How polymorphic markers contribute to genetic diseases in different populations? The study of inhibin A for premature ovarian insufficiency

ABSTRACT Objective To verify the incidence of the G679A mutation in exon 2 of the gene inhibin alpha (INHA), in women with secondary amenorrhea and diagnosis of premature ovarian insufficiency, and in controls. Methods A 5mL sample of peripheral blood was collected from all study participants in an EDTA tube and was used for DNA extraction. For the patient group, 5mL of blood were also collected in a tube containing heparin for karyotype, and 5mL were collected in a dry tube for follicle stimulant hormone dosage. All patient and control samples were initially submitted to analysis of the G679A variant in exon 2 of the INHA gene by PCR-RFLP technique. Samples from patients with premature ovarian insufficiency after PCR-RFLP were submitted to Sanger sequencing of the encoding exons 2 and 3. Sequencing was performed on ABI 3500 GeneticAnalyzer equipment and the results were evaluated by SeqA and Variant Reporter software. Results Samples of 70 women with premature ovarian insufficiency and 97 fertile controls were evaluated. The G769A variant was found in only one patient in the Premature Ovarian Insufficiency Group and in no control, and it appears to be rare in Brazilian patients with premature ovarian insufficiency. This polymorphism was previously associated to premature ovarian insufficiency in several populations worldwide. Conclusion There is genetic heterogeneity regarding the INHA gene in different populations, and among the causes of premature ovarian insufficiency.

摘要
目的 验证继发闭经且确诊为早发性卵巢功能不全（premature ovarian insufficiency, POI）的女性体内，抑制素α基因（inhibin alpha, INHA）外显子2的G679A突变发生率，并以健康人群作为对照开展对比分析。
方法 本研究所有受试者均采集5mL外周血置于乙二胺四乙酸（EDTA）抗凝管中，用于基因组DNA提取。针对患者组，额外采集5mL血液置于肝素抗凝管以开展核型分析，另采集5mL血液置于促凝管用于卵泡刺激素（follicle stimulating hormone, FSH）水平检测。所有患者与对照样本初始均通过聚合酶链反应-限制性片段长度多态性（PCR-RFLP）技术，对INHA基因外显子2的G679A变异位点进行检测。对于早发性卵巢功能不全患者中经PCR-RFLP检测后的样本，进一步采用桑格测序（Sanger sequencing）对其编码外显子2和3进行测序。测序实验在ABI 3500遗传分析仪（ABI 3500 GeneticAnalyzer）上完成，测序结果通过SeqA软件与Variant Reporter软件进行评估分析。
结果 本研究共纳入70例早发性卵巢功能不全女性受试者与97例生育功能正常的健康对照。仅在早发性卵巢功能不全组的1例患者中检出G769A变异，对照组未检出该变异，提示该变异在巴西早发性卵巢功能不全患者中较为罕见。此前已有研究证实，该多态性位点与全球多个人群的早发性卵巢功能不全发病存在关联。
结论 INHA基因在不同人群中存在遗传异质性，这亦是早发性卵巢功能不全的致病原因之一。