ESPE Abstracts (2021) 94 P1-50

1Genetics and Genomic Medicine, UCL Great Ormond Street Institute of Child Health, University College London, London, United Kingdom; 2Institute for Women’s Health, University College London, London, United Kingdom; 3GOSgene, Genetics and Genomic Medicine, UCL Great Ormond Street Institute of Child Health, University College London, London, United Kingdom; 4Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand; 5Laboratory of Development of the Gonads, UMR E008 Genetic Stability Stem Cells and Radiations, Université de Paris, Université Paris Saclay, CEA, Paris, France; 6Zayed Centre for Research into Rare Disease in Children, University College London, London, United Kingdom; 7Developmental Biology and Cancer, UCL Great Ormond Street Institute of Child Health, University College London, London, United Kingdom; 8Department of Endocrinology, Pontificia Universidad Católica de Chile, and Universidad San Sebastián, Santiago, Chile

Background: Primary ovarian insufficiency (POI) is genetically mediated in up to 30% of cases. Many genes associated with POI have roles in early ovary developmental processes, including meiosis.

Objectives: We investigated the genetic mechanism underlying early-onset POI in three young women presenting with absent puberty: two sisters from a consanguineous pedigree and a third unrelated proband.

Methods: Exome sequencing and filtering using an autosomal recessive model was performed in the affected patients and their unaffected family members. RNA sequencing and quantitative reverse transcriptase PCR were used to study the expression of key genes at critical stages of human fetal gonad development (Carnegie Stage (CS) 22/23, 9 weeks post conception (wpc), 11wpc, 15/16wpc, 19/20wpc) and in adult tissue. Expression data were matched to cell populations using publicly available single-cell RNA sequencing data. Immunohistochemistry was used to study protein expression. Patient peripheral leukocytes were used to investigate the effect of variants of interest on transcript stability and splicing. The cellular expression and localisation of variants were studied using in vitro HeLa/HEK-293 cell systems and co-immunoprecipitation studies. In silico modelling characterised wild-type and mutant proteins.

Results: Exome sequencing revealed pathogenic variants in YTHDC2 co-segregating with the POI phenotype: in the two sisters, a homozygous variant c. 2567C>G, p.P856R in the HA2 domain; in the third patient, a homozygous c.1129G>T, p.E377* variant. YTHDC2, an m6 A reader RNA helicase, and its partner protein MEIOC have been associated with meiotic progression in animal studies. We demonstrated higher YTHDC2 and MEIOC expression in the late (15/16wpc) compared to the early (CS22/23) fetal ovary (log2FC 1.14, p.adj 2.18E-08), coincident with the female meiotic peak. Higher YTHDC2 (log2FC 0.5, p.adj <0.05) and MEIOC (log2FC 5.2, p.adj <0.05) expression was seen in the ovary compared to testis at 15/16wpc. YTHDC2 was highly expressed in the adult testis, corresponding with male meiosis. Single-cell sequencing data localised YTHDC2 expression to human fetal ovary meiotic germ cells. Immunohistochemistry of a 20wpc human fetal ovary showed cytoplasmic staining of YTHDC2 in meiotic cells. Neither YTHDC2 variant affected transcript splicing nor stability. The p.P856R variant did not affect YTHDC2 cellular localization nor YTHDC2/MEIOC co-localization. In silico modelling demonstrated that the p.P856R variant results in a more stable, less flexible protein that affects downstream conformational kinetics of the HA2 domain.

Conclusion: We show that the m6A reader YTHDC2 plays a key role in meiosis regulation and human gonadal development and associate pathogenic variants within YTHDC2 with POI.

Volume 94

59th Annual ESPE (ESPE 2021 Online)

22 Sep 2021 - 26 Sep 2021

European Society for Paediatric Endocrinology 

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