目的:通过全外显子组测序(WES)对土耳其大型近亲家族进行分析,是否可以确定导致非阻塞性无精子症(NOA)的新的致病遗传变异,其特征是在原代精母细胞阶段停止?
结论:WES分析显示,在一个土耳其家族的三个受影响兄弟中,HORMAD1中存在纯合无义变异。
背景:使用高通量测序对小型或大型近亲家庭的患者队列进行研究,可以识别不同病理的遗传原因,包括不孕症。在过去的二十年里,已经发现了许多与人类男性不育有关的基因,但是,只有14个基因被鉴定为至少与男性分离的NOA或少精子症中度相关。
方法:该研究包括一个土耳其家庭,包括三个患有NOA的兄弟。两兄弟的核型正常,正常的激素水平和没有Yq微缺失。睾丸组织病理学分析显示精子发生在原代精母细胞阶段完全停止。
方法:我们招募了一个近亲土耳其家庭,父母是一级表亲,有七个孩子;三个儿子有NOA,两个有生育能力的儿子和两个没有信息的女儿。病人的唾液样本,他的两个受影响的兄弟,收集了父母和两个未受影响的兄弟(总共七个样本)。在WES之前,使用不孕症小组对索引患者进行了针对性的基因检测,其中包括133个不育基因。未发现致病变异。然后对可用的七个家族成员的DNA进行WES。使用内部管道进行生物信息学分析。对检测到的变体进行评分和排名,和拷贝数变异被调用和注释。通过细胞转染,然后进行免疫荧光或免疫印迹,研究了突变对蛋白质表达和定位的影响。
结果:WES揭示了纯合的无义变体chr1:150675797G>A;HORMAD1(NM_032132.5):c.1021C>T,p.Gln341*在外显子13中,这在所有三个受影响的兄弟中得到证实。HORMAD1编码含有HORMA结构域的蛋白1。父母以及两个肥沃的兄弟都是这种变体的携带者。这种变体可能导致产生缺乏核定位信号的截短蛋白;因此,用野生型和突变形式转染人类细胞,与绿色荧光蛋白融合。免疫印迹实验证实了截短的HORMAD1蛋白的产生,免疫荧光显微镜显示突变蛋白显示细胞质定位,而野生型蛋白位于细胞核。总之,我们的研究结果证实HORMAD1是人类减数分裂过程中必不可少的遗传因素.
结论:根据一种用于评估男性不育基因临床有效性的评分系统,这项研究将HORMAD1分类为显示与男性不育症有关的有限临床证据.然而,当只分析一个家族时,这样的评分是最大可能的评分,并且增加一名显示致病性或可能致病性变异的患者会立即将该分类更改为"中度".因此,本报告应促使其他研究人员对NOA患者进行这种遗传变异的筛查.
结论:识别与人类减数分裂过程有关的新遗传因素将有助于提高我们的基本知识,这反过来将允许更好的护理不孕症患者的管理。由于Hormad1-/-敲除的雌性小鼠也不育,HORMAD1也可能与人类女性不育症有关。我们的发现对患者及其家庭成员的遗传咨询具有直接意义。
背景:这项研究由疾病基金会资助(高通量测序和罕见疾病-2018,“罕见疾病基因组学”)。作者声明他们没有利益冲突。
背景:不适用。
Can the analysis of a large Turkish consanguineous family via whole exome sequencing (WES) identify novel causative genetic variation responsible for nonobstructive azoospermia (NOA) characterized by arrest at primary spermatocyte stage?
WES analysis revealed a homozygous nonsense variant in HORMAD1 in three affected brothers of a Turkish family.
Studying patient cohorts in small or large consanguineous families using high-throughput sequencing allows the identification of genetic causes of different pathologies, including infertility. Over the last two decades, a number of genes involved in human male infertility have been discovered, but only 14 genes have been identified as being at least moderately linked to isolated NOA or oligozoospermia in men.
The study included a Turkish family comprising three brothers with NOA. Two brothers had a normal karyotype, normal hormonal levels and no Yq microdeletion. The testicular histopathology analysis revealed the complete arrest of spermatogenesis at the primary spermatocyte stage.
We recruited a consanguineous Turkish family where parents were first-degree cousins and had seven children; three sons who had NOA, two sons who were fertile and two daughters for whom no information was available. Saliva samples from the index patient, his two affected brothers, parents and two nonaffected brothers (seven samples in total) were collected. Prior to WES, the index patient underwent targeted genetic testing using an infertility panel, which includes 133 infertility genes. No pathogenic variations were identified. WES was then performed on the DNA of the seven family members available. Bioinformatics analysis was performed using an in-house pipeline. Detected variants were scored and ranked, and copy number variants were called and annotated.The consequences of mutation on protein expression and localization were investigated by cell transfection followed by immunofluorescence or immunoblotting.
WES revealed a homozygous nonsense variant chr1:150675797G>A; HORMAD1 (NM_032132.5): c.1021C>T, p.Gln341* in exon 13, which was confirmed in all three affected brothers. HORMAD1 encodes the HORMA domain-containing protein 1. The parents as well as the two fertile brothers were carriers of this variant. This variant may lead to the production of a truncated protein lacking the nuclear localization signal; therefore, human cells were transfected with the wild-type and mutated form, in fusion with green fluorescent protein. Immunoblotting experiments confirmed the production of a truncated HORMAD1 protein, and immunofluorescence microscopy revealed that the mutated protein displayed cytoplasmic localization while the wild type protein located to the nucleus. Altogether, our findings validate HORMAD1 as an essential genetic factor in the meiotic process in human.
According to one scoring system used to evaluate the clinical validity of male infertility genes, this study would classify HORMAD1 as displaying limited clinical evidence of being involved in male infertility. However, such a score is the maximum possible when only one family is analyzed and the addition of one patient showing a pathogenic or likely pathogenic variant would immediately change this classification to \'moderate\'. Thus, this report should prompt other researchers to screen patients with NOA for this genetic variant.
Identification of new genetic factors involved in the human meiosis process will contribute to an improvement of our knowledge at the basic level, which in turn will allow the management of better care for infertile patients. Since Hormad1-/- knock-out female mice are also infertile, HORMAD1 could also be involved in human female infertility. Our findings have direct implications for the genetic counseling of patients and their family members.
The study was funded by Fondation Maladies Rares (High Throughput Sequencing and Rare Diseases-2018, \'GenOmics of rare diseases\'). The authors declare that they have no conflict of interest.
N/A.