Identification of a duplication within the GDF9 gene and novel candidate genes for primary ovarian insufficiency (POI) by a customized high-resolution array comparative genomic hybridization platform

Abstract

STUDY QUESTIONCan high-resolution array comparative genomic hybridization (CGH) analysis of DNA samples from women with primary ovarian insufficiency (POI) improve the diagnosis of the condition and identify novel candidate genes for POI?SUMMARY ANSWERA mutation affecting the regulatory region of growth differentiation factor 9 (GDF9) was identified for the first time together with several novel candidate genes for POI.WHAT IS KNOWN ALREADYMost patients with POI do not receive a molecular diagnosis despite a significant genetic component in the pathogenesis.STUDY DESIGN, SIZE, DURATIONWe performed a case–control study. Twenty-six patients were analyzed by array CGH for identification of copy number variants. Novel changes were investigated in 95 controls and in a separate population of 28 additional patients with POI. The experimental procedures were performed during a 1-year period.PARTICIPANTS/MATERIALS, SETTING, METHODSDNA samples from 26 patients with POI were analyzed by a customized 1M array-CGH platform with whole genome coverage and probe enrichment targeting 78 genes in sex development. By PCR amplification and sequencing, the breakpoint of an identified partial GDF9 gene duplication was characterized. A multiplex ligation-dependent probe amplification (MLPA) probe set for specific identification of deletions/duplications affecting GDF9 was developed. An MLPA probe set for the identification of additional cases or controls carrying novel candidate regions identified by array-CGH was developed. Sequencing of three candidate genes was performed.MAIN RESULTS AND THE ROLE OF CHANCEEleven unique copy number changes were identified in a total of 11 patients, including a tandem duplication of 475 bp, containing part of the GDF9 gene promoter region. The duplicated region contains three NOBOX-binding elements and an E-box, important for GDF9 gene regulation. This aberration is likely causative of POI. Fifty-four patients were investigated for copy number changes within GDF9, but no additional cases were found. Ten aberrations constituting novel candidate regions were detected, including a second DNAH6 deletion in a patient with POI. Other identified candidate genes were TSPYL6, SMARCC1, CSPG5 and ZFR2.LIMITATIONS, REASONS FOR CAUTIONThis is a descriptive study and no functional experiments were performed.WIDER IMPLICATIONS OF THE FINDINGSThe study illustrates the importance of analyzing small copy number changes in addition to sequence alterations in the genetic investigation of patients with POI. Also, promoter regions should be included in the investigation.STUDY FUNDING/COMPETING INTEREST(S)The study was supported by grants from the Swedish Research council (project no 12198 to A.W. and project no 20324 to A.L.H.), Stockholm County Council (E.I., A.W. and K.R.W.), Foundation Frimurare Barnhuset (A.N., A.W. and M.B.), Karolinska Institutet (A.N., A.L.H., E.I., A.W. and M.B.), Novo Nordic Foundation (A.W.) and Svenska Läkaresällskapet (M.B.). The funding sources had no involvement in the design or analysis of t...

A familial study of azoospermic men identifies three novel causative mutations in three new human azoospermia genes

“…A possible hint comes from a recent study that found co-occurrence of a large deletion in the human DNAH6 locus and primary ovarian insufficiency. 51 The high rate of amino acid substitutions during mammalian evolution of the DNAH6 mutated amino acid region might be mainly due to adaptive selection, a feature highly associated with some genes involved in reproduction. [52][53][54] Notably, dynein genes have been suggested as the main components of the force generating rapid prophase movements of the chromosomes (RPMs) during meiosis in various phylogenetic classes.…”

Genetics of primary ovarian insufficiency

“…Nevertheless, a large number of genes or genomic loci, affected by common CNVs or single nucleotide polymorphisms (SNPs) and identified by genome-wide association studied (GWA), may have a role in the disease susceptibility but can explain only a small proportion of the total heritability of POI (23). The analysis of a few cohorts of 46,XX POI patients by means of high throughput techniques, such as comparative genomic hybridization array (array-CGH) and SNP array, has led to the identification of CNVs affecting several X-linked and autosomal loci with a possible role in female fertility (24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34). Similarly, the recent application of whole-exome sequencing (WES) to a few POI multigenerational familial cases has succeeded in revealing rare single nucleotide variants affecting genes implicated in ovarian function (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48).…”

Effect of growth differentiation factor-9 C447T and G546A polymorphisms on the outcomes of in vitro fertilization

“…In addition, the elevated frequencies of certain GDF-9 heterozygous variants, including the C447T SNP, have been identified in European, Caucasian and Asian women with POF failure (26,28,37,38), however, not in New Zealander or Japanese woman (27,38). A tandem duplication of 475 bp in the GDF-9 promoter was previously identified as a causative factor of primary ovarian insufficiency (39). The GDF-9 variants have also been associated with the risk of PCOS (40) and have been associated with hirsutism scores and parity in patients with PCOS (41).…”