STUDY QUESTION:Are pathogenic variants in homeodomain-interacting protein kinase (HIPK4) associated with sperm head abnormalities that cause male infertility? SUMMARY ANSWER:HIPK4 is a novel candidate gene associated with sperm head defects and human male infertility. WHAT IS KNOWN ALREADY:Numerous genes have been described in which pathogenic variants cause male infertility due to multiple morphological abnormalities of the sperm flagella (MMAF), but the genetic basis of sperm head defects is less well understood. STUDY DESIGN SIZE DURATION:This study included four infertile brothers displaying varying degrees of quantitatively and/or qualitatively impaired spermatogenesis, their parents, and their fertile brother. We also queried the Male Reproductive Genomics (MERGE) cohort comprising exome/genome sequencing data of >3300 men. PARTICIPANTS/MATERIALS SETTING METHODS:We performed exome sequencing in all five brothers and their parents. To characterize sperm phenotypes, we carried out standard semen analysis, immunofluorescence staining, and transmission electron microscopy (TEM). Further, we evaluated the impact of the HIPK4 variant in cell culture experiments using HEK293T cells. MAIN RESULTS AND THE ROLE OF CHANCE:By analysing the exome data, we could not identify a common genetic cause in all four affected brothers. However, one of the affected brothers was compound heterozygous for two loss-of-function variants in DNAH17 (c.1076_1077dup p.(Lys360*) and c.7752+2T>A p.?), associated with markedly reduced sperm motility and MMAF. The variants' pathogenicity was further validated by TEM of flagellar cross sections revealing an outer dynein arm defect and axonemal disruption. In contrast, his three infertile brothers were homozygous for the start-loss variant c.1A>G in HIPK4. This gene is expressed during spermiogenesis and is reportedly involved in sperm head shaping in mice. Heterologous expression of (partial) HIPK4 variant cDNA showed that translation was being initiated at an alternative in-frame start codon located 35 amino acids downstream, resulting in an N-terminally truncated protein p.(Met1_Glu35del). The truncated HIPK4 protein lacks parts of its kinase domain and shows reduced protein stability. Corresponding with published mouse models, all three brothers displayed 100% abnormal sperm head morphology with variable defects. Importantly, one brother affected by HIPK4 variants fathered a child after successful ICSI, demonstrating a successful treatment option for HIPK4-related teratozoospermia. No further men from the MERGE cohort were affected by biallelic HIPK4 variants. Taken together, HIPK4 is an autosomal recessive candidate gene in which pathogenic variants are associated with sperm head defects and male infertility. LARGE-SCALE DATA:The reported variants in DNAH17 and HIPK4 have been published in ClinVar. LIMITATIONS REASONS FOR CAUTION:Independent replication is required to assess the phenotypic spectrum and the reproductive outcome associated with biallelic HIPK4 variants and to formally establish the gene-disease relationship for male infertility. WIDER IMPLICATIONS OF THE FINDINGS:This study raises awareness of the significant genetic heterogeneity of male infertility. The described family highlights that distinct genetic causes may underlie a seemingly similar phenotype. Exome sequencing of families is helpful to efficiently disentangle individual causes among affected family members. FUNDING:N.N., J.R., H.O., S.L., C.F., and F.T. were supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) within the Clinical Research Unit 'Male Germ Cells' (CRU326, project number 329621271). R.T.-W., N.N., J.R., H.O., and F.T. were supported by the Federal Ministry of Research, Technology and Space (BMFTR) as part of the project ReproTrack.MS (grant 01GR2303). S.A.K. was supported by the DFG Clinician Scientist programme CareerS Münster (project number 493624047). A.S.G. was supported by the Medical Faculty Münster via an Innovative Medical Research (IMF) grant (GA-122104). DISCLOSURES:The authors declare no conflicts of interest.

Pathogenic variants in KIAA0586/TALPID3 are associated with the ciliopathy Joubert syndrome (JS). We report individuals with KIAA0586/TALPID3 variants affected by primary and motile cilia defects leading to JS and chronic destructive airway disease. DNA variants were detected in three families by sequencing. In two unrelated families, a deep-intronic variant ( KIAA0586/TALPID3 :c.3990 + 3186G>A) activated a cryptic exon. We performed histological and functional analyses in native and air-liquid interface (ALI) cultured respiratory cells. Primary cilia lengths were measured in patient-derived fibroblasts. Our data associate KIAA0586/TALPID3 variants with a syndrome combining JS and chronic destructive airway disease, reduced number of motile cilia, disorganized basal body location, and ciliary clearance malfunction. Additionally, patient-derived cell lines showed primary cilia defects. Disease causing KIAA0586/TALPID3 variants, including a deep-intronic sequence variant, were associated with primary and motile cilia defects in JS patients. The combination of JS and respiratory symptoms should be considered indicative for KIAA0586/TALPID3 sequence alterations.

Germ cells accurately organize their chromosomes through the program of meiosis to successfully generate haploid gametes for fertilization1. Chromosomal pairing, which is essential for homologous recombination, is controlled by the cytoskeletal machinery of perinuclear microtubules that are organized by the centrosome2–4, but the underlying biomechanical regulation has remained incompletely understood. We have recently discovered the zygotene cilium in zebrafish oocytes, which mechanically anchors the centrosomal centrioles to facilitate chromosomal pairing dynamics and is essential for female meiosis5. Here, we show that cilia also regulate chromosomal dynamics and testis morphogenesis in the zebrafish male germline. Importantly, using germ cell-specific conditional elimination of zygotene cilia in mice, we establish a previously unrecognized ciliary function in chromosomal synapsis, germ cell development and fertility in mammals. Furthermore, we document the occurrence of cilia in human meiotic germ cells by identifying ciliary structures as well as gene expression signature of ciliary differentiation through single-cell transcriptomics. Finally, we have identified high impact variants in ciliary genes in women with premature ovarian insufficiency (POI) and men with crypto-/azoospermia, indicating that zygotene cilia defects may underlie their reproductive failure. Together, our findings uncover a strikingly conserved ciliary mechanism in the regulation of chromosomal dynamics in meiosis and implicate dysfunction of the zygotene cilium as an overlooked aetiology of human infertility.

Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type– and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the TUBB4B isotype that specifically perturbed centriole and cilium biogenesis. Distinct TUBB4B variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.

Hintergrund Die Primäre Ciliäre Dyskinesie (PCD) ist eine genetisch und klinisch heterogene Erkrankung, die durch eine Dysfunktion motiler Zilien gekennzeichnet ist. Die Diagnose der PCD basiert auf verschiedenen Tests, inklusive der Messung von nasalem Stickstoffmonoxid (nNO), Hochgeschwindigkeits-Videomikroskopie (HSVM), Transmissionselektronenmikroskopie (TEM), Immunfluoreszenzmikroskopie (IF) und genetischer Analyse.

Background and objectives: Primary ciliary dyskinesia (PCD, ORPHA:244) is a group of rare genetic disorders characterized by dysfunction of motile cilia. It is phenotypically and genetically heterogeneous, with more than 50 genes involved. Thanks to genetic, clinical, and functional characterization, immense progress has been made in the understanding and diagnosis of PCD. Nevertheless, it is underdiagnosed due to the heterogeneous phenotype and complexity of diagnosis. This review aims to help clinicians navigate this heterogeneous group of diseases. Here, we describe the broad spectrum of phenotypes associated with PCD and address pitfalls and difficult-to-interpret findings to avoid misinterpretation.Method: Review of literatureConclusion: PCD diagnosis is complex and requires integration of history, clinical picture, imaging, functional and structural analysis of motile cilia and, if available, genetic analysis to make a definitive diagnosis. It is critical that we continue to expand our knowledge of this group of rare disorders to improve the identification of PCD patients and to develop evidence-based therapeutic approaches.(c) 2023 Elsevier Masson SAS. All rights reserved.

Einführung Die Primäre Ciliäre Dyskinesie (PCD) ist eine heterogene Multisystemerkrankung, bei welcher es durch eine Dysfunktion motiler Zilien u.a. zu einer destruktiven Lungenerkrankung kommt. Der diagnostische Algorithmus der American Thoracic (ATS) und der European Respiratory Society (ERS) beinhaltet die Messung von nasalem Stickstoffmonoxid (nNO), die Transmissionselektronenmikroskopie, die Evaluation des Zilienschlages und die genetische Analyse, um eine PCD zu diagnostizieren.

Hydrocephalus is a common finding in newborns. In most cases, it is caused by intraventricular hemorrhage associated with prematurity, whereas in some patients the cause of hydrocephalus can be traced back to genetic changes, associated with disease syndromes such as RASopathies, lysosomal storage diseases, dystroglycanopathies, craniosynostosis but also ciliopathies. Ciliopathies are a group of diseases that can affect multiple organ systems due to dysfunction or the absence of cilia. Cilia are small organelles, extending from the cell surface. Nonmotile monocilia are ubiquitously present during cell development fulfilling chemosensory functions, whereas specialized epithelia such as the ependyma, lining the inner surface of the brain ventricles, exhibit multiciliated cells propelling fluids along the cell surface. This review highlights ciliopathies and their pathophysiology in congenital hydrocephalus. While nonmotile ciliopathies are often associated with severe prenatal hydrocephalus combined with other severe congenital brain malformations, motile ciliopathies, especially those associated with defects in multiciliogenesis can cause hydrocephalus and chronic lung disease.

Rationale: Primary ciliary dyskinesia (PCD) is a heterogeneous, multisystem disorder characterized by defective ciliary beating. Diagnostic guidelines of the American Thoracic Society and European Respiratory Society recommend measurement of nasal nitric oxide (nNO) for PCD diagnosis. Several studies demonstrated low nNO production rates in PCD individuals, but underlying causes remain elusive. Objectives: To determine nNO production rates in a well-characterized PCD cohort, including subgroup analyses with regard to ultrastructural and ciliary beating phenotypes. Methods: This study included 301 individuals assessed according to European Respiratory Society guidelines. Diagnostic cutoffs for nNO production rates for this study cohort and subgroups with normal and abnormal ultrastructure were determined. Diagnostic accuracy was also tested for the widely used 77 nl/min cutoff in this study cohort. The relationship between nNO production rates and ciliary beat frequencies (CBFs) was evaluated. Results: The study cohort comprised 180 individuals with definite PCD diagnosis, including 160 individuals with genetic diagnosis, 16 individuals with probable PCD diagnosis, and 105 disease controls. The 77 nl/min nNO cutoff showed a test sensitivity of 0.92 and specificity of 0.86. Test sensitivity was lower (0.85) in the subgroup of 47 PCD individuals with normal ultrastructure compared with 133 PCD individuals with abnormal ultrastructure (0.95). The optimal diagnostic cutoff for the nNO production rate for the whole study cohort was 69.8 nl/min (sensitivity, 0.92; specificity, 0.89); however, it was 107.8 nl/min (sensitivity, 0.89; specificity, 0.78) for the subgroup of PCD with normal ultrastructure. PCD individuals with normal ultrastructure compared with abnormal ultrastructure showed higher ciliary motility. Consistently, PCD individuals with higher CBFs showed higher nNO production rates. In addition, laterality defects occurred less frequently in PCD with normal ultrastructure. Conclusions: Measurements of nNO below the widely used 77 nl/min cutoff are less sensitive in detecting PCD individuals with normal ultrastructure. Our findings indicate that higher nNO production in this subgroup with a higher cutoff for the nNO production rate (107.8 nl/min) and higher residual ciliary motility is dependent on the underlying molecular PCD defect. Higher nNO production rates, higher residual CBFs, and the lower prevalence of laterality defects hamper diagnosis of PCD with normal ultrastructure. Adjusting the cutoff of nNO production rate to 107.8 nl/min might promote diagnosing PCD with normal ultrastructure.

Introduction: Primary ciliary dyskinesia (PCD) is a heterogeneous, multisystem disorder characterized by dysfunctional motile cilia. Diagnostic approaches of the American Thoracic (ATS) and European Respiratory Society (ERS) include nasal nitric oxide (nNO) measurement, transmission electron microscopy, evaluation of ciliary beating and genetic analyses for PCD diagnosis. Methods: All included subjects were evaluated according to ERS diagnostic guidelines. PCD individuals were categorized in subgroups with normal and abnormal ciliary ultrastructure. Diagnostic cutoffs for nNO-production rates as well as ciliary beat frequencies (CBFs) and laterality status were determined. Results: The study cohort comprised 180 PCD individuals (160 with genetic diagnosis) and 105 disease controls. The optimal diagnostic cutoff for the nNO-production rate for the whole PCD cohort was 69.8 nl/min (sensitivity 0.92, specificity 0.89), while it was 107.8 nl/min (sensitivity 0.89, specificity 0.78) for the subgroup of PCD with normal ultrastructure. This subgroup also showed higher ciliary motility and less laterality defects. Conclusions: Higher nNO-production rates, higher residual ciliary motility and lower prevalence of laterality defects make diagnosis of PCD with normal ultrastructure difficult. In distinct cases, only genetic analyses lead to PCD diagnosis. A frequent use of genetic testing and adjusting the cutoff for the nNO-production rate to 107.8 nl/min will promote diagnosing PCD with normal ultrastructure. We thank the PCD-affected families and the German support group. This work was supported by grants from the “Deutsche Forschungsgemeinschaft” (DFG) (e.g. CRU326 (RA3522/1-1; OM6/11) and "IZKF" Münster.

In order to identify the underlying genetic defect in a cohort of individuals with chronic destructive airway disease and congenital brain malformation, we performed whole exome sequencing. We identified homozygous loss of function variants in seven individuals from five non-related families in TP73. TP73 encodes for the tumor protein 73. As member of the TP53 family of transcription factors, it has been well studied in cancer research. All patients harboring mutations in TP73 were suffering from a chornic airway disease and lissencephaly. Since analysis in mice had shown a multiciliogenesis defect in TP73 deficient mice, we next asked whether the respiratory phenotype was caused by a mucociliary clearance disorder. To this end we used a ciliary beat assay using an air liquid interface culture of respiratory epithelia obtained from affected individuals and healthy controls. This analyses revealed that cilia were not able to generate a sufficient fluid flow, conistent with impaired mucociliary clearance. Transmission electron microscopy and immunofluorescence analysis of respiratory epithelia before and after cell culture showed reduced ciliary length and basal bodies mislocalized in the cytoplasm. Consitent with a defect in cell differentiation/proliferation a reduced cell layer and a reduced number of ciliated cells were identified. Our data show that autosomal recessive mutations in TP73 cause a motile ciliopathy due to a defect in multiciliated cell differentiation. References: Wallmeier J, Bracht D, Alsaif HS, Dougherty GW, Olbrich H, Cindric S, Dzietko M, Heyer C, Teig N, Thiels C, et al. Mutations in TP73 cause impaired mucociliary clearance and lissencephaly. Am J Hum Genet 2021;1–12

Airway mucociliary regeneration and function are key players for airway defense and are impaired in chronic obstructive pulmonary disease (COPD). Using transcriptome analysis in COPD-derived bronchial biopsies, we observed a positive correlation between cilia-related genes and microRNA-449 (miR449). In vitro, miR449 was strongly increased during airway epithelial mucociliary differentiation. In vivo, miR449 was upregulated during recovery from chemical or infective insults. miR0449−/− mice (both alleles are deleted) showed impaired ciliated epithelial regeneration after naphthalene and Haemophilus influenzae exposure, accompanied by more intense inflammation and emphysematous manifestations of COPD. The latter occurred spontaneously in aged miR449−/− mice. We identified Aurora kinase A and its effector target HDAC6 as key mediators in miR449-regulated ciliary homeostasis and epithelial regeneration. Aurora kinase A is downregulated upon miR449 overexpression in vitro and upregulated in miR449−/− mouse lungs. Accordingly, imaging studies showed profoundly altered cilia length and morphology accompanied by reduced mucociliary clearance. Pharmacological inhibition of HDAC6 rescued cilia length and coverage in miR449−/− cells, consistent with its tubulin-deacetylating function. Altogether, our study establishes a link between miR449, ciliary dysfunction, and COPD pathogenesis.

Motile cilia line the efferent ducts of the mammalian male reproductive tract. Several recent mouse studies have demonstrated that a reduced generation of multiple motile cilia in efferent ducts is associated with obstructive oligozoospermia and fertility issues. However, the sole impact of efferent duct cilia dysmotility on male infertility has not been studied so far either in mice or human. Using video microscopy, histological- and ultrastructural analyses, we examined male reproductive tracts of mice deficient for the axonemal motor protein DNAH5: this defect exclusively disrupts the outer dynein arm (ODA) composition of motile cilia but not the ODA composition and motility of sperm flagella. These mice have immotile efferent duct cilia that lack ODAs, which are essential for ciliary beat generation. Furthermore, they show accumulation of sperm in the efferent duct. Notably, the ultrastructure and motility of sperm from these males are unaffected. Likewise, human individuals with loss-of-function DNAH5 mutations present with reduced sperm count in the ejaculate (oligozoospermia) and dilatations of the epididymal head but normal sperm motility, similar to DNAH5 deficient mice. The findings of this translational study demonstrate, in both mice and men, that efferent duct ciliary motility is important for male reproductive fitness and uncovers a novel pathomechanism distinct from primary defects of sperm motility (asthenozoospermia). If future work can identify environmental factors or defects in genes other than DNAH5 that cause efferent duct cilia dysmotility, this will help unravel other causes of oligozoospermia and may influence future practices in genetic and fertility counseling as well as ART.

TP73 belongs to the TP53 family of transcription factors and has therefore been well studied in cancer research. Studies in mice, however, have revealed non-oncogenic activities related to multiciliogenesis. Utilizing whole-exome sequencing analysis in a cohort of individuals with a mucociliary clearance disorder and cortical malformation, we identified homozygous loss-of-function variants in TP73 in seven individuals from five unrelated families. All affected individuals exhibit a chronic airway disease as well as a brain malformation consistent with lissencephaly. We performed high-speed video microscopy, immunofluorescence analyses, and transmission electron microscopy in respiratory epithelial cells after spheroid or air liquid interface culture to analyze ciliary function, ciliary length, and number of multiciliated cells (MCCs). The respiratory epithelial cells studied display reduced ciliary length and basal bodies mislocalized within the cytoplasm. The number of MCCs is severely reduced, consistent with a reduced number of cells expressing the transcription factors crucial for multiciliogenesis (FOXJ1, RFX2). Our data demonstrate that autosomal-recessive deleterious variants in the TP53 family member TP73 cause a mucociliary clearance disorder due to a defect in MCC differentiation.

TP73 belongs to the TP53 family of transcription factors and has therefore been well studied in cancer research. Studies in mice, however, have revealed nononcogenic activities related to multiciliogenesis.

Primary ciliary dyskinesia (PCD) is a heterogeneous group of disorders affecting mainly the respiratory system, the male and female reproductive system as well as the establishment of left-right body asymmetry. PCD is diagnosed by nasal nitric oxide measurements, analysis of ciliary beating, transmission electron microscopy and genetic testing. To date, more than 40 genes have been published to cause PCD when mutated. In our center, we aim to decipher the genetic causes of PCD in human individuals. By using Sanger sequencing, panel and whole exome sequencing, we here report the genetic results in 1072 PCD-suspected individuals based on clinical symptoms out of a cohort of 4005 PCD-suspected individuals recruited mainly in Europe. 671 individuals carried mutations in PCD-associated genes. The most frequent mutated gene is DNAH5 with 151 individuals, followed by DNAH11 and CCDC40 with 62 individuals each, CCNO with 42 individuals and DNAI1 with 40 individuals. The least mutated genes are RSPH3, FOXJ1, CFAP53 and ENKUR (2 each) and SPEF2, DNAJB13 and C21orf59 with 1 individual each. Interestingly, we identified hotspot and/or founder mutations in 20 genes: DNAH5, DNAI1, CCDC39, CCDC40, C11orf70, DNAAF4, LRRC6, ZMYND10, SPAG1, CCDC114, CCDC151, ARMC4, CCDC103, CCDC164, CCNO, MCIDAS, RSPH1, RSPH9, RSPH4A and HYDIN. Screening for these mutations first will ease and reduce the cost of the genetic diagnosis of PCD. To confirm the identified loss-of-function mutations, we perform immunofluorescence analysis (IF) with antibodies targeting the protein encoded by the mutated gene and prove its absence from the ciliary axoneme. Genetic combined with IF analysis is a powerful combination to diagnose PCD in the majority of cases.