Biallelic mutations in spermatogenesis and centriole-associated 1 like (SPATC1L) cause acephalic spermatozoa syndrome and male infertility.

Acephalic spermatozoa syndrome is a rare type of teratozoospermia that severely impairs the reproductive ability of male patients, and genetic defects have been recognized as the main cause of acephalic spermatozoa syndrome. Spermatogenesis and centriole-associated 1 like (SPATC1L) is indispensable for maintaining the integrity of sperm head-to-tail connections in mice, but its roles in human sperm and early embryonic development remain largely unknown. Herein, we conducted whole-exome sequencing (WES) of 22 infertile men with acephalic spermatozoa syndrome. An in silico analysis of the candidate variants was conducted, and WES data analysis was performed using another cohort consisting of 34 patients with acephalic spermatozoa syndrome and 25 control subjects with proven fertility. We identified biallelic mutations in SPATC1L (c.910C>T:p.Arg304Cys and c.994G>T:p.Glu332X) from a patient whose sperm displayed complete acephalia. Both SPATC1L variants are rare and deleterious. SPATC1L is mainly expressed at the head-tail junction of elongating spermatids. Plasmids containing pathogenic variants decreased the level of SPATC1L in vitro. Moreover, none of the patient's four attempts at intracytoplasmic sperm injection (ICSI) resulted in a transplantable embryo, which suggests that SPATC1L defects might affect early embryonic development. In conclusion, this study provides the first identification of SPATC1L as a novel gene for human acephalic spermatozoa syndrome. Furthermore, WES might be applied for patients with acephalic spermatozoa syndrome who exhibit reiterative ICSI failures.

Novel IFT140 variants cause spermatogenic dysfunction in humans.

BACKGROUND: The intraflagellar transport protein 140 homolog (IFT140) is involved in the process of intraflagellar transport (IFT), a process that is essential for the formation and maintenance of most eukaryotic cilia and flagella. Variants IFT140 have been reported to account for ciliopathy but association with male fertility has never been described in humans. Here we report the identification of two novel variants of IFT140 which caused spermatogenic dysfunction and male infertility. METHODS: Whole-exome sequencing was performed in a 27-year-old infertile man presented with severe oligozoospermia, asthenozoospermia, and teratozoospermia (OAT) without other physical abnormality. Sanger sequencing was used to verify gene variants in the patient, his healthy brother, and their parents. Morphology and protein expression in the patient's sperm were examined by transmission electron microscopy (TEM) and immunofluorescence staining. Function of gene variants was predicted by online databases. RESULTS: Compound heterozygous variants of IFT140: exon16: c.1837G > A: p.Asp613Asn and exon31: c.4247G > A: p.Ser1416Asn were identified in the patient, both of which showed autosomal recessive inheritance in his family, and had extremely low allele frequency in the population. Morphological abnormalities of the head, nucleus, and tails and the absence of IFT140 from the neck and mid-piece of the patient's spermatozoa were observed. Mutation Taster database predicted a high probability of damage-causing by both variations. CONCLUSION: This study for the first time reported IFT140 variants that cause infertility in humans.

Novel Mutations in CFAP44 and CFAP43 Cause Multiple Morphological Abnormalities of the Sperm Flagella (MMAF).

Multiple morphological abnormalities of the sperm flagella (MMAF) is a rare disease that causes primary infertility. However, the genetic causes for approximately half of MMAF cases are unknown. Whole exome sequencing analysis of the 27 patients with MMAF identified several CFAP44 mutations (3 homozygous: c.2935_2944del: p.D979*, c.T1769A: p.L590Q, c.2005_2006del: p.M669Vfs*13; and putative compound heterozygous: c.G3262A: p.G1088S and c.C1718A: p.P573H.) and CFAP43 acceptor splice-site deletion (c.3661-2A>-) mutations in 5 and 1 patients, respectively. Real-time quantitative polymerase chain reaction assays also demonstrated that CFAP44 expression was very weak in patient (P)1 and P3, and CFAP43 expression was lower in P6 than in the control. Immunofluorescence analysis of CFAP43 showed lower CFAP43 protein expression levels in P6 than in the normal control. This study demonstrated that biallelic mutations in CFAP44 and CFAP43 cause MMAF. These results provide researchers with a new insight to understand the genetic etiology of MMAF and to identify new loci for genetic counselling of MMAF.

Biallelic mutations in PMFBP1 cause acephalic spermatozoa.

The majority of men with defects in spermatogenesis remain undiagnosed. Acephalic spermatozoa is one of the diseases causing primary infertility. However, the causes underlying over half of affected cases remain unclear. Here, we report by whole-exome sequencing the identification of homozygous and compound heterozygous truncating mutations in PMFBP1 of two unrelated individuals with acephalic spermatozoa. PMFBP1 was highly and specifically expressed in human and mouse testis. Furthermore, immunofluorescence staining in sperm from a normal control showed that PMFBP1 localizes to the head-flagella junction region, and the absence of PMFBP1 was confirmed in patients harboring PMFBP1 mutations. In addition, we generated Pmfbp1 knock-out (KO) mice, which we found recapitulate the acephalic sperm phenotype. Label-free quantitative proteomic analysis of testicular sperm from Pmfbp1 KO and control mice showed 124 and 35 proteins, respectively, increased or decreased in sperm from KO mice compared to that found in control mice. Gene ontology analysis indicates that the biological process of Golgi vesicle transport was the most highly enriched in differentially expressed proteins, indicating process defects related to Golgi complex function may disturb formation of the head-neck junction. Collectively, our data indicate that PMFBP1 is necessary for sperm morphology in both humans and mice, and that biallelic truncating mutations in PMFBP1 cause acephalic spermatozoa.

Patients with multiple morphological abnormalities of the sperm flagella harbouring CFAP44 or CFAP43 mutations have a good pregnancy outcome following intracytoplasmic sperm injection.

Multiple morphological abnormalities of the sperm flagella (MMAF) are a rare type of male infertility. Mutations in DNAH1, CFAP43 and CFAP44 are the main aetiology of the disorder. Previously, good intracytoplasmic sperm injection (ICSI) outcomes were reported for MMAF patients with DNAH1 mutations. However, the ICSI prognosis for MMAF patients with CFAP43 or CFAP44 mutations was not known. We designed a retrospective cohort study. Molecular genetic testing identified six MMAF patients with biallelic CFAP44 (CFAP44+ group) or CFAP43 mutations and 12 patients with homozygous or compound heterozygous DNAH1 mutations (DNAH1+ group). A control group consisted of age-matched, non-MMAF men. For MMAF patients carrying CFAP44 mutations, the recorded rates of fertilisation, transferable embryos, pregnancy and delivery after ICSI were 76.47%, 88.46%, 50.0% and 50.0% respectively. The fertilisation rate was significantly higher in the CFAP44+ group than in the DNAH1+ group (76.47% vs. 54.5%, p = 0.0196). There were no statistically significant differences in the rates of transferable embryos, implantation, clinical pregnancy and miscarriage between the CFAP44+ group and either the DNAH1+ group or the age-matched control group. Our results support a good ICSI prognosis for MMAF patients carrying CFAP44 or CFAP43 mutations.

Two cases of complex balanced autosomal translocations associated with severe oligozoospermia.

Complex balanced autosomal translocation is rare and can lead to impaired spermatogenesis in males; however, its effects on oligozoospermia have rarely been reported. We report here two cases of rare complex balanced translocation in men with infertility. The karyotype of the first case was 46,XY,der(1)t(1;12)(p22;p11.2)ins(9;1)(p24;q25q23),der(9)ins(9;1),der(12)t(1;12)·ish der(1)t(1;12)(RP11-636B1+;RP11-659D23+)ins(9;1)(RP11-118P13+),der(9)ins(9;1),der(12)t(1;12). And the patient showed severe oligozoospermia with adult schizophrenia without other abnormalities. The karyotype of the second patient was 46,XY,der(5)t(5;11)(q14;p11.2),der(11)t(11;18)(p11.2;q11.2),der(18)t(5,18)(q14;p11.3)add(18)(q11.2?)·ish der(5)t(5;11)(RP11-846K3+,RP11-89B9+),der(11)t(11;18)(RP11-89B9-,RP11-170L12+,RP11-469N6+),der(18)t(5;18)(RP11-125L2+,RP11-29M13+)add(18)(q11.2?), and the patient displayed severe oligozoospermia without other abnormalities. The two cases were verified by fluorescent in situ hybridization, and no abnormalities were found by genome-wide copy number variation analysis. To our knowledge, these two cases of complex autosomal karyotypes have not been reported previously. Although rare, these cases suggest that complex balanced translocations may be important causes of oligozoospermia. We speculate that the balanced translocation hinders germ cell meiosis and causes impaired spermatogenesis. Accordingly, the two reported patients have very low probabilities of giving birth to a normal child; therefore, we suggest choosing donor semen or adopting a child.

Identification of a novel mutation in FGFR1 gene in patients with Kallmann syndrome by high throughput sequencing.

Kallmann syndrome (KS) is a rare clinical and genetic heterogeneity disease, which is familial or sporadic. KS is known to have three patterns of inheritance: X linked recessive inheritance, autosomal dominant inheritance and rare autosomal recessive inheritance. Here, we report a sibling pedigree with autosomal dominant inheritance of KS, and we identified a novel heterozygous frameshift mutation c.299_300insCCGCAGACTCCGGCCTCTATGC (p.C101Rfs*17) in FGFR1 gene using whole-exome sequencing (WES). The mutation and affection status were cosegregated. The mutation is not present in the dbSNP, 1000 Genome, ExAC, and gnomAD databases. The discovery of this new mutation in the FGFR1 gene enriches the spectrum of FGFR1 mutations in patients with KS. ABBREVIATIONS: FGFR1: fibroblast growth factor receptor 1; HH: hypogonadotropic hypogonadism; KS: Kallmann syndrome; MRI: magnetic resonance imaging; WES: whole-exome sequencing.

TDRD6 is associated with oligoasthenoteratozoospermia by sequencing the patient from a consanguineous family.

Oligoasthenoteratozoospermia (OAT) is characterized as low sperm count, decreased sperm motility and structural abnormalities of the sperm head in the same patient. However, very few studies reported the genetic alterations associated with OAT. Here we report a 38-year-old patient with OAT from a consanguineous family, with 2-6 million/mL sperm density, 2.1-3.8% normal sperm morphology and immotile sperm. Whole-exome sequencing (WES) identified homozygous variant c.1259A>G:p.Y420C in the TDRD6 gene. TDRD6 is a testis-specific expressed protein that was localized to the chromatoid bodies in germ cells and played an important role in the nonsense-mediated decay pathway. This rare variant co-segregated with the OAT phenotype in this family. Bioinformatic analysis also suggested the variant a pathogenic mutation. Two intracytoplasmic sperm injection (ICSI) cycles were carried out in the patient's wife, but she did not become pregnant after embryo transfer. So the mutations in TDRD6 may be associated with human male infertility and early embryonic lethality.

Sperm-egg fusion disorder in a Chinese male patient was associated with a rare ADAM20 variant.

We report here a 28-year-old male with infertility. No abnormality was found in his semen examination. The couple achieved a successful pregnancy under the help of intracytoplasmic sperm injection during which we found that sperm could enter the zona pellucida, but could not fuse with the egg within the short insemination period. We then performed whole-exome sequencing technology on this patient and found a rare variant (c.641A>C:p.D214A) in ADAM20, which encoded a disintegrin and metalloprotease 20 protein. To further verify the pathogenicity of this variant, we analyzed ADAM20 protein expression in spermatozoa by immunostaining analysis, which showed a mis-localization of ADAM20 in the patient's spermatozoa. Therefore, we concluded that mutation in ADAM20 may be associated with sperm-egg fusion disorder in this patient.

Genetic contribution of SUN5 mutations to acephalic spermatozoa in Fujian China.

Acephalic spermatozoa is an extremely rare disease associated with primary infertility. A recent study showed that genetic alterations in the SUN5 gene lead to this disease, and SUN5 mutations could explain the disease in about half of the patients. Therefore, in the present study, to re-visit the genetic contribution of SUN5 mutations to acephalic spermatozoa, we recruited 15 unrelated affected individuals and screened the SUN5 gene for mutations by whole-exome sequencing (WES) and Sanger sequencing. Five of the 15 (33.33%) subjects were found to carry the same homozygous mutation in the SUN5 gene c.381delA (p.V128Sfs*7). Neither homozygous nor compound heterozygous mutations in SUN5 were found in the other 10 patients. The c.381delA mutation resulted in the truncation of the SUN5 protein and decreased the expression and altered the distribution of the outer dense fiber 1 (ODF1) protein. Thus, in our study SUN5 mutations accounted for only one-third of the patients in our cohort, which is lower than the percentage reported previously. Thus, our study suggests that the contribution of SUN5 mutations to acephalic spermatozoa might not be as high as described previously. These results will help in the genetic counseling of patients with acephalic spermatozoa.

A novel mutation in HAUS7 results in severe oligozoospermia in two brothers.

Severe oligozoospermia (SO) is a common disease resulting in male infertility; however, its pathophysiology remains unclear. Here, we report two brothers with SO. Whole-exome sequencing (WES) identified a hemizygous variant in HAUS7 (c.G386T:p.G129V), an X-linked gene. HAUS7 has been reported to play a role in the meiotic maturation and chromosome alignment of germ cells. The two patients inherited this variant from their mother, and this variant was considered to be a highly pathogenic mutation by in silico analysis. Moreover, in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) was carried out in both the proband's wife and the brother's wife, but they failed to become pregnant after the embryo transfers. Therefore, this novel mutation in HAUS7 gene may be associated with severe oligozoospermia.

A homozygous CEP135 mutation is associated with multiple morphological abnormalities of the sperm flagella (MMAF).

Multiple morphological abnormalities of the sperm flagella (MMAF) is a rare disease associated with primary infertility; however, ~50% of the genetic alterations associated with MMAF remain unclear. Here, we reported the case of a 30-year-old infertile male from a consanguineous family. Whole-exome sequencing identified a homozygous mutation in the CEP135 gene (c.A1364T:p.D455V), with CEP135 previously reported to play a role in centriole biogenesis and specifically central pair assembly. D455V-mutated proteins formed protein aggregates in the centrosome and the flagella, which might potentially affect the function of centriole assembly. Moreover, intracytoplasmic sperm injection was performed using sperm from this patient; however, pregnancy failed following embryo transfer. This represents the first report of a homozygous mutation of CEP135 associated with MMAF. These results provide researchers and clinicians with a deeper understanding of the gene involved with MMAF and will help predict and assess pregnancy outcomes associated with in vitro fertilization.

Use of targeted sequence capture and high-throughput sequencing identifies a novel PKD1 mutation involved in adult polycystic kidney disease.

Polycystic kidney disease (PKD) is a common inherited disease that is characterized by a progressive development of renal cysts. Approximately 85% of PKD cases are due to mutations in the polycystin 1 (PKD1) gene. Here, we report a pedigree containing nine patients with autosomal dominant PKD (ADPKD). Using targeted exome sequencing of PKD1 and PKD2 genes, we identified a novel heterozygous frameshift mutation c.3976_3977insCT (p.F1326Sfs*21) in the PKD1 gene that segregated between affected and unaffected family members. This mutation is currently not present in the 1000 Genomes Project nor ExAC databases and is therefore a novel PKD1 mutation involved in ADPKD. These results provide a novel sequence variant for the genetic analysis of this disease.

Genetic factors contributing to human primary ciliary dyskinesia and male infertility.

Primary ciliary dyskinesia (PCD) is an autosomal-recessive disorder resulting from the loss of normal ciliary function. Symptoms include neonatal respiratory distress, chronic sinusitis, bronchiectasis, situs inversus, and infertility. However, only 15 PCD-associated genes have been identified to cause male infertility to date. Owing to the genetic heterogeneity of PCD, comprehensive molecular genetic testing is not considered the standard of care. Here, we provide an update of the progress on the identification of genetic factors related to PCD associated with male infertility, summarizing the underlying molecular mechanisms, and discuss the clinical implications of these findings. Further research in this field will impact the diagnostic strategy for male infertility, enabling clinicians to provide patients with informed genetic counseling, and help to adopt the best course of treatment for developing directly targeted personalized medicine.

A Case of Bilateral Testicular Tumors Subsequently Diagnosed as Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency.

21-hydroxylase deficiency (21-OHD) caused congenital adrenal hyperplasia (CAH) is a group of autosomal recessive genetic disorders resulting from mutations in genes involved with cortisol (CO) synthesis in the adrenal glands. Testicular adrenal rest tumors (TARTs) are rarely the presenting symptoms of CAH. Here, we describe a case of simple virilizing CAH with TARTs, in a 15-year-old boy. The patient showed physical signs of precocious puberty. The levels of blood adrenocorticotropic hormone (ACTH), urinary 17-ketone steroids (17-KS), dehydroepiandrosterone sulfate (DHEA-S), and serum progesterone (PRGE) were elevated, whereas those of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and CO were reduced. Computed tomography (CT) of the adrenal glands and magnetic resonance imaging (MRI) of the testes showed a soft tissue density (more pronounced on the right side) and an irregularly swollen mass (more pronounced on the left side), respectively. Pathological examination of a specimen of the mass indicated polygonal/circular eosinophilic cytoplasm, cord-like arrangement of interstitial cells, and lipid pigment in the cytoplasm. Immunohistochemistry results precluded a diagnosis of Leydig cell tumors. DNA sequencing revealed a hackneyed homozygous mutation, I2g, on intron 2 of the CYP21A2 gene. The patient's symptoms improved after a three-month of dexamethasone therapy. Recent radiographic data showed reduced hyperplastic adrenal nodules and testicular tumors. A diagnosis of TART should be considered and prioritized in CAH patients with testicular tumors. Replacement therapy using a sufficient amount of dexamethasone in this case helps combat TART.