A novel follicle-stimulating hormone receptor mutation causing primary ovarian failure: a fertility application of whole exome sequencing
A novel follicle-stimulating hormone receptor mutation causing primary ovarian failure: a fertility application of whole exome sequencing
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DOI：
10.1093/humrep/dew025
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发表时间：
2016-04-01
影响因子：
6.1
通讯作者：
Vilain, Eric
中科院分区：
文献类型：
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作者：
Bramble, Matthew S.;Goldstein, Ellen H.;Vilain, Eric
研究问题：全外显子组测序（WES）和体外验证研究能否用于原发性卵巢功能衰竭和不孕症患者的遗传病因学研究？总结回答：WES发现了一种新的促卵泡激素受体（FSHR）突变，并通过体外流式细胞术研究显示，这种突变会影响膜运输。已知情况：WES可以诊断高达25-35%的疑似性发育障碍（DSD）患者。FSHR突变是一个非常罕见的原因46，XX性腺发育不全与原发性闭经由于高促性腺激素卵巢failure.Study设计，规模，持续时间：一个WES研究其次是流式细胞术研究的突变蛋白function.PARTICIPANTS/MATERIALS，设置，方法：研究对象是两个土耳其姐妹篇与高促性腺激素原发性闭经，他们的父母和两个未受影响的姐妹篇。受影响的兄弟姐妹和父母都进行了测序（trio-WES）。用含有野生型FSHR以及WES发现的新型FSHR变体的载体进行HEK 293 T细胞的瞬时转染。FSHR蛋白的细胞定位以及FSH刺激的环AMP（cAMP）的生产进行了评估，使用流式cytometry.Main结果和机会的作用：两个受影响的姐妹篇是纯合子的一个以前未报道的错义突变（c.1222G > T，p.Asp408Tyr）在FSHR的第二个跨膜结构域。建模预测破坏二级结构。流式细胞术显示细胞表面信号检测平均减少48%（P < 0.01）。FSH刺激的cAMP（FSHR的第二信使）的平均荧光信号在转染MUR的细胞中减少了50%（P < 0.01）。所有新的所谓的遗传变异可以在临床上报告只有作为“变异的不确定意义”，直到更多的患者具有相似的表型被发现与相同的variant.WIDER的影响更广泛的发现：我们报告的第一个WES发现的FSHR突变，通过定量流式细胞术验证。WES是诊断罕见遗传疾病的有价值的工具，流式细胞术允许定量表征据称的变体。WES辅助诊断允许针对疾病的潜在分子病因的治疗。未来的研究应侧重于针对FSHR破坏的药物和辅助生殖治疗，以便FSH抵抗患者可以通过个性化药物治疗。
STUDY QUESTION: Can whole exome sequencing (WES) and in vitro validation studies be used to find the causative genetic etiology in a patient with primary ovarian failure and infertility?SUMMARY ANSWER: A novel follicle-stimulating hormone receptor (FSHR) mutation was found by WES and shown, via in vitro flow cytometry studies, to affect membrane trafficking.WHAT IS KNOWN ALREADY: WES may diagnose up to 25-35% of patients with suspected disorders of sex development (DSD). FSHR mutations are an extremely rare cause of 46, XX gonadal dysgenesis with primary amenorrhea due to hypergonadotropic ovarian failure.STUDY DESIGN, SIZE, DURATION: A WES study was followed by flow cytometry studies of mutant protein function.PARTICIPANTS/MATERIALS, SETTING, METHODS: The study subjects were two Turkish sisters with hypergonadotropic primary amenorrhea, their parents and two unaffected sisters. The affected siblings and both parents were sequenced (trio-WES). Transient transfection of HEK 293T cells was performed with a vector containing wild-type FSHR as well as the novel FSHR variant that was discovered by WES. Cellular localization of FSHR protein as well as FSH-stimulated cyclic AMP (cAMP) production was evaluated using flow cytometry.MAIN RESULTS AND THE ROLE OF CHANCE: Both affected sisters were homozygous for a previously unreported missense mutation (c.1222G > T, p.Asp408Tyr) in the second transmembrane domain of FSHR. Modeling predicted disrupted secondary structure. Flow cytometry demonstrated an average of 48% reduction in cell-surface signal detection (P < 0.01). The mean fluorescent signal for cAMP (second messenger of FSHR), stimulated by FSH, was reduced by 50% in the mutant-transfected cells (P < 0.01).LIMITATIONS, REASONS FOR CAUTION: This is an in vitro validation. All novel purported genetic variants can be clinically reported only as 'variants of uncertain significance' until more patients with a similar phenotype are discovered with the same variant.WIDER IMPLICATIONS OF THE FINDINGS: We report the first WES-discovered FSHR mutation, validated by quantitative flow cytometry. WES is a valuable tool for diagnosis of rare genetic diseases, and flow cytometry allows for quantitative characterization of purported variants. WES-assisted diagnosis allows for treatments aimed at the underlying molecular etiology of disease. Future studies should focus on pharmacological and assisted reproductive treatments aimed at the disrupted FSHR, so that patients with FSH resistance can be treated by personalized medicine.