Validation of methylation-specific polymerase chain reaction method and evaluation of FMR1 gene pre-mutations in premature ovarian insufficiency

Background. Fragile X-associated premature ovarian insufficiency is clinically defined as a type of early ovarian failure with irregular menstrual cycles, increased follicle-stimulating hormone, premature menopause, and infertility. Genetically, the CGG trinucleotide repeat expansion in the 5'-untranslated region of the FMR1 gene is recognized as a causative factor. The aim of this study was to standardize molecular detection methods and estimate the number of repeats among patients with premature ovarian insufficiency. Methods. After obtaining a consent form and blood sampling, genomic DNA was extracted and treated with sodium bisulfite. The FMR1 gene is located on X chromosome and one allele is methylated by X-inactivation. As a result, the methylation-specific polymerase chain reaction (MS-PCR) was performed using two pairs of specific primers for methylated and non-methylated DNA yelding the products of 108+3n and 168+3n bp, respectively. Following clinical evaluations, 20 sporadic patients and two families with at least two patients were studied. Results. The mean level of follicle-stimulating hormone was 85.45±44.73UI/L in sporadic and 75.72±33.61UI/L in familial cases. Ultrasound examinations reported atrophic ovaries in sporadic and familial cases by 80% and 75%, respectively. The evaluation of the trinucleotide repeat expansions on agarose gels showed that two sporadic patients were carriers of FMR1 intermediate alleles (10%). No cases of pre-mutation or full mutation were observed in other sporadic or familial cases and the trinucleotide repeat expansions were estimated between 15 and 35. Conclusion. Due to the role of the FMR1 gene trinucleotide repeat expansion in women with premature ovarian insufficiency, a fast and cost-effective molecular detection method is of particular importance. This test will be beneficial not only in ovarian dysfunction, but also to identify pre-mutations that may expand in next generations leading to fragile X syndrome. Practical Implications. In young women, the detection of expanded CGG trinucleotide repeats in the 5'-untranslated region of the FMR1 gene along with genetic counseling will provide them to plan reproductive life.