Background: Several genetic defects (GHR, SHOX, GHSR, NPR2, IGFALS) have been reported in children classified as ISS. ISS children are GH sufficient and about one third of them show low IGF-I levels, suggesting some degree of GH insensitivity.
Objective: To explore potential genetic defects in ISS children suspicious of GH insensitivity, selected by low IGF-I levels and low response to IGF generation test.
Subjects and Methods: Levels of IGF-I, IGFBP-3, and ALS were determined in 42 ISS children (34 males; 2.19.4 years old, height <−2.5 S.D., stimulated GH > 4.8 μg/l). Those presenting IGF-I <50 ng/ml underwent an IGF generation test (GH doses 0.033 mg/kg.day), measuring IGF-I, IGFBP-3, and ALS. Sequencing of IGFALS gene in all participants, and whole exome sequencing (WES) in two selected cases, were performed. IGFALS gene variants were generated by site-directed mutagenesis and expressed in CHO cells. ALS variants were analyzed by Western immunoblot (WIB). STAT5b variants were characterized in HEK293T cells transiently transfected with plasmids containing c.DNA variants by a dual luciferase reporter assay.
Results: From eight children presenting IGF-I levels <50 ng/ml, one was compound heterozygous and 4 heterozygous for IGFALS variants (p.E35Gfs*17, p.P22L, p.R548W, and p.G506R). By WIB p.E35Gfs*17 was not detected, p.R548W was a hypomorphic variant, while p.P22L and p.G506R were variants of uncertain significance (VUS).Those three presenting the lower IGF-I response were further studied. Patient 1: the compound heterozygous child (p.L409F/ p.S490W) presented undetectable levels of IGFBP-3 and ALS (both IGFALS variants were not detected by WIB) was diagnosed as complete ALS deficient. The other two patients (patient 2, heterozygous carrier for p.R548W, and patient 3, IGFALS-WT) underwent WES. In patient 2, a 2.1 year-old boy, height 73.5 cm (−3.23 S.D.), weight 8.28 Kg (−2.5 S.D.), WES revealed a heterozygous novel STAT5B variant: c.1896G>T; p.K632N. In vitro studies demonstrate that p.K632N is an inactive variant, since it showed severe diminished reporter activity, under basal conditions and in response to GH treatment. In patient 3, a 6.3 year-old boy, height 103.8 cm (−2.57 S.D.), weight 17.5 Kg (−1.53 S.D.), WES analysis did not reveal potential pathogenic variants related with his phenotype.
Conclusions: Candidate gene approach combined with WES was useful to perform a genetic diagnosis of partial or complete ALS deficiency (one or two affected IGFALS alleles) or double heterozygotes (IGFALS and STAT5B genes) in children with apparent GH insensitivity.
27 - 29 Sep 2018
European Society for Paediatric Endocrinology