Whole-exome sequencing identifies mutations in FSIP2 as a recurrent cause of multiple morphological abnormalities of the sperm flagella
Whole-exome sequencing identifies mutations in FSIP2 as a recurrent cause of multiple morphological abnormalities of the sperm flagella
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DOI：
10.1093/humrep/dey264
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发表时间：
2018-10-01
影响因子：
6.1
通讯作者：
Coutton, Charles
中科院分区：
文献类型：
--
作者：
Martinez, Guillaume;Kherraf, Zine-Eddine;Coutton, Charles
研究问题：不育患者的全外显子组测序（WES）能否鉴定出导致精子鞭毛（MMAF）多种形态异常的新基因？总结回答：通过对78名MMAF表型不育男性的WES分析，在4名无关个体中发现了纤维鞘（FS）相互作用蛋白2（FSIP 2）基因的4个纯合突变。高通量测序技术的使用揭示了动力蛋白轴丝重链1（DNAH 1）基因的突变，纤毛和鞭毛相关蛋白43（CFAP 43）和44（CFAP 44）基因约占MMAF病例的三分之一，因此表明其他相关基因有待鉴定。这是一项回顾性遗传学研究，涉及2008年至2015年期间在三家生育诊所招募的78名MMAF表型患者。对照精子样本来自正常精子供体。对照组的等位基因频率来自大型公共数据库。对象/材料，环境，方法：对所有78名受试者进行WES。通过桑格测序确认所有鉴定的变体。在一组正常人和小鼠组织中通过定量RT-PCR评估所选候选基因（FSIP 2）的相对mRNA表达水平。为了表征患者精子中存在的结构和超微结构异常，对来自两名突变受试者和一名对照受试者的精子样本进行免疫荧光（IF）分析，并对来自一名突变受试者和一名对照受试者的精子样本进行透射电子显微镜（TEM）分析。我们确定了4名无关患者（4/78，5.1%）的FSIP 2基因，编码的蛋白质的精子FS和特异性表达在人类和小鼠睾丸中的纯合功能缺失突变。这些突变在对照序列数据库中均未报告。TEM分析显示与轴丝缺陷相关的FS完全解体。IF分析证实，在所有四名携带FSIP 2突变的患者中，轴丝的中心对微管和内外动力蛋白臂均异常。重要的是，与在MMAF和其他MMAF相关基因突变患者中观察到的情况相反，（DNAH 1、CFAP 43和CFAP 44），FSIP 2突变导致A-激酶锚定蛋白4（AKAP 4）缺失。生物样本数量少且缺乏可靠的抗-FSIP 2抗体阻止了FSIP 2蛋白质在精子中缺失的正式证明，FSIP 2突变的受试者的精子中FSIP 2蛋白质是不存在的。在人类中，以前与MMAF表型相关的基因编码轴丝相关蛋白（DNAH 1，CFAP 43和CFAP 44）。我们在这里表明，FSIP 2，蛋白质的精子FS，也是逻辑上与MMAF综合征，因为我们表明，它是必要的FS大会和整体轴丝和鞭毛生物发生。正如之前在小鼠和人中所提出的，我们的结果还表明，AKAP 4（与FSIP 2相互作用的主要蛋白之一）的缺陷将诱导MMAF表型。最后，这项工作进一步证明了WES测序是一种很好的策略，可以对严重男性不育表型患者进行基因诊断。研究资金/竞争兴趣：这项工作得到了以下赠款：由法国ANR和DGOS为PRTS 2014（14-CE 15）计划资助的“MAS-Flagella”项目和“鞭毛生长缺陷（FGD）患者的全基因组测序”项目，由基金会Maladies Rares资助，用于2012年的测序计划。作者没有利益冲突。
STUDY QUESTION: Can whole-exome sequencing (WES) of infertile patients identify new genes responsible for multiple morphological abnormalities of the sperm flagella (MMAF)?SUMMARY ANSWER: WES analysis of 78 infertile men with a MMAF phenotype permitted the identification of four homozygous mutations in the fibrous sheath (FS) interacting protein 2 (FSIP2) gene in four unrelated individuals.WHAT IS KNOWN ALREADY: The use of high-throughput sequencing techniques revealed that mutations in the dynein axonemal heavy chain 1 (DNAH1) gene, and in the cilia and flagella associated protein 43 (CFAP43) and 44 (CFAP44) genes account for approximately one-third of MMAF cases thus indicating that other relevant genes await identification.STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of 78 patients presenting a MMAF phenotype who were recruited in three fertility clinics between 2008 and 2015. Control sperm samples were obtained from normospermic donors. Allelic frequency for control subjects was derived from large public databases.PARTICIPANTS/MATERIALS, SETTING, METHODS: WES was performed for all 78 subjects. All identified variants were confirmed by Sanger sequencing. Relative mRNA expression levels for the selected candidate gene (FSIP2) was assessed by quantitative RT-PCR in a panel of normal human and mouse tissues. To characterize the structural and ultrastructural anomalies present in patients' sperm, immunofluorescence (IF) was performed on sperm samples from two subjects with a mutation and one control and transmission electron microscopy (TEM) analyses was performed on sperm samples from one subject with a mutation and one control.MAIN RESULTS AND THE ROLE OF CHANCE: We identified four unrelated patients (4/78, 5.1%) with homozygous loss of function mutations in the FSIP2 gene, which encodes a protein of the sperm FS and is specifically expressed in human and mouse testis. None of these mutations were reported in control sequence databases. TEM analyses showed a complete disorganization of the FS associated with axonemal defects. IF analyses confirmed that the central-pair microtubules and the inner and outer dynein arms of the axoneme were abnormal in all four patients carrying FSIP2 mutations. Importantly, and in contrast to what was observed in patients with MMAF and mutations in other MMAF-related genes (DNAH1, CFAP43 and CFAP44), mutations in FSIP2 led to the absence of A-kinase anchoring protein 4 (AKAP4).LIMITATIONS, REASONS FOR CAUTION: The low number of biological samples and the absence of a reliable anti-FSIP2 antibody prevented the formal demonstration that the FSIP2 protein was absent in sperm from subjects with a FSIP2 mutation.WIDER IMPLICATIONS OF THE FINDINGS: Our findings indicate that FSIP2 is one of the main genes involved in MMAF syndrome. In humans, genes previously associated with a MMAF phenotype encoded axonemal-associated proteins (DNAH1, CFAP43 and CFAP44). We show here that FSIP2, a protein of the sperm FS, is also logically associated with MMAF syndrome as we showed that it is necessary for FS assembly and for the overall axonemal and flagellar biogenesis. As was suggested before in mouse and man, our results also suggest that defects in AKAP4, one of the main proteins interacting with FSIP2, would induce a MMAF phenotype. Finally, this work reinforces the demonstration that WES sequencing is a good strategy to reach a genetic diagnosis for patients with severe male infertility phenotypes.STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the following grants: the 'MAS-Flagella' project financed by the French ANR and the DGOS for the program PRTS 2014 (14-CE15) and the 'Whole genome sequencing of patients with Flagellar Growth Defects (FGD)' project financed by the Fondation Maladies Rares for the program Sequencage a haut debit 2012. The authors have no conflict of interest.