Mutation Screening Of Patients Who Were Born From Related Parents With Multiple Morphological Abnormalities Of The Sperm Flagella

BackgroudMale infertility affects more than 20 million men worldwide and represents a real health concern.It is a typical multifactorial disorder with a strong genetic basis and additional etiological factors such as urogenital infections,immunological or endocrine diseases,attack from reactive oxygen species,or perturbations from endocrine disruptors.Multiple morphological abnormalities of the sperm flagella is a rare sperm movement obstacle of abnormal ultrastructure of sperm's flagella causing primary male infertility without anyother clinical feature.To date,despite substantial efforts made to identify genes specifically involved in male infertility by many teams worldwide,only a handful of genes have been formally correlated with human sperm defects.Objective1.To screen new mutations of patients who were born from related parents with multiple morphological abnormalities of the sperm flagella by the whole-exome sequencing.2.To investigate the pathogenic role of MMAF with DNAH1,CFAP43 and CFAP44 genes mutation during assembling the organization of the sperm flagella.MethodsThe 20 participants with multiple morphological abnormalities of the sperm flagella were recruited from the Reproductive Medicine Center of the First Affiliated Hospital of Anhui Medical University for ART treatments or clinical consultation,from January 2016 to October 2017.Peripheral blood and semen samples were collected from all subjects for the subsequent experiment,such as: DNA extraction,PCR,whole exome sequencing,Sanger sequencing,morphological analyze of the sperm,HE staining and transmission electron microscopy.This study was approved by the Ethics Committee of the first Affiliated Hospital of Anhui Medical University and all patients had signed the informed consent.ResultsIn the 20 cases of multiple sperm abnormalities of the spermatozoa from a close relative,the 8 MMAF patients were found to have a gene mutation,including three DNAH1 mutation sites,c.1832 T>C?p.L611P?,c.11726₁1727del?p.P3909fs?,c.12122 C>T?p.T4041L?,four CFAP43 mutation sites,and one CFAP44 mutation site,c.1140₁143del?p.Lys381Profs*??c.739A>T?p.Lys247*??c.1474G>C?p.Glu492Arg??c.4600C>G?p.Leu1534Val??c.4963C>T?p.Arg1655*?.Sanger sequencing further confirmed the reliability of the whole exome sequencing results.Under the transmission electron microscopy,the sperm flagellar axon of healthy males has a complete "9+2"?9 peripheral microtubule diplex and 2 central microtubules?ultrastructure,whereas patients with multiple malformations of the sperm tail often present as external the disorganization of microtubules,loss of central microtubules and radial axons.What we observed on the TEM are consistent with the results observed under the light microscope.Besides,the results of acetyl-Tubulin immunofluorescence and HE staining in the testis of Cfap44 knockout mice showed that the tubulin fluorescence signal in the Cfap44 knockout mice was extremely weak compared to wild-type mice,and the number of spermatozoa in HE stained testis was also significantly lower compared to wild-type mice.ConclusionOur results indicate that multiple morphological abnormalities of the sperm flagella is a recessive genetic disease,and we discovered a total of 8 new mutation sites in 20 patients with MMAF through the whole-exome sequencing technology,including 3 DNAH1 mutation sites?c.1832 T>C?,c.11726₁1727del,c.12122 C>T),four CFAP43 mutation sites?c.1140₁143del,c.739A>T,c.1474G>C,c.4600C>G?and one CFAP44 mutation site?c.4963C>T?.Besides the identified genes can lay a molecular foundation for the genetic counseling of patients in the future.