ESPE Abstracts

Introduction: The Sonic hedgehog (SHH) signaling pathway plays a crucial role in development of the forebrain and pituitary. Mutations in SHH signaling related genes are well known to be the cause of Holoprosencephaly (HPE), which results from developmental field defect or impaired midline cleavage of the embryonic forebrain, and is frequently associated with hypopituitarism. This study aimed to define the prevalence of congenital hypopituitarism (CH) in terms of seven SHH or HPE-related genes (GLI2, SHH, TGIF1, SIX3, ZIC2, GPR161, and CDON) among Japanese patients.

Subjects and Methods: We enrolled 120 (among them, 32 patients with midline defect) Japanese CH patients. The inclusion criteria were as follows: 1) short statue with severe GH deficiency (GH peak < 3 ng/mL) confirmed by hypoglycemic provocation test, or inadequate low serum GH at a time of severe hypoglycemia as neonate, and 2) anterior pituitary hypoplasia as detected by brain MRI. We sequenced all coding exons and flanking introns of 7 genes (GLI2, SHH, TGIF1, SIX3, ZIC2, GPR161, and CDON) by PCR-direct sequencing or next generation sequencing methods. The gene regulatory properties of mutant TGIF1 proteins were characterized in vitro.

Results: We identified a novel TGIF1 mutation, namely p.N235Y in one CH patient with cleft palate, and one recurrent TGIF1 mutation, namely p.R219C in non-syndromic CH patient. We also identified two novel GLI2 mutations in CH patients with or without midline defects. In vitro experiments showed that N235Y TGIF1 resulted in a decrease of repressing activity in Luciferase assay. Western blotting and subcellular localization revealed no significant difference between wild type and N235Y TGIF1. Electrophoretic mobility shift assays showed that the N235Y TGIF1 bound with slightly low efficiency to the wild type.

Discussion: The frequency of SHH or HPE-related gene mutations in patients with CH was 3.3% (4/120).