LRRC50

Abstract: Cilia perform essential motile and sensory functions central to many developmental and physiological processes. Disruption of their structure or function can have profound phenotypic consequences, and has been linked to left-right patterning and polycystic kidney disease. In a forward genetic screen for mutations affecting ciliary motility, we isolated zebrafish mutant hu255H. The mutation was found to disrupt an ortholog of the uncharacterized highly conserved human SDS22-like leucine-rich repeat (LRR)-containing protein LRRC50 (16q24.1) and Chlamydomonas Oda7p. Zebrafish lrrc50 is specifically expressed in all ciliated tissues. lrrc50hu255H mutants develop pronephric cysts with an increased proliferative index, severely reduced brush border, and disorganized pronephric cilia manifesting impaired localized fluid flow consistent with ciliary dysfunction. Electron microscopy analysis revealed ultrastructural irregularities of the dynein arms and misalignments of the outer-doublet microtubules on the ciliary axonemes, suggesting instability of the ciliary architecture in lrrc50hu255H mutants. The SDS22-like leucine-rich repeats present in Lrrc50 are necessary for proper protein function, since injection of a deletion construct of the first LRR did not rescue the zebrafish mutant phenotype. Subcellular distribution of human LRRC50-EGFP in MDCK and HEK293T cells is diffusely cytoplasmic and concentrated at the mitotic spindle poles and cilium. LRRC50 RNAi knock-down in human proximal tubule HK-2 cells thoroughly recapitulated the zebrafish brush border and cilia phenotype, suggesting conservation of LRRC50 function between both species. In summary, we present the first genetic vertebrate model for lrrc50 function and propose LRRC50 to be a novel candidate gene for human cystic kidney disease, involved in regulation of microtubule-based cilia and actin-based brush border microvilli.

Abstract: The genetic disorder primary ciliary dyskinesia (PCD) arises from dysmotility of cilia in the respiratory tract, brain ventricles, oviduct and the embryonic node. Patients have chronic obstructive pulmonary disease, reduced fertility and situs abnormalities. PCD is genetically heterogeneous with 12 genes causing ~40% of all cases, two encoding proteins (KTU, LRRC50) involved in cytosolic axonemal dynein co-assembly. We have identified mutations in the C19ORF51 gene located within a previously mapped PCD locus. C19ORF51 encodes a protein orthologous to PF22, a Chlamydomonas protein involved in the cytoplasmic assembly of outer dynein arms preceeding their import into the axoneme. Chlamydomonas pf22 cells display a disturbance in their cytoplasm of dynein heavy chain stabilityand the co-assembly of heavy with intermediate chains, both essential for dynein arm assembly. PF22 appears to act downstream of KTU and LRRC50 in the dynein preassembly pathway. PF22 knockdown in zebrafish causes a loss of dynein arms, cilia dysmotility, and a typical ciliopathy phenotype with axis curvature, pronephric cysts, hydrocephalus and situs inversus. We propose the existance of a conserved multi-step pathway for formation of assembly-competent dynein complexes, and that PF22 (now renamed DNAAF3, “dynein axonemal assembly factor 3”) mutations cause PCD with situs inversus due to deficient cytoplasmic dynein assembly.

Abstract: Cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and diffuse panbronchiolitis (DPB) are rare airway diseases CF is the most common life-shortening genetic disorder in Caucasians, caused by mutations in a single gene on the long arm of chromosome 7 that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) The predominant CFTR mutation is Phe508del, yet more than 2000 variants in this gene have been identified, which can be divided into six classes Class II mutations, including Phe508del, cause retention of a misfolded protein in the endoplasmic reticulum and subsequent degradation in the proteasome Patients with Class I, II, and III mutations, which are associated with loss of CFTR function, typically have a severe phenotype, whereas individuals with Class IV, V, and VI mutations, which retain residual CFTR function, have mild lung phenotypes and pancreatic sufficiency PCD is usually inherited in an autosomal recessive manner and is genetically heterogeneous Of the 30 mutations that are known to cause PCD, those affecting the DNAH5, DNAI1, DNAAF1 (LRRC50), LRRC6, CCDC39, CCDC40, and DNAH11 genes are found in 15–21%, 2–9%, 4–5%, 3%, 2–10%, 2–8%, and 6% of patients, respectively In terms of the relationship between phenotype and genotype, mutation of DNAH5, DNAI1, DNAI2, DNAL1, CCDC114, TXNDC3 (NME8), or ARMC4 results in loss of the outer dynein arms In regard to DPB, an interaction of environmental and genetic factors is thought to underpin the disease The most probable location for DPB susceptibility genes is thought to lie in a 200 kb major histocompatibility complex (MHC) class I region between HLA-A and HLA-B This contains the DPB critical region 1 gene (DPCR1, chromosome 6p2133), as well as MUC21, and the panbronchiolitis-related mucin-like genes 1 and 2 (PBMUCL1 and PBMCL2) The fact that DPCR1, MUC21, PBMUCL1, and PBMUCL2 are all mucin or mucin-like genes is highly relevant for the excessive airway mucus secretion that is typical in DPB In summary, CF and PCD are both hereditary disorders of mucociliary clearance that result in chronic upper and lower airways disease, while in DPB, it is thought that genetic factors may determine disease susceptibility

Abstract: Genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility in primary ciliary dyskinesia (PCD). The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Here, we demonstrate that large genomic deletions, as well as point mutations involving LRRC50, are responsible for a distinct PCD variant that is characterized by a combined defect involving assembly of the ODAs and IDAs. Functional analyses showed that LRRC50 deficiency disrupts assembly of distally and proximally DNAH5- and DNAI2-containing ODA complexes, as well as DNALI1-containing IDA complexes, resulting in immotile cilia. On the basis of these findings, we assume that LRRC50 plays a role in assembly of distinct dynein-arm complexes.