:p.Pro187Ser LHX4:p.Arg84His, p.Gln100His and p.Trp204Leu and GLI2:p.1404Lfs impair activation of the reporter gene, while the LHX3:p.Leu220Met and GLI2:p.L761P have WT activity on their respective assays. Identification of disease-causing variants in CH is complicated by phenotypic variation, incomplete penetrance, and VUS. Functional testing of potentially pathogenic variants is critical to arrive at a definitive molecular diagnosis. A full catalogue of variant effects in known causative genes would be invaluable for clinicians in order to simplify the interpretation of novel variants and reduce the diagnostic odyssey that families often experience.