ESPE Abstracts (2018) 89 FC4.1

Monogenic and Digenic Gene Mutations are Present in Children with Idiopathic Short Stature (ISS)

Nora María Sanguinetia, Laura Ramíreza, Ana Claudia Keselmana, Paula Alejandra Scagliaa, María Gabriela Ropelatoa, María Gabriela Ballerinia, Liliana Karabatasa, Sabina Domenéa, Lucía Martuccia, Débora Braslavskya, Estefania Landia, Hamilton Cassinellia, Bárbara Casalia, Graciela Del Reya, Patricia Pennisia, Héctor Jaspera, Martín Vázquezb, Rodolfo Reya, Horacio Domenéa, Mariana Gutiérreza & Ignacio Bergadáa

aCentro de Investigaciones Endocrinológicas ‘Dr. César Bergadá’ (CEDIE) CONICET - FEI - División de Endocrinología, Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina; bInstituto de Agrobiotecnología de Rosario (INDEAR), CONICET, Rosario, Argentina

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; 2.1–9.4 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/, 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.

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