Practical guide for the diagnosis and management of primary ciliary dyskinesia.

OBJECTIVE: Primary ciliary dyskinesia (PCD) is a relatively rare genetic disorder that affects approximately 1 in 20,000 people. Approximately 50 genes are currently known to cause PCD. In light of differences in causative genes and the medical system in Japan compared with other countries, a practical guide was needed for the diagnosis and management of Japanese PCD patients. METHODS: An ad hoc academic committee was organized under the Japanese Rhinologic Society to produce a practical guide, with participation by committee members from several academic societies in Japan. The practical guide including diagnostic criteria for PCD was approved by the Japanese Rhinologic Society, Japanese Society of Otolaryngology-Head and Neck Surgery, Japanese Respiratory Society, and Japanese Society of Pediatric Pulmonology. RESULTS: The diagnostic criteria for PCD consist of six clinical features, six laboratory findings, differential diagnosis, and genetic testing. The diagnosis of PCD is categorized as definite, probable, or possible PCD based on a combination of the four items above. Diagnosis of definite PCD requires exclusion of cystic fibrosis and primary immunodeficiency, at least one of the six clinical features, and a positive result for at least one of the following: (1) Class 1 defect on electron microscopy of cilia, (2) pathogenic or likely pathogenic variants in a PCD-related gene, or (3) impairment of ciliary motility that can be repaired by correcting the causative gene variants in iPS cells established from the patient's peripheral blood cells. CONCLUSION: This practical guide provides clinicians with useful information for the diagnosis and management of PCD in Japan.

Cystic Lung Changes, Bronchiectasis, and a Heterozygous-Primary Ciliary Dyskinesia-Associated Variant in the DNAH5 Gene: A Diagnostic Challenge.

BACKGROUND Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disease that can present at different ages with different phenotypes. Missed and delayed diagnoses are fairly common. Many variants in the DNAH5 gene have been described that confirm the diagnosis of PCD. Advances in medicine, especially in molecular genetics, have led to increasingly early discoveries of such cases, especially in those with nonclassical presentations. CASE REPORT This report describes a patient with bronchiectasis, lung cysts, finger clubbing, and failure to thrive who was misdiagnosed for several years as having asthma. Many differentials were suspected and worked up, including a suspicion of PCD. Genetic tests with whole-exome sequencing (WES) and whole-genome sequencing (WGS) detected a heterozygous, likely pathogenic, variant in the DNAH5 gene associated with PCD. CONCLUSIONS Despite a thorough workup done for this case, including a genetic workup, a PCD diagnosis was not established. We plan to reanalyze the WGS in the future, and with advent of technology and better coverage of genes, a genetic answer for this challenging case may resolve this diagnostic quandary in the future.

Whole-exome sequencing of Nigerian benign prostatic hyperplasia reveals increased alterations in apoptotic pathways.

BACKGROUND: Through whole-exome sequencing of 60 formalin-fixed paraffin-embedded Nigerian (NGRn) benign prostatic hyperplasia (BPH) samples, we identified germline and somatic alterations in apoptotic pathways impacting BPH development and progression. Prostate enlargement is a common occurrence in male aging; however, this enlargement can lead to lower urinary tract symptoms that negatively impact quality of life. This impact is disproportionately present in men of African ancestry. BPH pathophysiology is poorly understood and studies examining non-European populations are lacking. METHODS: In this study, NGRn BPH, normal prostate, and prostate cancer (PCa) tumor samples were sequenced and compared to characterize genetic alterations in NGRn BPH. RESULTS: Two hundred and two nonbenign, ClinVar-annotated germline variants were present in NGRn BPH samples. Six genes [BRCA1 (92%), HSD3B1 (85%), TP53 (37%), PMS2 (23%), BARD1 (20%), and BRCA2 (17%)] were altered in at least 10% of samples; however, compared to NGRn normal and tumor, the frequency of alterations in BPH samples showed no significant differences at the gene or variant level. BRCA2_rs11571831 and TP53_rs1042522 germline alterations had a statistically significant co-occurrence interaction in BPH samples. In at least two BPH samples, 173 genes harbored somatic variants known to be clinically actionable. Three genes (COL18A1, KIF16B, and LRP1) showed a statistically significant (p < 0.05) higher frequency in BPH. NGRn BPH also had five gene pairs (PKD1/KIAA0100, PKHD1/PKD1, DNAH9/LRP1B, NWD1/DCHS2, and TCERG1/LMTK2) with statistically significant co-occurring interactions. Two hundred and seventy-nine genes contained novel somatic variants in NGRn BPH. Three genes (CABP1, FKBP1C, and RP11-595B24.2) had a statistically significant (p < 0.05) higher alteration frequency in NGRn BPH and three were significantly higher in NGRn tumor (CACNA1A, DMKN, and CACNA2D2). Pairwise Fisher's exact tests showed 14 gene pairs with statistically significant (p < 0.05) interactions and four interactions approaching significance (p < 0.10). Mutational patterns in NGRn BPH were similar to COSMIC (Catalog of Somatic Mutations in Cancer) signatures associated with aging and dysfunctional DNA damage repair. CONCLUSIONS: NGRn BPH contained significant germline alteration interactions (BRCA2_rs11571831 and TP53_rs1042522) and increased somatic alteration frequencies (LMTK2, LRP1, COL18A1, CABP1, and FKBP1C) that impact apoptosis. Normal prostate development is maintained by balancing apoptotic and proliferative activity. Dysfunction in either mechanism can lead to abnormal prostate growth. This work is the first to examine genomic sequencing in NGRn BPH and provides data that fill known gaps in the understanding BPH and how it impacts men of African ancestry.

Gene signature developed for predicting early relapse and survival in early-stage pancreatic cancer.

BACKGROUND: The aim of this study was to construct a predictive signature integrating tumour-mutation- and copy-number-variation-associated features using machine learning to precisely predict early relapse and survival in patients with resected stage I-II pancreatic ductal adenocarcinoma. METHODS: Patients with microscopically confirmed stage I-II pancreatic ductal adenocarcinoma undergoing R0 resection at the Chinese PLA General Hospital between March 2015 and December 2016 were enrolled. Whole exosome sequencing was performed, and genes with different mutation or copy number variation statuses between patients with and without relapse within 1 year were identified using bioinformatics analysis. A support vector machine was used to evaluate the importance of the differential gene features and to develop a signature. Signature validation was performed in an independent cohort. The associations of the support vector machine signature and single gene features with disease-free survival and overall survival were assessed. Biological functions of integrated genes were further analysed. RESULTS: Overall, 30 and 40 patients were included in the training and validation cohorts, respectively. Some 11 genes with differential patterns were first identified; using a support vector machine, four features (mutations of DNAH9, TP53, and TUBGCP6, and copy number variation of TMEM132E) were further selected and integrated to construct a predictive signature (the support vector machine classifier). In the training cohort, the 1-year disease-free survival rates were 88 per cent (95 per cent c.i. 73 to 100) and 7 per cent (95 per cent c.i. 1 to 47) in the low-support vector machine subgroup and the high-support vector machine subgroup respectively (P < 0.001). Multivariable analyses showed that high support vector machine was significantly and independently associated with both worse overall survival (HR 29.20 (95 per cent c.i. 4.48 to 190.21); P < 0.001) and disease-free survival (HR 72.04 (95 per cent c.i. 6.74 to 769.96); P < 0.001). The area under the curve of the support vector machine signature for 1-year disease-free survival (0.900) was significantly larger than the area under the curve values of the mutations of DNAH9 (0.733; P = 0.039), TP53 (0.767; P = 0.024), and TUBGCP6 (0.733; P = 0.023), the copy number variation of TMEM132E (0.700; P = 0.014), TNM stage (0.567; P = 0.002), and differentiation grade (0.633; P = 0.005), suggesting higher predictive accuracy for prognosis. The value of the signature was further validated in the validation cohort. The four genes included in the support vector machine signature (DNAH9, TUBGCP6, and TMEM132E were novel in pancreatic ductal adenocarcinoma) were significantly associated with the tumour immune microenvironment, G protein-coupled receptor binding and signalling, cell-cell adhesion, etc. CONCLUSION: The newly constructed support vector machine signature precisely and powerfully predicted relapse and survival in patients with stage I-II pancreatic ductal adenocarcinoma after R0 resection.

DNALI1 deficiency causes male infertility with severe asthenozoospermia in humans and mice by disrupting the assembly of the flagellar inner dynein arms and fibrous sheath.

The axonemal dynein arms (outer (ODA) and inner dynein arms (IDAs)) are multiprotein structures organized by light, intermediate, light intermediate (LIC), and heavy chain proteins. They hydrolyze ATP to promote ciliary and flagellar movement. Till now, a variety of dynein protein deficiencies have been linked with asthenospermia (ASZ), highlighting the significance of these structures in human sperm motility. Herein, we detected bi-allelic DNALI1 mutations [c.663_666del (p.Glu221fs)], in an ASZ patient, which resulted in the complete loss of the DNALI1 in the patient's sperm. We identified loss of sperm DNAH1 and DNAH7 rather than DNAH10 in both DNALI1663_666del patient and Dnali1-/- mice, demonstrating that mammalian DNALI1 is a LIC protein of a partial IDA subspecies. More importantly, we revealed that DNALI1 loss contributed to asymmetries in the most fibrous sheath (FS) of the sperm flagellum in both species. Immunoprecipitation revealed that DNALI1 might interact with the cytoplasmic dynein complex proteins in the testes. Furthermore, DNALI1 loss severely disrupted the transport and assembly of the FS proteins, especially AKAP3 and AKAP4, during flagellogenesis. Hence, DNALI1 may possess a non-classical molecular function, whereby it regulates the cytoplasmic dynein complex that assembles the flagella. We conclude that a DNALI deficiency-induced IDAs injury and an asymmetric FS-driven tail rigid structure alteration may simultaneously cause flagellum immotility. Finally, intracytoplasmic sperm injection (ICSI) can effectively resolve patient infertility. Collectively, we demonstrate that DNALI1 is a newly causative gene for AZS in both humans and mice, which possesses multiple crucial roles in modulating flagellar assembly and motility.

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.

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.

FBB18 participates in preassembly of almost all axonemal dyneins independent of R2TP complex.

Assembly of dynein arms requires cytoplasmic processes which are mediated by dynein preassembly factors (DNAAFs). CFAP298, which is conserved in organisms with motile cilia, is required for assembly of dynein arms but with obscure mechanisms. Here, we show that FBB18, a Chlamydomonas homologue of CFAP298, localizes to the cytoplasm and functions in folding/stabilization of almost all axonemal dyneins at the early steps of dynein preassembly. Mutation of FBB18 causes no or short cilia accompanied with partial loss of both outer and inner dynein arms. Comparative proteomics using 15N labeling suggests partial degradation of almost all axonemal dynein heavy chains (DHCs). A mutant mimicking a patient variant induces particular loss of DHCα. FBB18 associates with 9 DNAAFs and 14 out of 15 dynein HCs but not with IC1/IC2. FBB18 interacts with RuvBL1/2, components of the HSP90 co-chaperone R2TP complex but not the holo-R2TP complex. Further analysis suggests simultaneous formation of multiple DNAAF complexes involves dynein folding/stability and thus provides new insights into axonemal dynein preassembly.

Updated Overall Survival and Exploratory Analysis From Randomized, Phase II EVAN Study of Erlotinib Versus Vinorelbine Plus Cisplatin Adjuvant Therapy in Stage IIIA Epidermal Growth Factor Receptor+ Non-Small-Cell Lung Cancer.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.The randomized, open-label, phase II EVAN study investigated the efficacy (disease-free survival [DFS] and 5-year overall survival [OS]) and safety of erlotinib versus vinorelbine/cisplatin as adjuvant chemotherapy after complete resection (R0) for stage III epidermal growth factor receptor (EGFR) mutation+ non-small-cell lung cancer. We describe the updated results at the 43-month follow-up. In EVAN, patients were randomly assigned (1:1) to erlotinib (n = 51) or vinorelbine/cisplatin (n = 51). The median follow-up was 54.8 and 63.9 months in the erlotinib and chemotherapy arms, respectively. With erlotinib, the respective 5-year DFS by Kaplan-Meier analysis was 48.2% (95% CI, 29.4 to 64.7) and 46.2% (95% CI, 27.6 to 62.9) in the intention-to-treat and per-protocol populations. The median OS was 84.2 months with erlotinib versus 61.1 months with chemotherapy (hazard ratio, 0.318; 95% CI, 0.151 to 0.670). The 5-year survival rates were 84.8% and 51.1% with erlotinib and chemotherapy, respectively. In whole-exome sequencing analysis, frequent genes with variants co-occurring at baseline were TP53, MUC16, FAM104B, KMT5A, and DNAH9. With erlotinib, a single-nucleotide polymorphism mutation in UBXN11 was associated with significantly worse DFS (P = .01). To our knowledge, this study is the first to demonstrate clinically meaningful OS improvement with adjuvant erlotinib compared with chemotherapy in R0 stage III EGFR+ non-small-cell lung cancer.

Novel compound heterozygous variants of DNAH17 in a Chinese infertile man with multiple morphological abnormalities of sperm flagella.

Multiple morphological abnormalities of the sperm flagellum (MMAF) have been reported to be an important cause of male infertility and reflect a heterogeneous genetic disorder. Previous studies have identified dozens of candidate pathogenic genes for MMAF, but the aetiology in approximately 50% of cases remains unexplained. The present study aimed to identify novel potentially pathogenic gene variants of MMAF. A Chinese family with a 32-year-old infertile proband presenting with MMAF was recruited, and sperm morphology of the patient was examined by Papanicolaou staining. Whole exome sequencing was performed on the proband and Sanger sequencing was used to identify genetic variants in the family. The frequencies of variants were assessed using public databases and the effects on protein structure and function were predicted by online bioinformatics tools. The patient exhibited asthenozoospermia and a MMAF phenotype. Novel compound heterozygous variants (c.5368C > T, p.R1790C and c.13183C > T, p.R4395W) of the DNAH17 gene were identified in the patient, and showed autosomal recessive inheritance in this family. These variants were very rare in the GnomAD database. The two mutated amino acids were located in a highly conserved region of the DNAH17 protein. In silico analysis revealed that the compound heterozygous variants may compromise the function of DNAH17. Our findings expand upon the spectrum of pathogenic DNAH17 variants that are responsible for MMAF, and provide new knowledge for genetic counselling of male infertility due to MMAF.

Dnah9 mutant mice and organoid models recapitulate the clinical features of patients with PCD and provide an excellent platform for drug screening.

Primary cilia dyskinesia (PCD) is a rare genetic disease caused by ciliary structural or functional defects. It causes severe outcomes in patients, including recurrent upper and lower airway infections, progressive lung failure, and randomization of heterotaxy. To date, although 50 genes have been shown to be responsible for PCD, the etiology remains elusive. Meanwhile, owing to the lack of a model mimicking the pathogenesis that can be used as a drug screening platform, thereby slowing the development of related therapies. In the current study, we identified compound mutation of DNAH9 in a patient with PCD with the following clinical features: recurrent respiratory tract infections, low lung function, and ultrastructural defects of the outer dynein arms (ODAs). Bioinformatic analysis, structure simulation assay, and western blot analysis showed that the mutations affected the structure and expression of DNAH9 protein. Dnah9 knock-down (KD) mice recapitulated the patient phenotypes, including low lung function, mucin accumulation, and increased immune cell infiltration. Immunostaining, western blot, and co-immunoprecipitation analyses were performed to clarify that DNAH9 interacted with CCDC114/GAS8 and diminished their protein levels. Furthermore, we constructed an airway organoid of Dnah9 KD mice and discovered that it could mimic the key features of the PCD phenotypes. We then used organoid as a drug screening model to identify mitochondrial-targeting drugs that can partially elevate cilia beating in Dnah9 KD organoid. Collectively, our results demonstrated that Dnah9 KD mice and an organoid model can recapture the clinical features of patients with PCD and provide an excellent drug screening platform for human ciliopathies.

Novel Compound Heterozygous Mutation in FSIP2 Causes Multiple Morphological Abnormalities of the Sperm Flagella (MMAF) and Male Infertility.

Multiple morphological abnormalities of the sperm flagella (MMAF), characteristic with bent, short, coiled, absent, and abnormal caliber flagella, is an important basis of male infertility. Genetic factors account for a large proportion of patients with MMAF. The fibrous sheath interacting protein 2 (FSIP2) has a significant function in the spermatogenesis and flagellar motility. In our study, a novel compound heterozygous mutation (c.1494C > A, p.C498* and c.11020_11024del, p.Tyr3675Cysfs*3) in FSIP2 gene was identified in an infertile male patient with MMAF. H&E staining presented typical MMAF phenotype and thick neck, midpiece in the patient's sperm cells. Transmission electron microscopy observation showed abnormal mitochondrial arrangement and disorganization and dysplastic of the fibrous sheath (FS), which were verified again under light microscopy. Immunofluorescence (IF) analysis of FISP2 expression showed that FSIP2 was absent in the flagellum of the patient's sperm cells. Our findings will be helpful to the precise diagnosis of MMAF and male infertility and enrich the mutational spectrum of FSIP2 gene.

The role of SPAG1 in the assembly of axonemal dyneins in human airway epithelia.

Mutations in SPAG1, a dynein axonemal assembly factor (DNAAF) that facilitates the assembly of dynein arms in the cytoplasm before their transport into the cilium, result in primary ciliary dyskinesia (PCD), a genetically heterogenous disorder characterized by chronic oto-sino-pulmonary disease, infertility and laterality defects. To further elucidate the role of SPAG1 in dynein assembly, we examined its expression, interactions and ciliary defects in control and PCD human airway epithelia. Immunoprecipitations showed that SPAG1 interacts with multiple DNAAFs, dynein chains and canonical components of the R2TP complex. Protein levels of dynein heavy chains (DHCs) and interactions between DHCs and dynein intermediate chains (DICs) were reduced in SPAG1 mutants. We also identified a previously uncharacterized 60 kDa SPAG1 isoform, through examination of PCD subjects with an atypical ultrastructural defect for SPAG1 variants, that can partially compensate for the absence of full-length SPAG1 to assemble a reduced number of outer dynein arms. In summary, our data show that SPAG1 is necessary for axonemal dynein arm assembly by scaffolding R2TP-like complexes composed of several DNAAFs that facilitate the folding and/or binding of the DHCs to the DIC complex.

Cytoplasmic factories for axonemal dynein assembly.

Axonemal dyneins power the beating of motile cilia and flagella. These massive multimeric motor complexes are assembled in the cytoplasm, and subsequently trafficked to cilia and incorporated into the axonemal superstructure. Numerous cytoplasmic factors are required for the dynein assembly process, and, in mammals, defects lead to primary ciliary dyskinesia, which results in infertility, bronchial problems and failure to set up the left-right body axis correctly. Liquid-liquid phase separation (LLPS) has been proposed to underlie the formation of numerous membrane-less intracellular assemblies or condensates. In multiciliated cells, cytoplasmic assembly of axonemal dyneins also occurs in condensates that exhibit liquid-like properties, including fusion, fission and rapid exchange of components both within condensates and with bulk cytoplasm. However, a recent extensive meta-analysis suggests that the general methods used to define LLPS systems in vivo may not readily distinguish LLPS from other mechanisms. Here, I consider the time and length scales of axonemal dynein heavy chain synthesis, and the possibility that during translation of dynein heavy chain mRNAs, polysomes are crosslinked via partially assembled proteins. I propose that axonemal dynein factory formation in the cytoplasm may be a direct consequence of the sheer scale and complexity of the assembly process itself.

Whole-exome sequencing reveals a combination of extremely rare single-nucleotide polymorphism of DNAH9 and RSPH1 genes in a Japanese fetus with situs viscerum inversus.

Randomization of left-right body asymmetry, situs viscerum inversus (heterotaxy), is commonly associated with primary ciliary dyskinesia (PCD) resulting from an abnormal ciliary structure, with approximately 50% of PCD patients exhibiting organ laterality defects. I herein report an intrauterine fetal death case, in which an autopsy revealed two lobes of the bilateral lungs as well as heterotaxy of abdominal organs (right-sided spleen and inversion of the alimentary and biliary organs). Whole-exome sequencing (WES) identified a heterozygous single-nucleotide change (c.12775T>C) in exon 68 of the DNAH9 gene, which is a rare single-nucleotide polymorphism (SNP) of rs746081639 and results in the amino acid change of p.C4259R. WES also identified a rare SNP of rs763089682 (c.121G>A) in the RSPH1 gene that causes a heterozygous amino acid alteration of p.G41R. The frequencies of both SNPs, C in rs746081639 and A in rs763089682, are 0.00000824, and a polyphen-2 analysis predicted these amino acid changes to be probably damaging, with a score of 1.000. The combination of extremely rare SNPs in DNAH9 and RSPH1 genes might have been the possible mechanism underlying the development of the laterality defect in the present case.

A Young Girl With Bronchiectasis and Elevated Sweat Chloride.

CASE PRESENTATION: A 14-year old girl presented with history of productive cough since the age of 3 years. For the past 6 years, she complained of chest tightness and wheezing. There was also nasal stuffiness and discharge for the past 6 years. She denied history of hemoptysis, ear discharge, or chest pain. There was no history of respiratory distress at the time of birth. Her brother also suffered from productive cough and wheezing since the age of 3 years. However, both her parents were asymptomatic.

Infertility due to defective sperm flagella caused by an intronic deletion in DNAH17 that perturbs splicing.

Artificial insemination in pig (Sus scrofa domesticus) breeding involves the evaluation of the semen quality of breeding boars. Ejaculates that fulfill predefined quality requirements are processed, diluted and used for inseminations. Within short time, eight Swiss Large White boars producing immotile sperm that had multiple morphological abnormalities of the sperm flagella were noticed at a semen collection center. The eight boars were inbred on a common ancestor suggesting that the novel sperm flagella defect is a recessive trait. Transmission electron microscopy cross-sections revealed that the immotile sperm had disorganized flagellar axonemes. Haplotype-based association testing involving microarray-derived genotypes at 41,094 SNPs of six affected and 100 fertile boars yielded strong association (P = 4.22 × 10-15) at chromosome 12. Autozygosity mapping enabled us to pinpoint the causal mutation on a 1.11 Mb haplotype located between 3,473,632 and 4,587,759 bp. The haplotype carries an intronic 13-bp deletion (Chr12:3,556,401-3,556,414 bp) that is compatible with recessive inheritance. The 13-bp deletion excises the polypyrimidine tract upstream exon 56 of DNAH17 (XM_021066525.1: c.8510-17_8510-5del) encoding dynein axonemal heavy chain 17. Transcriptome analysis of the testis of two affected boars revealed that the loss of the polypyrimidine tract causes exon skipping which results in the in-frame loss of 89 amino acids from DNAH17. Disruption of DNAH17 impairs the assembly of the flagellar axoneme and manifests in multiple morphological abnormalities of the sperm flagella. Direct gene testing may now be implemented to monitor the defective allele in the Swiss Large White population and prevent the frequent manifestation of a sterilizing sperm tail disorder in breeding boars.

Cilia and centrosomes: Ultrastructural and mechanical perspectives.

Cilia and centrosomes of eukaryotic cells play important roles in cell movement, fluid transport, extracellular sensing, and chromosome division. The physiological functions of cilia and centrosomes are generated by their dynamics, motions, and forces controlled by the physical, chemical, and biological environments. How an individual cilium achieves its beat pattern and induces fluid flow is governed by its ultrastructure as well as the coordination of associated molecular motors. Thus, a bottom-up understanding of the physiological functions of cilia and centrosomes from the molecular to tissue levels is required. Correlations between the structure and motion can be understood in terms of mechanics. This review first focuses on cilia and centrosomes at the molecular level, introducing their ultrastructure. We then shift to the organelle level and introduce the kinematics and mechanics of cilia and centrosomes. Next, at the tissue level, we introduce nodal ciliary dynamics and nodal flow, which play crucial roles in the organogenetic process of left-right asymmetry. We also introduce respiratory ciliary dynamics and mucous flow, which are critical for protecting the epithelium from drying and exposure to harmful particles and viruses, i.e., respiratory clearance function. Finally, we discuss the future research directions in this field.

FSIP2 can serve as a predictive biomarker for Clear Cell Renal Cell Carcinoma prognosis.

Purpose: To characterize the role of fibrous sheath interacting protein 2 (FSIP2) in the survival outcomes and prognosis of clear cell renal cell carcinoma (ccRCC) patients, which is currently not well understood. Methods: The Oncomine and CCLE databases were used to investigate the differential expression of FSIP2 in ccRCC versus other cancer types. Levels of FSIP2 in 85 ccRCC patients were assessed by immunohistochemical analysis; clinicopathological features related to FSIP2 expression were examined in these patients finally, disease-free survival and overall survival were estimated by survival analysis to elucidate the impact of FSIP2 expression in ccRCC patients. Results: Analysis using the Oncomine database revealed significant upregulation of the FSIP2 gene in papillary RCC, compared to that in normal tissues. Additionally, FSIP2 expression was found to be significantly associated with abnormal platelet count, positive distant metastasis, and death as the incidence of distant metastasis and death were higher in patients with FSIP2 expression compared to those without FSIP2 expression. Survival analysis revealed that FSIP2 expression was significantly related to shorter disease-free survival and overall survival. Meanwhile, patients with FSIP2 expression had worse prognosis than those without FSIP2 expression. Conclusions: FSIP2 expression is associated with poor survival outcomes and poor prognosis in ccRCC patients. FSIP2 may therefore serve as a potential predictive biomarker of ccRCC prognosis.

Novel dynein axonemal assembly factor 1 mutations identified using whole­exome sequencing in patients with primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous disorder caused by dysfunction of the cilia and flagella; however, causative genetic defects have not been detected in all patients with PCD. Seven Chinese Han patients with Kartagener syndrome were enrolled onto the present study. Transmission electron microscopy (TEM) was performed to evaluate the cilial defects and whole­exome sequencing was used to analyze relevant genetic variations in all patients. In two of the seven patients with PCD, four novel dynein axonemal assembly factor 1 (DNAAF1) mutations were identified (NM_178452.6:c.3G>A, c.124+1G>C, c.509delG and c.943A>T) in three alleles. Both of these patients had long­standing infertility. Their chest computed tomography results showed bronchiectasis, lung infections and situs inversus, and paranasal computed tomography revealed sinusitis. Semen analysis of the male patient showed poor sperm motility. TEM showed defects in the inner and outer dynein arms in both patients. The DNAAF1 sequences of family members were then analyzed. Bioinformatics analysis indicated that these mutations may be the cause of the cilial defects in these two probands. Thus, the present study identified novel PCD­causing mutations in DNAAF1 in two patients with PCD. These genetic variations were predicted to alter DNAAF1 amino acid residues and lead to loss of function, thereby inhibiting cilia­mediated motility. Accordingly, the two probands had PCD symptoms, and one of them died due to PCD­associated complications.