High-resolution array-CGH analysis on 46,XX patients affected by early onset primary ovarian insufficiency discloses new genes involved in ovarian function

Abstract

Study question: Can high resolution array-CGH analysis on a cohort of women showing a primary ovarian insufficiency (POI) phenotype in young age identify copy number variants (CNVs) with a deleterious effect on ovarian function?

Summary answer: This approach has proved effective to clarify the role of CNVs in POI pathogenesis and to better unveil both novel candidate genes and pathogenic mechanisms.

What is known already: POI describes the progression toward the cessation of ovarian function before the age of 40 years. Genetic causes are highly heterogeneous and despite several genes being associated with ovarian failure, most of genetic basis of POI still needs to be elucidated.

Study design, size, duration: The current study included 67 46,XX patients with early onset POI (<19 years) and 134 control females recruited between 2012 and 2016 at the Medical Cytogenetics and Molecular Genetics Lab, IRCCS Istituto Auxologico Italiano.

Participants/materials, setting, methods: High resolution array-CGH analysis was carried out on POI patients' DNA. Results of patients and female controls were analyzed to search for rare CNVs. All variants were validated and subjected to a gene content analysis and disease gene prioritization based on the present literature to find out new ovary candidate genes. Case-control study with statistical analysis was carried out to validate our approach and evaluate any ovary CNVs/gene enrichment. Characterization of particular CNVs with molecular and functional studies was performed to assess their pathogenic involvement in POI.

Main results and the role of chance: We identified 37 ovary-related CNVs involving 44 genes with a role in ovary in 32 patients. All except one of the selected CNVs were not observed in the control group. Possible involvement of the CNVs in POI pathogenesis was further corroborated by a case-control analysis that showed a significant enrichment of ovary-related CNVs/genes in patients (P = 0.0132; P = 0.0126). Disease gene prioritization identified both previously reported POI genes (e.g. BMP15, DIAPH2, CPEB1, BNC1) and new candidates supported by transcript and functional studies, such as TP63 with a role in oocyte genomic integrity and VLDLR which is involved in steroidogenesis.

Large scale data: ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/); accession numbers SCV000787656 to SCV000787743.

Limitations, reasons for caution: This is a descriptive analysis for almost all of the CNVs identified. Inheritance studies of CNVs in some non-familial sporadic cases was not performed as the parents' DNA samples were not available. Addionally, RT-qPCR analyses were carried out in few cases as RNA samples were not always available and the genes were not expressed in blood.

Wider implications of the findings: Our array-CGH screening turned out to be efficient in identifying different CNVs possibly implicated in disease onset, thus supporting the extremely wide genetic heterogeneity of POI. Since almost 50% of cases are negative rare ovary-related CNVs, array-CGH together with next generation sequencing might represent the most suitable approach to obtain a comprehensive genetic characterization of POI patients.

Study funding/competing interest(s): Supported by Italian Ministry of Health grants 'Ricerca Corrente' (08C203_2012) and 'Ricerca Finalizzata' (GR-2011-02351636, BIOEFFECT) to IRCCS Istituto Auxologico Italiano.

Keywords: TP63; VLDLR; array-CGH; ovarian dysgenesis; primary ovarian insufficiency.

Figure 1

Flowchart of the study design.

Figure 2

Schematic view of genes according to their distribution among POI association scores from 1 (low) to 8 (high) and related functional categories.

Figure 3

Case-control statistical analysis. (A) Pearson Chi-q test. Significant P-values (<0.05) are in bold. (B) Wilcoxon rank sum test without continuity correction. Mean and standard deviation (SD) are represented for both cases and controls. Significant P-values (<0.05) are in bold. (C and D) Ideogram view showing the results of Chi-q and Wilcoxon tests, respectively.

Figure 4

Molecular characterization of TP63 gene duplication. (A) array-CGH profile output. (B) Duplication view according to UCSC genome browser; intragenic duplication is represented by a blue bar from IVS1 to IVS9. (C) TP63 isoforms are shown with the respective duplicated portion of 161 kb based on array-CGH results. (D) Duplication breakpoint characterization on gDNA performed by LR-PCR and sequencing with specific primers for the duplicated allele is shown (direct tandem orientation is considered); an overlapping sequence of 11 bp between the breakpoint junctions in IVS11 and IVS1 is shown. (E) Wild-type mRNA and mutated transcript with the relative sequences are shown. RT-qPCR analysis revealed a statistically significant TP63 overexpression in patients compared to controls which is indicative of the amount of the aberrant transcript. (F) Wild-type and hypothetical aberrant Tp63 proteins are shown.

Figure 5

Molecular characterization of 9p24.2 deletion. (A) array-CGH profile output. (B) Deletion view according to UCSC genome browser; deletion is represented by a red bar from VLDLR 3’UTR to downstream. Primers used for LR-PCR analysis are shown and the relative output is enlarged below the red bar. Regulatory elements included are shown (E1, E2, enhancers; INS, insulator). (C) Luciferase assay experiments using both HeLa and COV434 cell lines. Ideograms showing the chemiluminescent emission for each construct are depicted. (D) RT-qPCR analysis highlighted in patient a dramatic downregulation of VLDLR mRNA compared to the average of controls. (E) The heterozygosity assay specific for rs6148 in VLDLR exon 14 showed a mono-allelic expression.