A novel mutation in DNAH17 is present in a patient with multiple morphological abnormalities of the flagella.

Asthenoteratospermia is characterized by malformed spermatozoa with motility defects, which results in male infertility. Multiple morphological abnormalities of the sperm flagella (MMAF) is a hallmark of asthenoteratospermia. The genetic causes of MMAF, however, are unknown in about one-third of cases. Which other MMAF-associated genes are waiting to be discovered? Whole-exome sequencing was conducted to identify causative genes in a man with MMAF. Immunofluorescence staining and western blot were applied to assess the pathogenicity of the identified variant. Intracytoplasmic sperm injection (ICSI) was used to assist fertilization for the patient with MMAF. Sanger sequencing of the family demonstrated that the infertile man carried a homozygous DNAH17 variant (c. 4810C>T [p.R1604C]). The obviously decreased DNAH17 expression was observed in HEK293T cells transfected with MUT-DNAH17 plasmid compared with cells with WT-DNAH17 plasmid. Immunofluorescence analysis showed that this mutation induced significant decrease in DNAH17 expression, which negatively affected the DNAH8 expression in the patient's spermatozoa. Moreover, the outcome of ICSI in the patient was unsuccessful. Our study revealed a novel homozygous missense mutation in DNAH17 involved in MMAF phenotype. The finding of the novel mutation in DNAH17 enriches the gene variant spectrum of MMAF, further contributing to diagnosis, genetic counselling and prognosis for male infertility.

Zheng R ，Sun Y ，Jiang C ，Chen D ，Yang Y ，Shen Y ... -  《-》

DNAH17 is associated with asthenozoospermia and multiple morphological abnormalities of sperm flagella.

Multiple morphological abnormalities of the sperm flagella (MMAF) is one kind of severe asthenozoospermia, which is caused by dysplastic development of sperm flagella. In our study, we sought to investigate the novel gene mutations leading to severe asthenozoospermia and MMAF. The patient's spermatozoa were tested by Papanicolaou staining and transmission electron microscopy. Whole exome sequencing was performed on the patient with severe asthenozoospermia and MMAF. Sanger sequencing verified the mutations in the family. The expression of DNAH17 was detected by immunofluorescence and Western blot. Spermatozoa sample from the patient showed severe asthenozoospermia and MMAF. We detected biallelic mutations (c.C4445T, p.A1482V and c.C6857T, and p.S2286L) in DNAH17 (MIM:610063). The protein expression of DNAH17 was almost undetectable in spermatozoa from the patient with the biallelic mutations. These results demonstrated that DNAH17 may be involved in severe asthenozoospermia and MMAF.

Sha Y ，Wei X ，Ding L ，Mei L ，Huang X ，Lin S ，Su Z ，Kong L ，Zhang Y ，Ji Z ... -  《-》

A novel CCDC39 mutation causes multiple morphological abnormalities of the flagella in a primary ciliary dyskinesia patient.

Male infertility is a widespread symptom in patients with primary ciliary dyskinesia (PCD). PCD-related male infertility is often caused by asthenozoospermia, with barely normal sperm morphology. Multiple morphological abnormalities of the sperm flagella (MMAF) are a major cause of asthenozoospermia, characterized by various malformed morphologies of sperm flagella. To date, a limited number of genes have been suggested to be involved in the pathogenesis of both PCD and MMAF. What other genes associated with both PCD and MMAF are waiting to be discovered? Whole-exome sequencing (WES) was performed to identify the pathogenic mutation associated with MMAF in a PCD patient. Peripheral venous blood and semen samples were collected from the PCD patient. Transmission electron microscopy (TEM), immunofluorescence staining and western blotting were conducted to confirm the pathogenicity of the identified mutation. A novel homozygous mutation in CCDC39, c.983 T>C (p. Leu328Pro), was identified in two PCD-affected siblings of a consanguineous family showing a typical PCD phenotype, while the proband was infertile, which is associated with characterized MMAF. Furthermore, TEM revealed the abnormal ultrastructure of the patient's sperm flagella. Moreover, immunofluorescence staining revealed that CCDC39 was almost undetectable in the spermatozoa, which was further confirmed by western blotting. The outcome of intracytoplasmic sperm injection (ICSI) in the patient with the CCDC39 mutation was also favourable. This study demonstrates that a novel loss-of-function mutation of CCDC39 is involved in the pathogenesis of PCD and MMAF and initially reported that ICSI treatment has a good outcome. Therefore, the novel variant of CCDC39 contributes to the genetic diagnosis, counselling and treatment of male infertility in PCD patients with MMAF phenotype.

Chen D ，Liang Y ，Li J ，Zhang X ，Zheng R ，Wang X ，Zhang H ，Shen Y ... -  《-》

Novel loss-of-function variants in DNAH17 cause multiple morphological abnormalities of the sperm flagella in humans and mice.

Multiple morphological abnormalities of the flagella (MMAF) is a genetically heterogeneous disorder leading to male infertility. Recent studies have revealed that DNAH17 variants are associated with MMAF, yet there is no functional evidence in support of their pathnogenicity. Here, we recruited two consanguineous families of Pakistani and Chinese origins, respectively, diagnosed with MMAF. Whole-exome sequencing identified novel homozygous DNAH17 variants, which led to loss of DNAH17 proteins, in the patients. Transmission electron microscope analyses revealed completely disorganized axonemal structure as the predominant anomaly and increased frequencies of missings of microtubule doublet(s) 4-7 in sperm flagella of patients. Similar to those found in patients, Dnah17-/- mice also displayed MMAF phenotype along with completely disorganized axonemal structures. Clusters of disorganized microtubules and outer dense fibers were observed in developing spermatids, indicating impaired sperm flagellar assembly. Besides, we also noticed many elongating spermatids with a deformed nuclear shape and abnormal step 16 spermatids that failed to spermiate, which subsequently underwent apoptosis in Dnah17-null mice. These findings present direct evidence establishing that DNAH17 is a MMAF-related gene in humans and mice, extend the clinical interpretations of DNAH17 variants, and highlight an essential and complex role of DNAH17 in spermatogenesis.

Zhang B ，Khan I ，Liu C ，Ma A ，Khan A ，Zhang Y ，Zhang H ，Kakakhel MBS ，Zhou J ，Zhang W ，Li Y ，Ali A ，Jiang X ，Murtaza G ，Khan R ，Zubair M ，Yuan L ，Khan M ，Wang L ，Zhang F ，Wang X ，Ma H ，Shi Q ... -  《-》

Bi-allelic DNAH8 Variants Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Primary Male Infertility.

Sperm malformation is a direct factor for male infertility. Multiple morphological abnormalities of the flagella (MMAF), a severe form of asthenoteratozoospermia, are characterized by immotile spermatozoa with malformed and/or absent flagella in the ejaculate. Previous studies indicated genetic heterogeneity in MMAF. To further define genetic factors underlying MMAF, we performed whole-exome sequencing in a cohort of 90 Chinese MMAF-affected men. Two cases (2.2%) were identified as carrying bi-allelic missense DNAH8 variants, variants which were either absent or rare in the control human population and were predicted to be deleterious by multiple bioinformatic tools. Re-analysis of exome data from a second cohort of 167 MMAF-affected men from France, Iran, and North Africa permitted the identification of an additional male carrying a DNAH8 homozygous frameshift variant. DNAH8 encodes a dynein axonemal heavy-chain component that is expressed preferentially in the testis. Hematoxylin-eosin staining and electron microscopy analyses of the spermatozoa from men harboring bi-allelic DNAH8 variants showed a highly aberrant morphology and ultrastructure of the sperm flagella. Immunofluorescence assays performed on the spermatozoa from men harboring bi-allelic DNAH8 variants revealed the absent or markedly reduced staining of DNAH8 and its associated protein DNAH17. Dnah8-knockout male mice also presented typical MMAF phenotypes and sterility. Interestingly, intracytoplasmic sperm injections using the spermatozoa from Dnah8-knockout male mice resulted in good pregnancy outcomes. Collectively, our experimental observations from humans and mice demonstrate that DNAH8 is essential for sperm flagellar formation and that bi-allelic deleterious DNAH8 variants lead to male infertility with MMAF.

Liu C ，Miyata H ，Gao Y ，Sha Y ，Tang S ，Xu Z ，Whitfield M ，Patrat C ，Wu H ，Dulioust E ，Tian S ，Shimada K ，Cong J ，Noda T ，Li H ，Morohoshi A ，Cazin C ，Kherraf ZE ，Arnoult C ，Jin L ，He X ，Ray PF ，Cao Y ，Touré A ，Zhang F ，Ikawa M ... -  《-》