Characterization of pathogenic genetic variants in Russian patients with primary ciliary dyskinesia using gene panel sequencing and transcript analysis.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a group of rare genetically heterogeneous disorders caused by defective cilia and flagella motility. The clinical phenotype of PCD patients commonly includes chronic oto-sino-pulmonary disease, infertility, and, in about half of cases, laterality defects due to randomization of left-right body asymmetry. To date, pathogenic variants in more than 50 genes responsible for motile cilia structure and assembly have been reported in such patients. While multiple population-specific mutations have been described in PCD cohorts from different countries, the data on genetic spectrum of PCD in Russian population are still extremely limited. RESULTS: The present study provides a comprehensive clinical and genetic characterization of 21 Russian families with PCD living in various country regions. Anomalies of ciliary beating in patients` respiratory epithelial cells were confirmed by high-speed video microscopy. In the most cases, custom-designed panel sequencing allowed to uncover causative variants in well-known or rarely mentioned PCD-related genes, including DNAH5, DNAH11, CFAP300, LRRC6, ZMYND10, CCDC103, HYDIN, ODAD4, DNAL1, and OFD1. The variations comprised common mutations, as well as novel genetic variants, some of which probably specific for Russian patients. Additional targeted analysis of mRNA transcripts from ciliated cells enabled us to specify functional effects of newly identified genetic variants in DNAH5 (c.2052+3G>T, c.3599-2A>G), HYDIN (c.10949-2A>G, c.1797C>G), and ZMYND10 (c.510+1G>C) on splicing process. In particular, the splice site variant c.2052+3G>T, detected in four unrelated families, resulted in skipping of exon 14 in DNAH5 transcripts and, according to haplotype analysis of affected probands, was proposed as an ancestral founder mutation in Udmurt population. CONCLUSIONS: The reported data provide a vital insight into genetic background of primary ciliary dyskinesia in the Russian population. The findings clearly illustrate the utility of gene panel sequencing coupled with transcriptional analysis in identification and clinical interpretation of novel genetic variants.

[Clinical and genetic characteristics of a case of primary ciliary dyskinesia caused by new frameshift mutation of the DNAH5 gene].

OBJECTIVE: To investigate the clinical and genetic characteristics of a case of primary ciliary dyskinesia (PCD). METHODS: We collected the clinical data on a case of PCD treated in the Department of Reproductive Medicine of Linyi People's Hospital in July 2020, detected the genes of the patient by whole-exome sequencing (WES), verified the candidate mutations by Sanger sequencing, and predicted the protein structure of the mutant gene by SWISS-MODEL. RESULTS: The proband was found with the clinical phenotypes of chronic rhinitis, bronchiectasis, visceral transposition and male infertility. WES revealed a homozygous frameshift variation of c.12890dup (p.N4297Kfs*13) in exon 74 of the DNAH5 gene, which led to the premature termination of polypeptide chain synthesis and affected the gene function. SWISS-MODEL prediction showed that some of the amino acid residues were deleted after mutation, resulting in a 3D conformational change of the protein. This variation was not recorded in the ClinVar, gnomAD and OMIM databases and, according to the relevant guidelines of the American College of Genetics and Genomics, was classified as a pathogenic variation (PVS1+PM2_P+PM3_P). CONCLUSION: The homozygous variation of the DNAH5 gene c.12890dup (p.N4297Kfs*13) may be the cause of the clinical phenotype of this case of PCD, and the above findings have enriched the variation spectrum of the DNAH5 gene.

A novel splicing mutation DNAH5 c.13,338 + 5G > C is involved in the pathogenesis of primary ciliary dyskinesia in a family with primary familial brain calcification.

BACKGROUND: Primary ciliary dyskinesia (PCD) is an autosomal recessive hereditary disease characterized by recurrent respiratory infections. In clinical manifestations, DNAH5 (NM_001361.3) is one of the recessive pathogenic genes. Primary familial brain calcification (PFBC) is a neurodegenerative disease characterized by bilateral calcification in the basal ganglia and other brain regions. PFBC can be inherited in an autosomal dominant or recessive manner. A family with PCD caused by a DNAH5 compound heterozygous variant and PFBC caused by a MYORG homozygous variant was analyzed. METHODS: In this study, we recruited three generations of Han families with primary ciliary dyskinesia combined with primary familial brain calcification. Their clinical phenotype data were collected, next-generation sequencing was performed to screen suspected pathogenic mutations in the proband and segregation analysis of families was carried out by Sanger sequencing. The mutant and wild-type plasmids were constructed and transfected into HEK293T cells instantaneously, and splicing patterns were detected by Minigene splicing assay. The structure and function of mutations were analyzed by bioinformatics analysis. RESULTS: The clinical phenotypes of the proband (II10) and his sister (II8) were bronchiectasis, recurrent pulmonary infection, multiple symmetric calcifications of bilateral globus pallidus and cerebellar dentate nucleus, paranasal sinusitis in the whole group, and electron microscopy of bronchial mucosa showed that the ciliary axoneme was defective. There was also total visceral inversion in II10 but not in II8. A novel splice variant C.13,338 + 5G > C and a frameshift variant C.4314delT (p. Asn1438lysfs *10) were found in the DNAH5 gene in proband (II10) and II8. c.347_348dupCTGGCCTTCCGC homozygous insertion variation was found in the MYORG of the proband. The two pathogenic genes were co-segregated in the family. Minigene showed that DNAH5 c.13,338 + 5G > C has two abnormal splicing modes: One is that part of the intron bases where the mutation site located is translated, resulting in early translation termination of DNAH5; The other is the mutation resulting in the deletion of exon76. CONCLUSIONS: The newly identified DNAH5 splicing mutation c.13,338 + 5G > C is involved in the pathogenesis of PCD in the family, and forms a compound heterozygote with the pathogenic variant DNAH5 c.4314delT lead to the pathogenesis of PCD.

Novel pathogenic variants of DNAH5 associated with clinical and genetic spectra of primary ciliary dyskinesia in an Arab population.

Introduction: Primary ciliary dyskinesia (PCD) is caused by the dysfunction of motile cilia resulting in insufficient mucociliary clearance of the lungs. This study aimed to map novel PCD variants and determine their pathogenicity in PCD patients in Kuwait. Methods: Herein, we present five PCD individuals belonging to a cohort of 105 PCD individuals recruited from different hospitals in Kuwait. Genomic DNAs from the family members were analysed to screen for pathogenic PCD variants. Transmission electron microscopy (TEM) and immunofluorescence (IF) analyses were performed on the nasal biopsies to detect specific structural abnormalities within the ciliated cells. Results: Genetic screening and functional analyses confirmed that the five PCD individuals carried novel pathogenic variants of DNAH5 causing PCD in three Arabic families. Of these, one multiplex family with two affected individuals showed two novel homozygous missense variants in DNAH5 causing PCD with situs inversus; another multiplex family with two affected individuals showed two newly identified compound heterozygous variants in DNAH5 causing PCD with situs solitus. In addition, novel heterozygous variants were identified in a child with PCD and situs solitus from a singleton family with unrelated parents. TEM analysis demonstrated the lack of outer dynein arms (ODAs) in all analysed samples, and IF analysis confirmed the absence of the dynein arm component of DNAH5 from the ciliary axoneme. Conclusion: The newly identified pathogenic variants of DNAH5 are associated with PCD as well as variable pulmonary clinical manifestations in Arabic families.

Human Genetics of Defects of Situs.

Defects of situs are associated with complex sets of congenital heart defects in which the normal concordance of asymmetric thoracic and abdominal organs is disturbed. The cellular and molecular mechanisms underlying the formation of the embryonic left-right axis have been investigated extensively in the past decade. This has led to the identification of mutations in at least 33 different genes in humans with heterotaxy and situs defects. Those mutations affect a broad range of molecular components, from transcription factors, signaling molecules, and chromatin modifiers to ciliary proteins. A substantial overlap of these genes is observed with genes associated with other congenital heart diseases such as tetralogy of Fallot and double-outlet right ventricle, d-transposition of the great arteries, and atrioventricular septal defects. In this chapter, we present the broad genetic heterogeneity of situs defects including recent human genomics efforts.

Next-Generation Sequencing-Based Copy Number Variation Analysis in Chinese Patients with Primary Ciliary Dyskinesia Revealed Novel DNAH5 Copy Number Variations.

Primary ciliary dyskinesia (PCD) is a rare disorder characterized by extensive genetic heterogeneity. However, in the genetic pathogenesis of PCD, copy number variation (CNV) has not received sufficient attention and has rarely been reported, especially in China. Next-generation sequencing (NGS) followed by targeted CNV analysis was used in patients highly suspected to have PCD with negative results in routine whole-exome sequencing (WES) analysis. Quantitative real-time polymerase chain reaction (qPCR) and Sanger sequencing were used to confirm these CNVs. To further characterize the ciliary phenotypes, high-speed video microscopy analysis (HSVA), transmission electron microscopy (TEM), and immunofluorescence (IF) analysis were used. Patient 1 (F1: II-1), a 0.6-year-old girl, came from a nonconsanguineous family-I. She presented with situs inversus totalis, neonatal respiratory distress, and sinusitis. The nasal nitric oxide level was markedly reduced. The respiratory cilia beat with reduced amplitude. TEM revealed shortened outer dynein arms (ODA) of cilia. chr5:13717907-13722661del spanning exons 71-72 was identified by NGS-based CNV analysis. Patient 2 (F2: IV-4), a 37-year-old man, and his eldest brother Patient 3 (F2: IV-2) came from a consanguineous family-II. Both had sinusitis, bronchiectasis and situs inversus totalis. The respiratory cilia of Patient 2 and Patient 3 were found to be uniformly immotile, with ODA defects. Two novel homozygous deletions chr5:13720087_13733030delinsGTTTTC and chr5:13649539_1 3707643del, spanning exons 69-71 and exons 77-79 were identified by NGS-based CNV analysis. Abnormalities in DNA copy number were confirmed by qPCR amplification. IF showed that the respiratory cilia of Patient 1 and Patient 2 were deficient in dynein axonemal heavy chain 5 (DNAH5) protein expression. This report identified three novel DNAH5 disease-associated variants by WES-based CNV analysis. Our study expands the genetic spectrum of PCD with DNAH5 in the Chinese population. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-023-00130-0.

Identification and validation of a prognostic-related mutant gene DNAH5 for hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide and has a poor prognosis. Thus, there is a need for an effective biomarker to improve and predict the prognosis of HCC. Methods: RNA sequencing data, simple nucleotide variation data, and clinical data of HCC patients from The Cancer Genome Atlas (TCGA) to identify mutant genes, simple nucleotide variation data, and clinical data of HCC patients from the International Cancer Genome Consortium (ICGC) to validate the prognostic value of mutant genes were the data sources of the present study. To identify the overall survival (OS)-related mutant genes, a Kaplan-Meier (KM) analysis was conducted. We carried out univariate Cox and multivariate Cox regression analyses to identify the independent prognostic factors. We also conducted a correlation analysis of immune cells and mutant genes. To explore the molecular mechanisms of mutant genes, we conducted a gene set enrichment analysis (GSEA). A nomogram was constructed to help predict the prognosis of HCC. In addition, we explored the expression profile of mutant genes in HCC based on a TCGA dataset, an ICGC dataset, and our own HCC tissue samples. Results: We identified and validated a mutant gene, dynein axonemal heavy chain 5 (DNAH5), which was negatively related to the OS of HCC patients. Univariate Cox and multivariate Cox regression analyses revealed that the mutant gene DNAH5 could act as an independent prognostic factor for HCC. Most pathways of the mutant gene DNAH5 were involved in cancer development and progression based on GSEA analysis. The mutant gene DNAH5 was negatively correlated with monocytes, naive CD4 T cells, activated dendritic cells, and activated mast cells. In addition, the mRNA and protein levels of DNAH5 had a significantly higher level of expression in the tissue samples of patients with HCC. A nomogram consisting of the pathological stage, DNAH5, and tumor mutation burden (TMB) performed well. Conclusion: The mutant gene DNAH5 had a significantly higher level of expression in the tissue samples of patients with HCC, could act as an independent prognostic factor for HCC, and is a potential new immunotherapy target for HCC.

Clinical and Genetic Characterization of Patients with Primary Ciliary Dyskinesia in Southwest Saudi Arabia: A Cross Sectional Study.

BACKGROUND: Primary ciliary dyskinesia (PCD, MIM 244400) is an inherited ciliopathy disorder characterized by recurrent sinopulmonary infections, subfertility, and laterality defects. The true incidence of PCD in Saudi Arabia is not known, but it is likely underdiagnosed due to the high prevalence of consanguineous marriages. In this study, we aim to study the clinical and genetic characteristics of PCD patients in the southwestern region of Saudi Arabia to provide guidance to clinicians and researchers studying PCD. METHODS: This was a cross-sectional study conducted between 2019 and 2023 in Abha Maternity and Children's Hospital. Twenty-eight patients with clinically diagnosed PCD were recruited. The diagnosis of PCD was confirmed via whole-exome sequencing. RESULTS: A total of 28 patients from 20 families were identified and recruited for this study. The median age of patients was 7.5 years (IQR = 3, 13 years). The people of different sexes were evenly distributed, and 18 patients (64%) had neonatal respiratory distress (NRD). The median age of diagnosis was 5.5 years (IQR = 2, 11 years), while the age when the first symptoms appeared was 3 months old (IQR = 1, 6 months). The prevalence of a chronic wet cough, chronic rhinosinusitis, ear infections were 100% (n = 28), 78.6% (n = 22), and 67.9% (19), respectively. The most common gene in our study was DNAH5, which represented 17.9% (five out of twenty-eight) of the cases. Furthermore, the remaining pathogenic variants included: 14.3% with RSPH9 in four individuals (three families), 14.3% with DNAI2 in four individuals (two families), and 10.7% with LRRC56 in three individuals (one family). The most common findings on the chest CT scans were consolidation (seen in all patients), mucus plugging (seen in 95%), and bronchiectasis (seen in 77%). In the patients with bronchiectasis, the most commonly affected lobes were the right lower lobe (88%) and left lower lobe (76%). The patients with PCD and situs inversus were more likely to experience NRD than the patients with PCD and situs solitus. The median PICADAR score in the patients with PCD and situs inversus (median: 11.5; Q1: 10-Q3: 12.5) was significantly higher compared to those with PCD and situs solitus (median: 7.5; Q1: 5.8-Q3: 8) (U = 10.5; p < 0.001). CONCLUSION: This study provides preliminary data on the clinical and genetic characteristics of PCD patients in the southwestern region of Saudi Arabia. We found that DNAH5 and RSPH9 genes were the most common genes among the studied population. Furthermore, PCD should be considered for each child with early NRD and laterality defects, and further confirmatory tests are recommended. These findings also highlight the need for greater awareness of the disease in daily clinical practice to facilitate early diagnosis and avoid irreversible lung damage.

Novel Pathogenic DNAH5 Variants in Primary Ciliary Dyskinesia: Association with Visceral Heterotaxia and Neonatal Cholestasis.

The dynein axonemal heavy chain 5 gene codes for a subunit of axonemal dynein necessary for ciliary motor function. Though research has elucidated the consequences of some variants in this gene, it is still unclear whether many variants in the DNAH5 locus are benign or pathogenic due to the rarity of primary ciliary dyskinesia (PCD, of which Kartagener's syndrome is a subset). Here, we introduce the case of an infant boy presenting with the classical findings of PCD along with visceral heterotaxia and neonatal cholestasis. Genetic testing indicated that the patient is a compound heterozygote with a pathogenic c.8498G > A (known as pathogenic) on the maternally derived allele and two variants of uncertain significance, c.1206T > A and c.7800T > G, on the paternally derived allele. As PCD is autosomal recessive, we conclude that one, or both, of these paternally derived variants are pathogenic. To our knowledge, this is the first time that the clinical implications of c.1206T > A (p.Asn402Lys) and c.7800T > G (p.Ile2600Met) are documented. Furthermore, we use this case as an example to recommend clinicians to assess for PCD and laterality defects when presented with severe infantile cholestasis. While the association of cholestasis with PCD is relatively uncommon, PCD is a risk factor for increased prevalence of biliary atresia and infections, both of which are known causes of cholestasis in early infancy.

Multiomics Analysis of a DNAH5-Mutated PCD Organoid Model Revealed the Key Role of the TGF-ß/BMP and Notch Pathways in Epithelial Differentiation and the Immune Response in DNAH5-Mutated Patients.

Dynein axonemal heavy chain 5 (DNAH5) is the most mutated gene in primary ciliary dyskinesia (PCD), leading to abnormal cilia ultrastructure and function. Few studies have revealed the genetic characteristics and pathogenetic mechanisms of PCD caused by DNAH5 mutation. Here, we established a child PCD airway organoid directly from the bronchoscopic biopsy of a patient with the DNAH5 mutation. The motile cilia in the organoid were observed and could be stably maintained for an extended time. We further found abnormal ciliary function and a decreased immune response caused by the DNAH5 mutation through single-cell RNA sequencing (scRNA-Seq) and proteomic analyses. Additionally, the directed induction of the ciliated cells, regulated by TGF-ß/BMP and the Notch pathway, also increased the expression of inflammatory cytokines. Taken together, these results demonstrated that the combination of multiomics analysis and organoid modelling could reveal the close connection between the immune response and the DNAH5 gene.

Genomic and transcriptomic analyses reveal genetic adaptation to cold conditions in the chickens.

Under the pressure of natural and artificial selection, domestic animals, including chickens, have evolved unique mechanisms of genetic adaptations such as high-altitude adaptation, hot and arid climate adaptation, and desert adaptation. Here, we investigated the genetic basis of cold tolerance in chicken by integrating whole-genome and transcriptome sequencing technologies. Genome-wide comparative analyses of 118 chickens living in different latitudes showed 46 genes and several pathways that may be involved in cold adaptation. The results of the functional enrichment analysis of differentially expressed genes proved the important role of metabolic pathways and immune-related pathways in cold tolerance in chickens. The subsequent integration of whole genome and transcriptome sequencing technology further identified six genes - dnah5 (dynein axonemal heavy chain 5), ptgs2 (prostaglandin-endoperoxide synthase 2), inhba (inhibin beta A subunit), irx2 (iroquois homeobox 2), ensgalg00000054917, and ensgalg00000046652 - requiring more detailed studies. In addition, we also discovered different allele frequency distributions of five SNPs (single nucleotide polymorphisms) within ptgs2 and nine SNPs within dnah5 in chickens in different latitudes, suggesting strong selective pressure of these two genes in chickens. We provide a novel insight into the genetic adaptation in chickens to cold environments, and provide a reference for evaluating and developing adaptive chicken breeds in cold environments.

DNAH5 gene and its correlation with linc02220 expression and sperm characteristics.

BACKGROUND: Numerous pieces of evidence show that many environmental and genetic factors can cause male infertility. Much research in recent years has investigated the function of long non-coding RNAs (lncRNAs) in fertility. The main objective of the current study was to investigate the expression of Dynein Axonemal Heavy Chain 5 (DNAH5) as a gene that plays an essential role in sperm motility in individuals with asthenozoospermia and terato-asthenozoospermia. Alterations in linc02220 expression (located close to the DNAH5 gene), its action potential in DNAH5 regulating, and the correlation between their expression and normal sperm morphology and motility were also examined. METHOD AND MATERIAL: This study examined the semen of 31 asthenozoospermia individuals (AZ), 33 terato-asthenozoospermia (TAZ) individuals, and 33 normospermia (NZ) individuals with normal sperm as a control group. The expression levels of DNAH5 and linc02220 in the sperm samples were analyzed by real-time PCR. RESULTS: Gene expression analysis revealed a significant association between DNAH5 expression and sperm motility and morphology (p < 0.0001). The DNAH5 expression levels in the TAZ and AZ groups were also significantly reduced; however, linc02220 was significantly upregulated in both TAZ and AZ groups compared to the NZ group (p < 0.0001). DNAH5 expression in the TAZ and AZ groups was negatively correlated with linc02220 expression, thus, DNAH5 downregulation was associated with linc02220 overexpression (p < 0.05). CONCLUSIONS: The gene linc02220 could be a potential regulatory target for DNAH5, and both could affect sperm's normal motility and morphology.

Novel compound heterozygous mutations of DNAH5 identified in a pediatric patient with Kartagener syndrome: case report and literature review.

BACKGROUND: Kartagener syndrome is a subtype of primary ciliary dyskinesia that may exhibit various symptoms including neonatal respiratory distress and frequent infections of the lung, sinus and middle ear because of the impaired function of motile cilia. In addition to typical symptoms of primary ciliary dyskinesia, patients with Kartagener syndrome also show situs inversus. It is an autosomal recessive disorder which is mostly caused by mutations in DNAH5. Kartagener syndrome is often underdiagnosed due to challenges in the diagnosis process. As next-generation sequencing becomes widely used in clinical laboratories, genetic testing provides an accurate approach to the diagnosis of Kartagener syndrome. CASE PRESENTATION: A 7-year-old female patient presented with runny nose of 6 years duration and recurrent cough with phlegm of 2 years duration. Kartagener syndrome was diagnosed through diagnostic tests such as nasal nitric oxide (NO) concentration and transmission electron microscopy, and after performing other exams that corroborated the diagnosis, such as computed tomography, bronchoscopy and hearing test. Whole-exome sequencing was performed for the patient and both parents. The pediatric patient was diagnosed as Kartagener syndrome with the typical symptoms of ciliary dyskinesia including bronchiectasis, sinusitis, conductive hearing loss and situs inversus along with a reduced nasal NO concentration and ciliary abnormalities. The patient carried two novel compound heterozygous mutations in DNAH5, NM_001369:c.12813G > A (p. Trp4271Term) and NM_001369:c.9365delT (p. Leu3122Term). Both mutations lead to premature stop codons and thus are pathogenic. The p. Trp4271Term and p. Leu3122Term mutations were inherited from the father and the mother of the patient individually. A literature review was also conducted to summarize DNAH5 mutations in pediatric patients with Kartagener syndrome across different ethnic groups. CONCLUSIONS: Our study provides a good example of the diagnosis of Kartagener syndrome in pediatric patients using a series of diagnostic tests combined with genetic testing. Two novel loss-of-function mutations in DNAH5 were identified and validated in a pediatric patient with Kartagener syndrome.

Deciphering balanced translocations in infertile males by next-generation sequencing to identify candidate genes for spermatogenesis disorders.

Male infertility affects about 7% of the general male population. Balanced structural chromosomal rearrangements are observed in 0.4-1.4% of infertile males and are considered as a well-established cause of infertility. However, underlying pathophysiological mechanisms still need to be clarified. A strategy combining standard and high throughput cytogenetic and molecular technologies was applied in order to identify the candidate genes that might be implicated in the spermatogenesis defect in three male carriers of different balanced translocations. Fluorescence in situ hybridization (FISH) and whole-genome paired-end sequencing were used to characterize translocation breakpoints at the molecular level while exome sequencing was performed in order to exclude the presence of any molecular event independent from the chromosomal rearrangement in the patients. All translocation breakpoints were characterized in the three patients. We identified four variants: a position effect on LACTB2 gene in Patient 1, a heterozygous CTDP1 gene disruption in Patient 2, two single-nucleotide variations (SNVs) in DNAH5 gene and a heterozygous 17q12 deletion in Patient 3. The variants identified in this study need further validation to assess their roles in male infertility. This study shows that beside the mechanical effect of structural rearrangement on meiosis, breakpoints could result in additional alterations such as gene disruption or position effect. Moreover, additional SNVs or copy number variations may be fortuitously present and could explain the variable impact of chromosomal rearrangements on spermatogenesis. In conclusion, this study confirms the relevance of combining different cytogenetic and molecular techniques to investigate patients with spermatogenesis disorders and structural rearrangements on genomic scale.

Motility of efferent duct cilia aids passage of sperm cells through the male reproductive system.

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.

Randomization of Left-right Asymmetry and Congenital Heart Defects: The Role of DNAH5 in Humans and Mice.

Background - Nearly one in 100 live births presents with congenital heart defects (CHD). CHD are frequently associated with laterality defects, such as situs inversus totalis (SIT), a mirrored positioning of internal organs. Body laterality is established by a complex process: monocilia at the embryonic left-right organizer (LRO) facilitate both the generation and sensing of a leftward fluid flow. This induces the conserved left-sided Nodal signaling cascade to initiate asymmetric organogenesis. Primary ciliary dyskinesia (PCD) originates from dysfunction of motile cilia, causing symptoms such as chronic sinusitis, bronchiectasis and frequently SIT. The most frequently mutated gene in PCD, DNAH5 is associated with randomization of body asymmetry resulting in SIT in half of the patients; however, its relation to CHD occurrence in humans has not been investigated in detail so far. Methods - We performed genotype / phenotype correlations in 132 PCD patients carrying disease-causing DNAH5 mutations, focusing on situs defects and CHD. Using high speed video microscopy-, immunofluorescence-, and in situ hybridization analyses, we investigated the initial steps of left-right axis establishment in embryos of a Dnah5 mutant mouse model. Results - 65.9% (87 / 132) of the PCD patients carrying disease-causing DNAH5 mutations had laterality defects: 88.5% (77 / 87) presented with SIT, 11.5% (10 / 87) presented with situs ambiguus; and 6.1% (8 / 132) presented with CHD. In Dnah5mut/mut mice, embryonic LRO monocilia lack outer dynein arms resulting in immotile cilia, impaired flow at the LRO, and randomization of Nodal signaling with normal, reversed or bilateral expression of key molecules. Conclusions - For the first time, we directly demonstrate the disease-mechanism of laterality defects linked to DNAH5 deficiency at the molecular level during embryogenesis. We highlight that mutations in DNAH5 are not only associated with classical randomization of left-right body asymmetry but also with severe laterality defects including CHD.

Identification of Pathogenic Mutations and Investigation of the NOTCH Pathway Activation in Kartagener Syndrome.

Primary ciliary dyskinesia (PCD), a rare genetic disorder, is mostly caused by defects in more than 40 known cilia structure-related genes. However, in approximately 20-35% of patients, it is caused by unknown genetic factors, and the inherited pathogenic factors are difficult to confirm. Kartagener syndrome (KTS) is a subtype of PCD associated with situs inversus, presenting more complex genetic heterogeneity. The aim of this study was to identify pathogenic mutations of candidate genes in Chinese patients with KTS and investigate the activation of the heterotaxy-related NOTCH pathway. Whole-exome sequencing was conducted in five patients with KTS. Pathogenic variants were identified using bioinformatics analysis. Candidate variants were validated by Sanger sequencing. The expression of the NOTCH pathway target genes was detected in patients with KTS. We identified 10 KTS-associated variants in six causative genes, namely, CCDC40, DNAH1, DNAH5, DNAH11, DNAI1, and LRRC6. Only one homozygote mutation was identified in LRRC6 (c.64dupT). Compound heterozygous mutations were found in DNAH1 and DNAH5. Six novel mutations were identified in four genes. Further analyses showed that the NOTCH pathway might be activated in patients with KTS. Overall, our study showed that compound heterozygous mutations widely exist in Chinese KTS patients. Our results demonstrated that the activation of the NOTCH pathway might play a role in the situs inversus pathogenicity of KTS. These findings highlight that Kartagener syndrome might be a complex genetic heterogeneous disorder mediated by heterozygous mutations in multiple PCD- or cilia-related genes.

Clinical and Genetic Analysis of Children with Kartagener Syndrome.

Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder characterized by dysfunction of motile cilia causing ineffective mucus clearance and organ laterality defects. In this study, two unrelated Portuguese children with strong PCD suspicion underwent extensive clinical and genetic assessments by whole-exome sequencing (WES), as well as ultrastructural analysis of cilia by transmission electron microscopy (TEM) to identify their genetic etiology. These analyses confirmed the diagnostic of Kartagener syndrome (KS) (PCD with situs inversus). Patient-1 showed a predominance of the absence of the inner dynein arms with two disease-causing variants in the CCDC40 gene. Patient-2 showed the absence of both dynein arms and WES disclosed two novel high impact variants in the DNAH5 gene and two missense variants in the DNAH7 gene, all possibly deleterious. Moreover, in Patient-2, functional data revealed a reduction of gene expression and protein mislocalization in both genes' products. Our work calls the researcher's attention to the complexity of the PCD and to the possibility of gene interactions modelling the PCD phenotype. Further, it is demonstrated that even for well-known PCD genes, novel pathogenic variants could have importance for a PCD/KS diagnosis, reinforcing the difficulty of providing genetic counselling and prenatal diagnosis to families.

A Japanese Case of Primary Ciliary Dyskinesia with DNAH5 Mutations.

A 33-year-old woman presented with a productive cough from childhood. She had suffered from repeated bacterial pneumonia. Her clinical and imaging findings revealed chronic sinusitis, bronchiectasis and situs inversus. We suspected primary ciliary dyskinesia (PCD) and performed a bronchial mucosal biopsy. The ciliary beat pattern according to high-speed video microscopy was complete loss. Electron microscopic findings of cilia showed defect of outer dynein arm (ODA). A genetic examination detected compound heterozygous mutations of DNAH5 that encode ODA components. There are few reports of genetic mutation analyses in Japanese PCD patients. We herein report a PCD patient with DNAH5 mutations and review the related literature.

A novel compound heterozygous mutation in DNAH5 in a Korean neonate with primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disease affecting motile cilia. A female neonate was hospitalized with respiratory distress 72 hours after birth and showed concurrent situs inversus. She was identified to have compound heterozygous mutations in DNAH5: c.5647C>T, p.Arg1883Ter (nonsense mutation) and c.10810dupA, p.Ile3604AsnfsTer2 (frameshift mutation). Sanger sequencing confirmed that they were inherited from her father and mother, respectively, and she was diagnosed with PCD. The c.10810dupA is a novel DNAH5 mutation that has never been reported. To the best of our knowledge, this is the first report describing DNAH5 mutations in a Korean patient with PCD.