CSB-PGBD3 Mutations Cause Premature Ovarian Failure

Abstract

Premature ovarian failure (POF) is a rare, heterogeneous disorder characterized by cessation of menstruation occurring before the age of 40 years. Genetic etiology is responsible for perhaps 25% of cases, but most cases are sporadic and unexplained. In this study, through whole exome sequencing in a non-consanguineous family having four affected members with POF and Sanger sequencing in 432 sporadic cases, we identified three novel mutations in the fusion gene CSB-PGBD3. Subsequently functional studies suggest that mutated CSB-PGBD3 fusion protein was impaired in response to DNA damage, as indicated by delayed or absent recruitment to damaged sites. Our data provide the first evidence that mutations in the CSB-PGBD3 fusion protein can cause human disease, even in the presence of functional CSB, thus potentially explaining conservation of the fusion protein for 43 My since marmoset. The localization of the CSB-PGBD3 fusion protein to UVA-induced nuclear DNA repair foci further suggests that the CSB-PGBD3 fusion protein, like many other proteins that can cause POF, modulates or participates in DNA repair.

Fig 1. CSB-PGBD3 mutations identified in the index family with POF and sporadic cases.

(A) Pedigree of the index family, ascertained through Ⅲ 5. Whole exome sequencing was performed in POF patientsⅡ3, Ⅱ11 and Ⅲ5; a genetically obligate male carrier (Ⅱ5); and one normal family member Ⅱ9. Sanger sequencing of CSB-PGBD3 was performed in other family members when DNA was available. (B) Genomic structure of CSB-PGBD3 and chromatograms of three mutations identified in present study. PGBD3 was inserted into intron 5 of CSB to make the fusion gene CSB-PGBD3. CSB exons are in yellow, PGBD3 exon in gray, and the three mutations indicated in red, and the corresponding chromatograms are shown. (C) Alignment of the coding strand of CSB-PGBD3 in 15 eutherian mammals from Ensembl database shows conservation of nucleotides 2237 and 3166 in primates and 643 in mammals.

Fig 2. The cellular localization of PGBD3 and CSB-PGBD3.

(A) Immunohistochemistry using anti-PGBD3 antibody (Abnova PAB21786) and (B) in situ hybridization using probe targeting at CSB-PGBD3 mRNA were performed in rhesus monkey ovary, which showed that CSB-PGBD3 was expressed exclusively in nuclei of oocytes from primordial to antral follicles. (C) Western blot performed with the use of anti-PGBD3 antibody (Abnova PAB21786) in human heart tissue, COV434 cells, adult ovary tissue and granulosa cells showed that PGBD3 and CSB-PGBD3 fusion protein were expressed in human ovary but not in granulosa cells. (Scale bars: 100 um.)

Fig 3. Delayed recruitment of CSB-PGBD3 mutants at DNA lesion.

(A) DNA damage was induced by laser micro-irradiation in the nuclei of U2OS cells expressing wild type and mutant CSB-PGBD3-RFP. (B) OGG1-GFP was recruited to the DNA damage site along with CSB-PGBD3-RFP. (C) Fluorescence accumulation curve showed the recruitment of mutant p.G746D and p.V1056I to damaged sites were weaker compared with wild type, and mutant p.E215X could not accumulate at the damaged site completely. (D) Percentage of reactive cells after laser micro-irradiation was less for mutants compare with wild type (*p<0.001). (E) Western blot for wild type and mutant CSB-PGBD3-eGFP protein attached to chromatin in HeLa cells after H₂O₂ treatment. Mutant p.G746D and p.V1056I bound onto chromatin with a delayed time course compared with wild type. The quick recruitment and separation of p.E215X was elusive. (F) Immunoprecipitation experiments showed the truncated protein p.E215X failed to interact with RNApol II with or without treatment. (G) and (H) showed the clonogenic survival of CSB-PGBD3 tranfected cells suffering UV irradiation. The percent survival relative to the control was significantly higher for wild type compared with the mutants. (Scale bars: 5um)