Copy number variation analysis detects novel candidate genes involved in follicular growth and oocyte maturation in a cohort of premature ovarian failure cases

Abstract

Study question: Can spontaneous premature ovarian failure (POF) patients derived from population-based biobanks reveal the association between copy number variations (CNVs) and POF?

Summary answer: CNVs can hamper the functional capacity of ovaries by disrupting key genes and pathways essential for proper ovarian function.

What is known already: POF is defined as the cessation of ovarian function before the age of 40 years. POF is a major reason for female infertility, although its cause remains largely unknown.

Study design, size, duration: The current retrospective CNV study included 301 spontaneous POF patients and 3188 control individuals registered between 2003 and 2014 at Estonian Genome Center at the University of Tartu (EGCUT) biobank.

Participants/materials, setting, methods: DNA samples from 301 spontaneous POF patients were genotyped by Illumina HumanCoreExome (258 samples) and HumanOmniExpress (43 samples) BeadChip arrays. Genotype and phenotype information was drawn from the EGCUT for the 3188 control population samples, previously genotyped with HumanCNV370 and HumanOmniExpress BeadChip arrays. All identified CNVs were subjected to functional enrichment studies for highlighting the POF pathogenesis. Real-time quantitative PCR was used to validate a subset of CNVs. Whole-exome sequencing was performed on six patients carrying hemizygous deletions that encompass genes essential for meiosis or folliculogenesis.

Main results and the role of chance: Eleven novel microdeletions and microduplications that encompass genes relevant to POF were identified. For example, FMN2 (1q43) and SGOL2 (2q33.1) are essential for meiotic progression, while TBP (6q27), SCARB1 (12q24.31), BNC1 (15q25) and ARFGAP3 (22q13.2) are involved in follicular growth and oocyte maturation. The importance of recently discovered hemizygous microdeletions of meiotic genes SYCE1 (10q26.3) and CPEB1 (15q25.2) in POF patients was also corroborated.

Limitations, reasons for caution: This is a descriptive analysis and no functional studies were performed. Anamnestic data obtained from population-based biobank lacked clinical, biological (hormone levels) or ultrasonographical data, and spontaneous POF was predicted retrospectively by excluding known extraovarian causes for premature menopause.

Wider implications of the findings: The present study, with high number of spontaneous POF cases, provides novel data on associations between the genomic aberrations and premature menopause of ovarian cause and demonstrates that population-based biobanks are powerful source of biological samples and clinical data to reveal novel genetic lesions associated with human reproductive health and disease, including POF.

Study funding/competing interest: This study was supported by the Estonian Ministry of Education and Research (IUT20-43, IUT20-60, IUT34-16, SF0180027s10 and 9205), Enterprise Estonia (EU30020 and EU48695), Eureka's EUROSTARS programme (NOTED, EU41564), grants from European Union's FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, SARM, |EU324509) and Horizon 2020 innovation programme (WIDENLIFE, 692065), Academy of Finland and the Sigrid Juselius Foundation.

Keywords: CNV; POF; SNP genotyping; copy number variation; menopause; population-based biobank; premature ovarian failure.

Figure 1

Schematic representation of study design. A total of 301 spontaneous premature ovarian failure (POF) patients were enrolled in the study. After single nucleotide polymorphism (SNP) genotyping and copy number variation (CNV) calling, genome-wide CNV load analysis was performed per individual to estimate the effect of CNV load in the genome on time of menopause. Subsequently, all the detected CNVs were compared against the EGCUT control population (n = 3188) to identify nonoverlapping CNV regions (CNVRs) (n = 220), for which functional enrichment and association studies were performed. After the following interpretation of each CNVR, six individuals with hemizygous deletions were also selected for whole-exome sequencing (WES) to determine whether identified deletions can result in unmasking recessive mutations or other POF-associated variants in the exome.