Biallelic mutations in Sperm flagellum 2 cause human multiple morphological abnormalities of the sperm flagella (MMAF) phenotype

Male patients with multiple morphological abnormalities of the sperm flagella (MMAF) are infertile and exhibit absent, short, coiled, bent and/or irregular sperm flagella. Mutations in the SPEF2 gene reduce sperm motility and cause sperm tail defects in animal models and humans. In the present study, we performed a genetic analysis on an MMAF patient and identified novel biallelic mutations in the SPEF2 gene. The biallelic mutations were confirmed by Sanger sequencing and in silico analysis revealed that, these variations were deleterious. The expression of truncated SPEF2 protein was reduced significantly in the patient's spermatozoa. The spermatozoa harbored biallelic mutations and showed severe ultrastructural defects in the axoneme and mitochondrial sheath. Our data suggest that biallelic mutations in SPEF2 can cause severe sperm flagellum defects, thus providing a novel candidate genetic pathogen for the human MMAF phenotype.     

Loss-of-function mutation in DNAH8 induces asthenoteratospermia associated with multiple morphological abnormalities of the sperm flagella

Asthenozoospermia is a common cause of male infertility associated with the reduced motility and/or abnormal morphology of spermatozoa, although its etiology remains incompletely understood. Multiple morphological abnormalities of the sperm flagella (MMAF) is one of the main causes of asthenozoospermia. However, the MMAF-associated genes identified to date cannot explain all the human MMAF cases. Herein, a loss-of-function mutation of DNAH8 was identified in an asthenozoospermia patient with MMAF. Moreover, the negative effect of this mutation on DNAH8 expression was confirmed by immunofluorescence staining and western blotting. Remarkably, it is the first time that DNAH8 is suggested to be associated with human MMAF. Our findings provide strong evidence that a loss-of-function mutation in DNAH8 can cause male infertility with MMAF and that DNAH8 is essential for sperm flagellar formation.     

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.     

A novel homozygous CFAP65 mutation in humans causes male infertility with multiple morphological abnormalities of the sperm flagella

Multiple morphological abnormalities of the sperm flagella (MMAF) is a rare autosomal recessive inherited disorder associated with male infertility. To date, 14 genetic causative genes have been identified in MMAF, which can only explain the genetic causes of ~60% of MMAF cases. Here, we report a man with primary infertility, who had a typical MMAF phenotype. Whole-exome sequencing (WES) was performed on the patient and a homozygous mutation (c. 2675 G>A [p. Trp892*]) was identified in cilia and flagella-associated protein 65 (CFAP65) gene, which is primarily expressed in the testis. Another loss-of-function mutation of CFAP65 has been detected in a MMAF patient, and the orthologue of CFAP65 also plays a vital role in sperm motility in chickens. Our experimental observations on human subjects suggested that CFAP65 is involved in sperm flagellum structure and assembly and that loss-of-function mutations could lead to male infertility in humans by causing the MMAF phenotype.     

Whole exome sequencing of men with multiple morphological abnormalities of the sperm flagella reveals novel homozygous QRICH2 mutations

Multiple morphological anomalies of the sperm flagella (MMAF syndrome) is a severe male infertility phenotype which has so far been formally linked to the presence of biallelic mutations in nine genes mainly coding for axonemal proteins overexpressed in the sperm flagellum. Homozygous mutations in QRICH2, a gene coding for a protein known to be required for stabilizing proteins involved in sperm flagellum biogenesis, have recently been identified in MMAF patients from two Chinese consanguineous families. Here, in order to better assess the contribution of QRICH2 in the etiology of the MMAF phenotype, we analyzed all QRICH2 variants from whole exome sequencing data of a cohort of 167 MMAF-affected subjects originating from North Africa, Iran, and Europe. We identified a total of 14 potentially deleterious variants in 18 unrelated individuals. Two unrelated subjects, representing 1% of the cohort, carried a homozygous loss-of-function variant: c.3501C>G [p.Tyr1167Ter] and c.4614C>G [p.Tyr1538Ter], thus confirming the implication of QRICH2 in the MMAF phenotype and human male infertility. Sixteen MMAF patients (9.6%) carried a heterozygous QRICH2 potentially deleterious variant. This rate was comparable to what was observed in a control group (15.5%) suggesting that the presence of QRICH2 heterozygous variants is not associated with MMAF syndrome.     

Loss-of-function mutations in centrosomal protein 112 is associated with human acephalic spermatozoa phenotype

Acephalic spermatozoa, characterized by the headless sperm in the ejaculate, is a rare type of teratozoospermia. Here, we recruited two infertile patients with an acephalic spermatozoa phenotype to investigate the genetic pathology of acephalic spermatozoa. Whole-exome sequencing analysis was performed and found mutations in CEP112 in the two patients: homozygous mutation c.496C > T:p.(Arg166X) in exon 5 from P1; and the biallelic mutations c.2074C > T:p.(Arg692Trp) in exon 20 and c.2104C > T:p.(Arg702Cys) in exon 20 from P2. Sanger sequencing confirmed the CEP112 mutations in the two patients. In silico analysis revealed that these CEP112 mutations are deleterious and rare, and all the mutations impact the coiled-coil domain of CEP112, which may affect the protein function. The c.496C > T:p.Arg166X resulted in a truncated CEP112, which was verified by the mutation expression plasmid. The CEP112 expression was significantly reduced in the P2, suggesting the biallelic mutations c.2074C > T and c.2104C > T may affect the function and stability of CEP112. Therefore, we speculate that the loss-of-function mutations in CEP112 may be account for the human acephalic spermatozoa phenotype.     

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.     

A novel mutation in ALS2 associated with severe and progressive infantile onset of spastic paralysis

Infantile onset ascending spastic paralysis (IAHSP) is a type of recessively inherited spastic paraplegia. We investigated the clinical and genetic cause of a recessively inherited disorder in two siblings manifesting severe spasticity in the lower limbs which hindered their gait. A novel homozygous nonsense mutation c.1918 C?>?T (p.Arg640*) was identified after whole-exome sequencing within ALS2 in the DNA of both patients. The obligate carriers were heterozygous for the mutation and other unaffected members were homozygous for the wild type allele. The variant was absent from 100 control chromosomes and all public databases. This report extends the allelic heterogeneity of ALS2 mutations and emphasizes the importance of genetic testing for diagnosis of pediatric disorders.     

Three cases of genetic defects affecting sperm tail: a FISH study

Submicroscopic alterations in the cytoskeletal structure of sperm flagellum are associated with severely reduced or completely absent motility in subfertile or infertile men. Sometimes these alterations can be related to well known genotypic defects when the same anomaly affects the whole sperm population. Transmission electron microscopy (TEM) is the only tool able to specifically characterize the morphological features of genetic sperm defects. In this study, the frequencies of aneuploid and diploid spermatozoa were identified in three patients showing specific flagellar anomalies, each of them affecting the whole sperm population: dysplasia of the fibrous sheath, primary ciliary dyskinesia and absence of fibrous sheath. All these defects were highlighted by TEM. Fluorescence in situ hybridization (FISH) analysis was performed on decondensed sperm nuclei for chromosomes 18, X and Y, highlighting higher diploidies and sex chromosome disomies in cases of dysplasia of the fibrous sheath and primary ciliary dyskinesia, in agreement with other reports. We have also described FISH results in spermatozoa with absence of fibrous sheath. In this case, the only one reported due to the rarity of this defect, the aneuploidies and diploidies were within normal range. These data contribute to the growing evidence that genetic sperm defects of sperm flagella are generally correlated with meiotic segregation derangement. For this reason, genetic counseling is advisable, although all the genes involved and the possible mechanisms of these mutations have not yet been fully characterized.     

CCDC65, encoding a component of the axonemal Nexin-Dynein regulatory complex,is required for sperm flagellum structure in humans

Sperm flagella share an evolutionary conserved microtubule-based structure with motile cilia expressed at the surface of several cell types, such as the airways epithelial cells. As a result, male infertility can be observed as an isolated condition or a syndromic trait, illustrated by Primary Cilia Dyskinesia (PCD). We report two unrelated patients showing multiple morphological abnormalities of the sperm flagella (MMAF) and carrying distinct homozygous truncating variants in the PCD-associated gene CCDC65. We characterized one of the identified variants (c.1208del; p.Asn403Ilefs*9), which induces the near absence of CCDC65 protein in patient sperm. In Chlamydomonas, CCDC65 ortholog (DRC2, FAP250) is a component of the Nexin-Dynein Regulatory complex (N-DRC), which interconnects microtubule doublets and coordinates dynein arms activity. In sperm cells from the patient, we also show the loss of GAS8, another component of the N-DRC, supporting a structural/functional link between the two proteins. Our work indicates that, similarly to ciliary axoneme, CCDC65 is required for sperm flagellum structure. Importantly, our work provides first evidence that mutations in the PCD-associated gene CCDC65 also cause asthenozoospermia.     

Autosomal dominant polycystic kidney disease caused by somatic and germline mosaicism

Autosomal dominant polycystic kidney disease (ADPKD) is a heterogeneous genetic disorder caused by loss of function mutations of PKD1 or PKD2 genes. Although PKD1 is highly polymorphic and the new mutation rate is relatively high, the role of mosaicism is incompletely defined. Herein, we describe the molecular analysis of ADPKD in a 19‐year‐old female proband and her father. The proband had a PKD1 truncation mutation c.10745dupC (p.Val3584ArgfsX43), which was absent in paternal peripheral blood lymphocytes (PBL). However, very low quantities of this mutation were detected in the father's sperm DNA, but not in DNA from his buccal cells or urine sediment. Next generation sequencing (NGS) analysis determined the level of this mutation in the father's PBL, buccal cells and sperm to be ～3%, 4.5% and 10%, respectively, consistent with somatic and germline mosaicism. The PKD1 mutation in ～10% of her father's sperm indicates that it probably occurred early in embryogenesis. In ADPKD cases where a de novo mutation is suspected because of negative PKD gene testing of PBL, additional evaluation with more sensitive methods (e.g. NGS) of the proband PBL and paternal sperm can enhance detection of mosaicism and facilitate genetic counseling.     

Value of transmission electron microscopy for primary ciliary dyskinesia diagnosis in the era of molecular medicine: Genetic defects with normal and non-diagnostic ciliary ultrastructure

ABSTRACT

Primary ciliary dyskinesia (PCD) is a genetic disorder causing chronic oto-sino-pulmonary disease. No single diagnostic test will detect all PCD cases. Transmission electron microscopy (TEM) of respiratory cilia was previously considered the gold standard diagnostic test for PCD, but 30% of all PCD cases have either normal ciliary ultrastructure or subtle changes which are non-diagnostic. These cases are identified through alternate diagnostic tests, including nasal nitric oxide measurement, high-speed videomicroscopy analysis, immunofluorescent staining of axonemal proteins, and/or mutation analysis of various PCD causing genes. Autosomal recessive mutations in DNAH11 and HYDIN produce normal TEM ciliary ultrastructure, while mutations in genes encoding for radial spoke head proteins result in some cross-sections with non-diagnostic alterations in the central apparatus interspersed with normal ciliary cross-sections. Mutations in nexin link and dynein regulatory complex genes lead to a collection of different ciliary ultrastructures; mutations in CCDC65, CCDC164, and GAS8 produce normal ciliary ultrastructure, while mutations in CCDC39 and CCDC40 cause absent inner dynein arms and microtubule disorganization in some ciliary cross-sections. Mutations in CCNO and MCIDAS cause near complete absence of respiratory cilia due to defects in generation of multiple cellular basal bodies; however, the scant cilia generated may have normal ultrastructure. Lastly, a syndromic form of PCD with retinal degeneration results in normal ciliary ultrastructure through mutations in the RPGR gene. Clinicians must be aware of these genetic causes of PCD resulting in non-diagnostic TEM ciliary ultrastructure and refrain from using TEM of respiratory cilia as a test to rule out PCD.     

Mutation in The Nuclear‐Encoded Mitochondrial Isoleucyl–tRNA Synthetase IARS2 in Patients with Cataracts,Growth Hormone Deficiency with Short Stature,Partial Sensorineural Deafness,and Peripheral Neuropathy or with Leigh Syndrome

Mutations in the nuclear‐encoded mitochondrial aminoacyl–tRNA synthetases are associated with a range of clinical phenotypes. Here, we report a novel disorder in three adult patients with a phenotype including cataracts, short‐stature secondary to growth hormone deficiency, sensorineural hearing deficit, peripheral sensory neuropathy, and skeletal dysplasia. Using SNP genotyping and whole‐exome sequencing, we identified a single likely causal variant, a missense mutation in a conserved residue of the nuclear gene IARS2, encoding mitochondrial isoleucyl–tRNA synthetase. The mutation is homozygous in the affected patients, heterozygous in carriers, and absent in control chromosomes. IARS2 protein level was reduced in skin cells cultured from one of the patients, consistent with a pathogenic effect of the mutation. Compound heterozygous mutations in IARS2 were independently identified in a previously unreported patient with a more severe mitochondrial phenotype diagnosed as Leigh syndrome. This is the first report of clinical findings associated with IARS2 mutations.     

Systematic genetic study of Alzheimer disease in Latin America: Mutation frequencies of the amyloid β precursor protein and presenilin genes in Colombia*

Nearly all mutations in the presenilin 1 (PSEN1), presenilin 2 (PSEN2), and amyloid β precursor protein (APP) genes lead to early‐onset Alzheimer disease (EOAD, onset age at or before 65 years). In order to assess the genetic contribution of these genes in a series of Colombian AD cases, we performed a systematic mutation analysis in 11 autosomal dominant, 23 familial, and 42 sporadic AD patients (34% with age of onset ≤ 65 years). No APP missense mutations were identified. In three autosomal dominant cases (27.2%), two different PSEN1 missense mutations were identified. Both PSEN1 mutations are missense mutations that occurred in early‐onset autosomal AD cases: an I143T mutation in one case (onset age 30 years) and an E280A mutation in two other cases (onset ages 35 and 42 years). In addition, a novel PSEN1 V94M mutation was present in one early‐onset AD case without known family history (onset age 53 years) and absent in 53 controls. The E318G polymorphism was present in five AD cases and absent in controls. In PSEN2, two different silent mutations were detected, including one not reported elsewhere (P129). The majority of the Colombian AD cases, predominantly late‐onset, were negative for PSEN and APP mutations. © 2001 Wiley‐Liss, Inc.     

A novel mutation combining with rs66612022 in a Chinese pedigree suggests a new pathogenesis to osteogenesis imperfecta via whole genome sequencing

Osteogenesis imperfecta (OI) is a rare heritable disease with systemic connective tissue disorder. Most of the patients represent autosomal dominant form of OI, and are usually resulting from the mutations in type I collagen genes. However, the gene mutations reported previously only account for ∼70% of the OI cases. Here, in a Chinese OI family, we examined seven patients and nine normal individuals using the whole genome sequencing and molecular genetic analysis. The mutation of rs66612022 (COL1A2:p.Gly328Ser) related to glycine substitution was found in the seven patients. Moreover, we identified a novel missense mutation (HMMR:p.Glu2Gln). Interestingly, the individuals of this family with both the mutations were suffering from OI, while the others carried one or none of them are normal. The mutations of COL1A2 and HMMR and their combined effect on OI would further expand the genetic spectrum of OI.     

Differential diagnosis of vacuolar myopathies in the NGS era

Altered autophagy accompanied by abnormal autophagic (rimmed) vacuoles detectable by light and electron microscopy is a common denominator of many familial and sporadic non‐inflammatory muscle diseases. Even in the era of next generation sequencing (NGS), late‐onset vacuolar myopathies remain a diagnostic challenge. We identified 32 adult vacuolar myopathy patients from 30 unrelated families, studied their clinical, histopathological and ultrastructural characteristics and performed genetic testing in index patients and relatives using Sanger sequencing and NGS including whole exome sequencing (WES). We established a molecular genetic diagnosis in 17 patients. Pathogenic mutations were found in genes typically linked to vacuolar myopathy (GNE, LDB3/ZASP, MYOT, DES and GAA), but also in genes not regularly associated with severely altered autophagy (FKRP, DYSF, CAV3, COL6A2, GYG1 and TRIM32) and in the digenic facioscapulohumeral muscular dystrophy 2. Characteristic histopathological features including distinct patterns of myofibrillar disarray and evidence of exocytosis proved to be helpful to distinguish causes of vacuolar myopathies. Biopsy validated the pathogenicity of the novel mutations p.(Phe55*) and p.(Arg216*) in GYG1 and of the p.(Leu156Pro) TRIM32 mutation combined with compound heterozygous deletion of exon 2 of TRIM32 and expanded the phenotype of Ala93Thr‐caveolinopathy and of limb‐girdle muscular dystrophy 2i caused by FKRP mutation. In 15 patients no causal variants were detected by Sanger sequencing and NGS panel analysis. In 12 of these cases, WES was performed, but did not yield any definite mutation or likely candidate gene. In one of these patients with a family history of muscle weakness, the vacuolar myopathy was eventually linked to chloroquine therapy. Our study illustrates the wide phenotypic and genotypic heterogeneity of vacuolar myopathies and validates the role of histopathology in assessing the pathogenicity of novel mutations detected by NGS. In a sizable portion of vacuolar myopathy cases, it remains to be shown whether the cause is hereditary or degenerative.     

Novel homozygous ENPP1 mutation causes generalized arterial calcifications of infancy,thrombocytopenia, and cardiovascular and central nervous system syndrome

Generalized arterial calcifications of infancy (GACI) is caused by mutations in ENPP1. Other ENPP1‐related phenotypes include pseudoxanthoma elasticum, hypophosphatemic rickets, and Cole disease. We studied four children from two Bedouin consanguineous families who presented with severe clinical phenotype including thrombocytopenia, hypoglycemia, hepatic, and neurologic manifestations. Initial working diagnosis included congenital infection; however, patients remained without a definitive diagnosis despite extensive workup. Consequently, we investigated a potential genetic etiology. Whole exome sequencing (WES) was performed for affected children and their parents. Following the identification of a novel mutation in the ENPP1 gene, we characterized this novel multisystemic presentation and revised relevant imaging studies. Using WES, we identified a novel homozygous mutation (c.556G > C; p.Gly186Arg) in ENPP1 which affects a highly conserved protein domain (somatomedin B2). ENPP1‐associated genetic diseases exhibit phenotypic heterogeneity depending on mutation type and location. Follow‐up clinical characterization of these families allowed us to revise and detect new features of systemic calcifications, which established the diagnosis of GACI, expanding the phenotypic spectrum associated with ENPP1 mutations. Our findings demonstrate that this novel ENPP1 founder mutation can cause a fatal multisystemic phenotype, mimicking severe congenital infection. This also represents the first reported mutation affecting the SMB2 domain, associated with GACI.     

Exome Sequencing Identifies a Novel Nonsense Mutation of MYO6 as the Cause of Deafness in a Brazilian Family

We investigated 313 unrelated subjects who presented with hearing loss to identify the novel genetic causes of this condition in Brazil. Causative GJB2/GJB6 mutations were found in 12.7% of the patients. Among the familial cases (100/313), four were selected for exome sequencing. In one case, two novel heterozygous variants were found and were predicted to be pathogenic based on bioinformatics tools, that is, p.Ser906* (MYO6) and p.Arg42Cys (GJB3). We confirmed that this nonsense MYO6 mutation segregated with deafness in this family. Only the proband and her unaffected mother exhibited the GJB3 mutation, which is in the same amino acid of a known Erythrokeratodermia variabilis mutation. None of the patients exhibited this skin disease, but the proband exhibited a more severe hearing loss. Hence, the GJB3 mutation was considered to be a variant of uncertain significance. In conclusion, we described a novel nonsense MYO6 mutation that was responsible for the hearing loss in a Brazilian family. This mutation resides in the neck domain of myosin‐VI after the motor domain. Thus, our data give further support for genotype‐phenotype correlations, which state that when the motor domain of the protein is functioning, the hearing loss is milder and has a later onset. The three remaining families without mutations in the known genes suggest that there are still deafness genes to be revealed.