A Homozygous Fancm Frameshift Mutation Causes Male Infertility In A Consanguineous Pakistani Family

Spermatogenesis is a sophisticated biological process that ultimately generates mature spermatozoa,which includes PGCs specification,migration,proliferation and differentiation;gonocytes proliferation,quiescence and migration;SSCs self-renewal and differentiation;spermatocyte meiosis and spermiogenesis.Any abnormalility in this process will lead to defective sperm morphology,impaired sperm motility or reduced sperm number（clinically known as teratozoospermia asthenozoospermia,oligospermia）or even absence of sperm（azoospermia）,which ultimately lead to male infertility.Male infertility affects approximately 7%of men and presents most severely with spermatogenic failure.Genetic factors may be the major underlying cause of spermatogenic failure,in the majority of cases,the etiology remains undetermined.We studied a Pakistani family having three infertile brothers,born to first-cousin parents.Two brothers were diagnosed with oligoasthenospermia and one with azoospermia.Whole exome sequencing and Sanger sequencing revealed a pathogenic mutation of Fanconi anemia（FA）core complex member FANCM（c.1946₁958del,p.P648Lfs*16）,which was homozygous in the patients and heterozygous in their parents,following Mendelian inheritance.Because FANCM participates in interstrand crosslink（ICL）repair,we tested lymphocytes from patients for hypersensitivity to chromosomal breaks induced by mitomycin C（MMC）.Compared to their father,lymphocytes from the patients displayed deficient repair of chromosomal breaks,functionally indicating the existence of the mutation,and its disruption of FANCM function in the patients.The frameshift mutation produces a truncated protein that lacked functional domains required for FANCD2 foci and FANCD2 monoubiquitination,which impaired the FA pathway activation,resulted in accumulation of DNA breaks and increased chromosome instability,and consequently compromised cell survival in cultured 293T cells.We generated a mouse model with a Fancm mutation（Fancm△C/△C）nearly equivalent to that in our patients,which displayed spermatogenic failure and significantly reduced fertility,recapitulating the phenotype in our patients.Fancm△C/△C male mice showed drastically decreased sperm count,impaired sperm motility,massive loss of germ cells of multiple stages,spermatogenic arrest at round spermatid stage.Further analysis of Fancm△C/△C male mice revealed increased sensitivity of spermatogonia to MMC,compromised spermatogonial proliferation and defectiveDNA break repair in round spermatids,while meiotic prophase I progression is normal Interestingly,the patients and adult Fancm△C/△C mice do not have any birth defect,bone marrow failure,cancer or tumor.Taken together,we first report that the recessive FANCM mutation causes spermatogenic failure and results in male infertility rather than bone marrow failure,i.e.an atypical FA（named as FA-M）,providing guidance for diagnosis and genetic counselling of male infertility and FA.