ESPE Abstracts

Objectives: We describe 5 pedigrees with a novel phenotype including GHD associated with primary ovarian insufficiency (POI) and investigate the underlying molecular basis.

Patients and Methods: 6 Turkish patients (5F, 1M) born to 5 consanguineous pedigrees with severe GHD were identified. All females had POI; the male had normal puberty. All had severe postnatal growth retardation (height -4.4 to -8.9 SDS at presentation) with clinical GHD, low normal prolactin concentrations (0.2-4.6ng/ml), undetectable basal IGF-I and IGFBP-3, and absent GH responses to provocation. Brain MRI showed anterior pituitary hypoplasia. Serum FSH was elevated in the eldest girl (FSH 24.8U/L, LH 0.7U/L) at age 8 years. Spontaneous puberty ensued at age 14 years (FSH 86.6U/L, LH 32.1U/L), but arrested at Tanner stage 3 with undetectable oestrogen. The other four affected females had elevated gonadotropins from age 3 years. Pelvic ultrasonography/MRI showed a small uterus along with small/undetectable ovaries in all girls. We performed whole exome sequencing (WES) on 6 affected patients and one unaffected parent. Expression of Rnpc3/RNPC3 was analysed by in situ hybridization on murine/human embryonic sections. RT-PCR was used to test splicing efficiency. Crispr/Cas9 was used to generate mice carrying the p.L483F mutation in the conserved murine Rnpc3 RRM2 domain.

Results: All affected patients present a novel homozygous missense variant (p.L483F) in RNPC3 (unaffected father heterozygous). RNPC3 encodes a 65K protein component of the U12-type spliceosome and required for U11/U12 small nuclear ribonucleoprotein (snRNP) formation and U12-type introns splicing. RNPC3 mutations were previously described in isolated GHD patients (Argente J et al., Embo Mol Med 2014). Abnormal U12 intron processing of preprohormone convertases SPCS2 and SPCS3 and actin-related ARPC5L genes (GH dysfunction candidates) and NUP107 (female-specific gonadal dysgenesis candidate) was observed in patient fibroblasts compared to controls. In both mouse and human, Rnpc3/RNPC3 was expressed in the telencephalon, diencephalon, trigeminal ganglia, hypothalamus and Rathke's pouch. Female homozygous mutant mice displayed a 20% reduction in pituitary GH content (P=0.016). Histological examination of sexually mature ovaries revealed no abnormalities.

Conclusion: A novel homozygous missense mutation (p.L483F) in RNPC3 was associated with GHD in humans and mice. Female human patients also present ovarian dysgenesis, in keeping with elevated FSH concentrations in previous patients (1). Our findings provide novel insights into the role of RNPC3 in ovarian function and emphasize a critical role of minor spliceosome in the processing of genes required for pituitary and ovarian development and function.