Introduction
Premature ovarian failure (POF) is characterized by hyper gonadotropic ovarian deficiency with primary or secondary amenorrhoea (1). It affects 0.0001% of females by the age of 20 years, 0.001% by 30 years and 0.01% by 40 years, showing the one major cause of female infertility (2).
Ovarian function is regulated by a combined stimulus of gonadotropins, follicular stimulating hormone (FSH), luteinizing hormone, and local ovarian factors such as inhibins, activins, bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9). A familial accumulation of the POF syndrome has been observed in between 4 and 31% of the patients with POF which suggests dominant genetic etiology (3).
BMP15 and GDF9 belong to transforming growth factor-β (TGF-β) superfamily (4, 5). Genes belonging to TGF β superfamily regulate many aspects of development by activating transmembrane serine/threonine kinase receptors (6). BMP15 has been shown to stimulate granulosa cell growth and promotes the progression of folliculogenesis from the primary stage to the follicle stimulating hormone (FSH) dependent stage (7-9). Mutations in BMP15 gene have been found as a culprit for POF in several worldwide cohorts with a variable prevalence between 1.5 and 12% (10-12). However, Zhang et al. (13) and Ledig et al. (14) failed to find any association between BMP15 and POF.
GDF9 is also expressed in the oocyte and its products can form noncovalent heterodimers acting in a synergistic manner on the function of surrounding follicular granulosa cells (15). GDF9 gene variations in humans described so far in different ethnicities are all heterozygous, affect exclusively the pro-region with a prevalence of 1.4%, and are not detected in the control samples (16-18).
Due to these conflicting results of various studies, this study was planned to evaluate the association between growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes mutations with premature ovarian failure.
Methods
Subjects: Patients with a diagnosis of POF were recruited from the Department of Obstetrics and Gynaecology, Sri Aurobindo Medical Collage and PG Institute, Indore during the period between September 2012 and March 2015. A total of 52 patients with idiopathic premature ovarian failure or insufficiency subjects with 46XX karyotype were enrolled in the present study. The patients had a mean age of 29.82±6.0 years (range, 17-39 years). Less than 40 year old healthy individuals visiting the department for routine investigations were selected as the control group. All the control individuals had a regular menstrual cycle, and normal reproductive hormone levels and chromosomes.
The inclusion criteria of all participants were ≤40 years of age, duration of amenorrhea ≥6 months, serum FSH level ≥40 IU/l on two or more occasions with the presence of amenorrhea. Individuals with a history of ovarian surgery, radiotherapy, chemotherapy or other factors, which may damage ovarian functions, were excluded. The present study was approved by the Ethics Review Committee of the Sri Aurobindo Medical Collage and PG Institute. Written informed consent was obtained from all participants following a detailed description of the potential benefits of the investigation.
DNA extraction, sequencing and analysis: All the laboratory investigations were performed in Central Research Laboratory of the institute; 5 ml venous blood sample was drawn in EDTA vacutainer. Genomic DNA was extracted from the blood using a QIAamp DNA isolation kit according to the manufacturer's instructions, and the concentration was determined using Qubit Fluorometer (Life Technologies). PCR amplification of the GDF9 and BMP15 genes was performed using the primers shown in table 1, in a 50 μl reaction volume containing 50 nmol DNA template, 200 nmol dNTP, 10 pmol forward and reverse primers, 1 U Taq DNA polymerase and ddH2O, under the PCR amplification conditions shown in table 1. The PCR products were purified by DNA purification kit and subjected to direct sequencing using big dye terminator kit 3.1 (Applied Biosystems).
The sequencing chromatograms were assessed using the ABI sequence scanner program and the sequences of the GDF9 and BMP15 genes were aligned to those registered in Gene Bank (http://www.ncbi.nlm.nih.gov/genbank/) for the identification of mutant loci.
To determine the potentially deleterious effect of the amino acid changes, Provean software (http://provean.jcvi.org/index.php), SIFT software (http://sift.jcvi.org) and Polyphen 2 software (http://genetics.bwh.harvard.edu/pph2/) were used. Provean software predicts whether an amino acid substitution or indel has an impact on the biological function of a protein (19). The SIFT software predicts whether an amino acid substitution affects protein function based on sequence homology and the physical properties of amino acids (20). PolyPhen-2 (Polymorphism Phenotyping v2) predicts possible impact of amino acid substitutions on the structure and function of human proteins using straightforward physical and evolutionary comparative considerations (21).
Results
Sequencing data collection and analysis were successfully performed for the GDF9 and BMP15 genes in all the cases and controls, which included non-familial POF cases; no variant was observed in the coding region of the GDF9 gene of any case and control sample. Four variants of BMP15 gene were observed in cases which included two missense substitutions, one in exon I and the other in exon II position (Table 2). FS 788-ins-TCT was present in 3 cases. Three novel variants were observed in our cohort namely, c. 510-512del T, c.165A>T (p.Glu55Asp) and c.538 G>T (p.Aln180Ser). c.510-512del T is the frameshift variant resulting in elongated chain and by dominant negative effect it impairs the function of normal protein. The c.165A>T (p.Glu.55Asp) is a missense variant and changes the glutamic acid to asparagine at position 55. Provean score for this missense variant was -0.61(cut off score >-2.5) and SIFT prediction score was 0.108 (Cut off >0.05). Both of these scores predicted the neutral effect of this missense variant. This variant was predicted to be benign with a score of 0.007 (sensitivity: 0.96; specificity: 0.75) by PolyPhen 2 software.
In other patients of POF, c.538 G>T (p. Aln 180 Ser) missense variant was observed. This variant also has neutral effect on the protein function as predicted by Provean (0.17) and SIFT score (0.418). PolyPhen 2 also predicted this variant as benign with score of 0.156 (sensitivity: 0.92; specificity: 0.87). The clinical characteristics of the patients carrying BMP15 variants are detailed in table 3. No variant in BMP15 gene was observed in the control group.
Discussion
POF is mostly portrayed as a heterogeneous genetic disorder but its etiology still remains elusive. Studies have illuminated the comprehensive role of two oocyte derived growth factors, GDF9 and BMP15, as the main driving force for the proliferation and progression of somatic follicle cells (22). The BMP15 gene plays a vital role in early human folliculogenesis and it is characterized as a strong candidate gene for POF. BMP15 is a member of the large superfamily of the transforming growth factor β (TGFβ) proteins involved in diverse cellular processes during embryonic development and tissue formation (23). The main roles of BMP15 include a) the promotion of follicle maturation from the primordial gonadotropin independent phases of folliculogenesis; b) regulation of follicular GC sensitivity to FSH action; c) prevention of GC apoptosis; d) promotion of oocyte developmental competence; and e) regulation of ovulation quota (24, 25).
In the present study, four variants in BMP15 gene were identified of which three variants were novel.
c.165A>T (p.Glu55Asp) and c.538 G>T (p.Aln180Ser) variants has no deleterious effect as predicted by in silico analysis.
Previous studies reported different variant C.538G>A (p.Aln180Thr) at the same position at which we observed C.538G>T (p.Aln180Ser) (11,26). Similar to our prediction previous reports also shows no deleterious effect of this variant.
788insTCT variant was observed in three patients in our study. This variant has been identified in patients presenting with POF in many previous studies (6, 12, 26, 28). This insertion may be considered as a polymorphism as it was previously identified in controls as well (6, 11, 27, 28). This variant was not found in any of the control subjects.
No variant was observed in GDF9 gene in either patients or controls. This is in contrast to the study done by dixit et al. (17) and Laissue et al. (11) which show the GDF9 variants in POF patients.
Conclusion
In conclusion, the present study provided evidence for naturally occurring variants in association with POF and points to the massive importance of BMP15 as a vital candidate gene, which should also be studied in other populations. Although software assessment of the variants reveals their neutral effect, their functional implication cannot be ruled out altogether. Experimental studies should be conducted to determine the real implication of those variations.
Acknowledgement
This study was financed by the Department of Science and Technology, New Delhi, India.
Conflict of Interest
The authors report no declarations of interest. Source of funding was DST, New Delhi, India.