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PurposeThis study aimed to identify the genetic causes of male infertility and primary ciliary dyskinesia (PCD)/PCD-like phenotypes in three unrelated Han Chinese families.MethodsWe conducted whole-exome sequencing of three patients with male infertility and PCD/PCD-like phenotypes from three unrelated Chinese families. Ultrastructural and immunostaining analyses of patient spermatozoa and respiratory cilia and in vitro analyses were performed to analyze the effects of SPEF2 variants. Intracytoplasmic sperm injection (ICSI) was administered to three affected patients.ResultsWe identified four novel SPEF2 variants, including one novel homozygous splicing site variant [NC_000005.10(NM_024867.4): c.4447 + 1G > A] of the SPEF2 gene in family 1, novel compound heterozygous nonsense variants [NC_000005.10(NM_024867.4): c.1339C > T (p.R447*) and NC_000005.10(NM_024867.4): c.1645G > T (p.E549*)] in family 2, and one novel homozygous missense variant [NC_000005.10(NM_024867.4): c.2524G > A (p.D842N)] in family 3. All the patients presented with male infertility and PCD/likely PCD. All variants were present at very low levels in public databases, predicted to be deleterious in silico prediction tools, and were further confirmed deleterious by in vitro analyses. Ultrastructural analyses of the spermatozoa of the patients revealed the absence of the central pair complex in the sperm flagella. Immunostaining of the spermatozoa and respiratory cilia of the patients validated the pathogenicity of the SPEF2 variants. All patients carrying SPEF2 variants underwent one ICSI cycle and delivered healthy infants.ConclusionOur study reported four novel pathogenic variants of SPEF2 in three male patients with infertility and PCD/PCD-like phenotypes, which not only extend the spectrum of SPEF2 mutations but also provide information for genetic counseling and treatment of such conditions.

Primary ciliary dyskinesia (PCD) is a rare hereditary orphan condition that results in variable phenotypes, including infertility. About 50 gene variants are reported in the scientific literature to cause PCD, and among them, dynein axonemal assembly factor 4 (DNAAF4) has been recently reported. DNAAF4 has been implicated in the preassembly of a multiunit dynein protein essential for the normal function of locomotory cilia as well as flagella. In the current study, a single patient belonging to a Chinese family was recruited, having been diagnosed with PCD and asthenoteratozoospermia. The affected individual was a 32-year-old male from a nonconsanguineous family. He also had abnormal spine structure and spinal cord bends at angles diagnosed with scoliosis. Medical reports, laboratory results, and imaging data were investigated. Whole-exome sequencing, Sanger sequencing, immunofluorescence analysis, hematoxylin-eosin staining, and in silico functional analysis, including protein modeling and docking studies, were used. The results identified DNAAF4 disease-related variants and confirmed their pathogenicity. Genetic analysis through whole-exome sequencing identified two pathogenic biallelic variants in the affected individual. The identified variants were a hemizygous splice site c.784-1G>A and heterozygous 20.1 Kb deletion at the DNAAF4 locus, resulting in a truncated and functionless DNAAF4 protein. Immunofluorescence analysis indicated that the inner dynein arm was not present in the sperm flagellum, and sperm morphological analysis revealed small sperm with twisted and curved flagella or lacking flagella. The current study found novel biallelic variants causing PCD and asthenoteratozoospermia, extending the range of DNAAF4 pathogenic variants in PCD and associated with the etiology of asthenoteratozoospermia. These findings will improve our understanding of the etiology of PCD.

Primary ciliary dyskinesia is a rare autosomal recessive disorder that causes oto-sino-pulmonary disease. We report a case of pulmonary infection related to mimivirus in a 10-year-old boy with primary ciliary dyskinesia that was identified using molecular techniques. Our findings indicate that the lineage C of mimivirus may cause pneumonia in humans.

PurposePrimary ciliary dyskinesia (PCD), which commonly causes male infertility, is an inherited autosomal recessive disorder. This study aimed to investigate the clinical manifestations and screen mutations associated with the dynein axonemal assembly factor 2 (DNAAF2) gene in a Han Chinese family with PCD.MethodsA three-generation family with PCD was recruited in this study. Eight family members underwent comprehensive medical examinations. Genomic DNA was extracted from the participants’ peripheral blood, and targeted next-generation sequencing technology was used to perform the mutation screening. The DNAAF2 expression was analyzed by immunostaining and Western blot.ResultsThe proband exhibited the typical clinical features of PCD. Spermatozoa from the proband showed complete immotility but relatively high viability. Two novel compound heterozygous mutations in the DNAAF2 gene, c.C156A [p.Y52X] and c.C26A [p.S9X], were identified. Both nonsense mutations were detected in the proband, whereas the other unaffected family members carried either none or only one of the two mutations. The two nonsense heterozygous mutations were not detected in the 600 ethnically matched normal controls or in the Genome Aggregation Database. The defect of the DNAAF2 and the outer dynein arms and inner dynein arms were notably observed in the spermatozoa from the proband by immunostaining.ConclusionThis study identified two novel compound heterozygous mutations of DNAAF2 leading to male infertility as a result of PCD in a Han Chinese family. The findings may enhance the understanding of the pathogenesis of PCD and improve reproductive genetic counseling in China.

Primary ciliary dyskinesia (PCD) is a rare inherited ciliopathy in which respiratory cilia are stationary or dyskinetic. The clinical presentation of PCD is highly non-specific since it includes infections and disorders of the upper (otitis and rhinosinusitis) and lower (neonatal respiratory distress, bronchitis, pneumonia and bronchiectasis) airways, starting in early life. Clinical examination alone does not allow a PCD diagnosis, which relies on several concordant tests, since none are sensitive or specific enough alone. Despite being the most sensitive and specific test to diagnose PCD, digital high-speed videomicroscopy (DHSV) is not sufficiently standardized, preventing its use with complete confidence as a confirmatory diagnostic test for PCD, or its inclusion in a diagnostic algorithm. Since the 2017 ERS recommendations for PCD diagnosis, three main issues remain to be solved in order to optimize DHSV ciliary beating evaluation: the problem in defining an accurate sensitivity and specificity as there is no gold standard method to diagnose all PCD cases, a lack of standardization in the operating procedure for processing respiratory samples, and in the choice of measured parameters (self-operating or not). The development of new automated analysis approaches is promising and will require full clinical validation.

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.

Background Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder characterized by dysfunction of motile cilia. While approximately 50 genes have been identified, around 25% of PCD patients remain genetically unexplained; elucidating the pathogenicity of specific variants remains a challenge. Methods Whole exome sequencing (WES) and Sanger sequencing were conducted to identify potential pathogenic variants of PCD. Minigene assays were performed to evaluate the pathogenicity of variants. Transmission electron microscopy (TEM) and high‐speed video analysis (HSVA) were conducted to analyze the function of cilia in respiratory epithelial cells. Results We identified two variants of DNAAF3: c.557G>A, p.G186E in exon 5, and c.1364G>A, p.G455D at the terminal nucleotide of exon 10 in a 16‐year‐old male patient. Through a minigene assay, we demonstrated that the c.1364G>A variant led to a four‐nucleotide skipping. The cilia in epithelial ciliary cells of the proband were almost immotile. The absence of outer dynein arms and inner dynein arms was also observed. Conclusions Our study identified two compound heterozygous variants of DNAAF3, a pathogenic gene for PCD, and proved that a novel missense variant c.1364G>A affects splicing. Our findings not only expanded the spectrum of mutations in the DNAAF3 gene but also highlighted the importance of investigating variants of uncertain significance (VUS) for comprehensive genetic diagnoses. Our study identified two compound heterozygous variants of DNAAF3, a pathogenic gene for PCD, and proved that a novel missense variant c.1364G>A affects splicing. Through a minigene assay, we demonstrated that the c.1364G>A variant led to a four‐nucleotide skipping. Our findings not only expanded the spectrum of mutations in the DNAAF3 gene but also highlighted the importance of investigating variants of uncertain significance (VUS) for comprehensive genetic diagnoses.

Introduction: Primary ciliary dyskinesia (PCD) is a rare disease classically transmitted as an autosomal recessive trait and characterised by recurrent airway infections due to abnormal ciliary structure and function. To date, only two autosomal genes, DNAI1 and DNAH5 encoding axonemal dynein chains, have been shown to cause PCD with defective outer dynein arms. Here, we investigated one non-consanguineous family in which a woman with retinitis pigmentosa (RP) gave birth to two boys with a complex phenotype combining PCD, discovered in early childhood and characterised by partial dynein arm defects, and RP that occurred secondarily. The family history prompted us to search for an X linked gene that could account for both conditions. Results: We found perfect segregation of the disease phenotype with RP3 associated markers (Xp21.1). Analysis of the retinitis pigmentosa GTPase regulator gene (RPGR) located at this locus revealed a mutation (631_IVS6+9del) in the two boys and their mother. As shown by study of RPGR transcripts expressed in nasal epithelial cells, this intragenic deletion, which leads to activation of a cryptic donor splice site, predicts a severely truncated protein. Conclusion: These data provide the first clear demonstration of X linked transmission of PCD. This unusual mode of inheritance of PCD in patients with particular phenotypic features (that is, partial dynein arm defects and association with RP), which should modify the current management of families affected by PCD or RP, unveils the importance of RPGR in the proper development of both respiratory ciliary structures and connecting cilia of photoreceptors.

Background: In primary ciliary dyskinesia (PCD) there is no single diagnostic test. Different predictive tools have been proposed to guide referral of high-risk patients for further diagnostic workup. We aimed to test clinical index (CI) on a large unselected cohort and compare its characteristics with other widely used tools—PICADAR and NA-CDCF. Methods: CI, PICADAR, and NA-CDCF scores were calculated in 1401 patients with suspected PCD referred to our center. Their predictive characteristics were analyzed using receiver operating characteristics (ROC) curves and compared to each other. Nasal nitric oxide (nNO) was measured in 569 patients older than 3 years. Results: PCD was diagnosed in 67 (4.8%) patients. CI, PICADAR, and NA-CDCF scores were higher in PCD than in nonPCD group (all p < 0.001). The area under the ROC curve (AUC) for CI was larger than for NA-CDCF (p = 0.005); AUCPICADAR and AUCNA-CDCF did not differ (p = 0.093). An overlap in signs and symptoms among tools was identified. PICADAR could not be assessed in 86 (6.1%) patients without chronic wet cough. For CI laterality or congenital heart defects assessment was not necessary. nNO further improved predictive power of all three tools. Conclusion: CI is a feasible predictive tool for PCD that may outperform PICADAR and NA-CFCD.

The core axoneme structure of both the motile cilium and sperm tail has the same ultrastructural 9 + 2 microtubular arrangement. Thus, it can be expected that genetic defects in motile cilia also have an effect on sperm tail formation. However, recent studies in human patients, animal models and model organisms have indicated that there are differences in components of specific structures within the cilia and sperm tail axonemes. Primary ciliary dyskinesia (PCD) is a genetic disease with symptoms caused by malfunction of motile cilia such as chronic nasal discharge, ear, nose and chest infections and pulmonary disease (bronchiectasis). Half of the patients also have situs inversus and in many cases male infertility has been reported. PCD genes have a role in motile cilia biogenesis, structure and function. To date mutations in over 40 genes have been identified cause PCD, but the exact effect of these mutations on spermatogenesis is poorly understood. Furthermore, mutations in several additional axonemal genes have recently been identified to cause a sperm-specific phenotype, termed multiple morphological abnormalities of the sperm flagella (MMAF). In this review, we discuss the association of PCD genes and other axonemal genes with male infertility, drawing particular attention to possible differences between their functions in motile cilia and sperm tails.