Primary Ovarian Insufficiency and Azoospermia in Carriers of a Homozygous PSMC3IP Stop Gain Mutation

Abstract

Context: The etiology of primary ovarian insufficiency (POI) remains unknown in most cases.

Objective: We sought to identify the genes causing POI.

Design: The study was a familial genetic study.

Setting: The study was performed at two academic institutions.

Patients: We identified a consanguineous Yemeni family in which four daughters had POI. A brother had azoospermia.

Intervention: DNA was subjected to whole genome sequencing. Shared regions of homozygosity were identified using Truploidy and prioritized using the Variant Annotation, Analysis, and Search Tool with control data from 387 healthy subjects. Imaging and quantification of protein localization and mitochondrial function were examined in cell lines.

Main outcome: Homozygous recessive gene variants shared by the four sisters.

Results: The sisters shared a homozygous stop gain mutation in exon 6 of PSMC3IP (c.489 C>G, p.Tyr163Ter) and a missense variant in exon 1 of CLPP (c.100C>T, p.Pro34Ser). The affected brother also carried the homozygous PSMC3IP mutation. Functional studies demonstrated mitochondrial fragmentation in cells infected with the CLPP mutation. However, no abnormality was found in mitochondrial targeting or respiration.

Conclusions: The PSMC3IP mutation provides additional evidence that mutations in meiotic homologous recombination and DNA repair genes result in distinct female and male reproductive phenotypes, including delayed puberty and primary amenorrhea caused by POI (XX gonadal dysgenesis) in females but isolated azoospermia with normal pubertal development in males. The findings also suggest that the N-terminal missense mutation in CLPP does not cause substantial mitochondrial dysfunction or contribute to ovarian insufficiency in an oligogenic manner.

Figure 1.

Pedigree of a Yemeni family with homozygous mutations in PSMC3IP and CLPP. The women affected with POI are indicated by filled circles. Ages and genotypes confirmed with Sanger sequencing are indicated for PSMC3IP and CLPP. CLPP genotype c.100C>T results in p.Pro34Ser. PSMC3IP genotype c.489C>G results in a stop gain mutation, p.Tyr163Ter.

Figure 2.

Minimal regions of intersection of LOH for all four probands using a window size of 1 megabase were identified on chromosomes 6, 10, 17, 19, 21, and X (black bars), as determined using Truploidy (P < 0.05). Candidate gene locations (red) with variants shared by four affected sisters and found in LOH regions are indicated.

Figure 3.

(A) pLenti-CLPP-mGFP with WT CLPP and (B–D) pLenti-CLPP-mGFP with Mut (c.100C>T) CLPP (green) infected cells; (E) WT CLPP-GFP and (F–H) Mut CLPP-GFP infected cells labeled with mitotracker (red) and (I) WT CLPP-GFP and (J–L) Mut CLPP-GFP merged and stained with Hoechst DNA stain (blue). CLPP localized in mitochondria in both CLPP WT and CLPP Mut. CLPP Mut were found with both normal appearing (B, F, J) and fragmented appearing (C, D, G, H, K, L) mitochondria.

Figure 4.

Western blot demonstrating CLPP-GFP Mut (p.Pro34Ser) and WT localization and size in the mitochondrial fraction. A representative Western blot of the CLPP-GFP and HSP60 protein bands in mitochondria shown.

Figure 5.

Model of PSMC3IP protein domains and functions, with corresponding human amino acid position noted below. C-terminal deletions modeled previously at amino acid 143 and 190 indicated in italics (33). Deletion at amino acid 143 removes the single strand DNA binding site, and the deletions at 143 and 190 remove the RAD51 and DMC1 interaction sites. The location of the stop gain mutation in the family described at amino acid 163 is underlined and would also remove the single strand DNA and RAD51/DMC1 interaction sites. The previously identified frameshift mutation at amino acid 201 is also underlined and impairs the RAD51 and DMC1 interaction sites (30, 33).