A biallelic loss of function variant in HORMAD1 within a large consanguineous Turkish family is associated with spermatogenic arrest.

Study Question: 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?

Summary Answer: WES analysis revealed a homozygous nonsense variant in HORMAD1 in three affected brothers of a Turkish family.

What Is Known Already: 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.

Study Design, Size, Duration: 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.

Participants/materials, Setting, Methods: 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.

Main Results And The Role Of Chance: 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.

Limitations, Reasons For Caution: 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.

Wider Implications Of The Findings: 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.

Study Funding/competing Interest(s): 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.

Trial Registration Number: N/A.