Loss-of-function mutations in the human ortholog of Chlamydomonas reinhardtii ODA7 disrupt dynein arm assembly and cause primary ciliary dyskinesia.

Cilia and flagella are evolutionarily conserved structures that play various physiological roles in diverse cell types. Defects in motile cilia result in primary ciliary dyskinesia (PCD), the most prominent ciliopathy, characterized by the association of respiratory symptoms, male infertility, and, in nearly 50% of cases, situs inversus. So far, most identified disease-causing mutations involve genes encoding various ciliary components, such those belonging to the dynein arms that are essential for ciliary motion. Following a candidate-gene approach based on data from a mutant strain of the biflagellated alga Chlamydomonas reinhardtii carrying an ODA7 defect, we identified four families with a PCD phenotype characterized by the absence of both dynein arms and loss-of-function mutations in the human orthologous gene called LRRC50. Functional analyses performed in Chlamydomonas reinhardtii and in another flagellated protist, Trypanosoma brucei, support a key role for LRRC50, a member of the leucine-rich-repeat superfamily, in cytoplasmic preassembly of dynein arms.

A heterozygous mutation disrupting the SPAG16 gene results in biochemical instability of central apparatus components of the human sperm axoneme.

The SPAG16 gene encodes two major transcripts, one for the 71-kDa SPAG16L, which is the orthologue of the Chlamydomonas rheinhardtii central apparatus protein PF20, and a smaller transcript, which codes for the 35-kDa SPAG16S nuclear protein that represents the C-terminus (exons 11-16) of SPAG16L. We have previously reported that a targeted mutation in exon 11 of the Spag16 gene impairs spermatogenesis and prevents transmission of the mutant allele in chimeric mice. In the present report, we describe a heterozygous mutation in exon 13 of the SPAG16 gene, which causes a frame shift and premature stop codon, affording the opportunity to compare mutations with similar impacts on SPAG16L and SPAG16S for male reproductive function in mice and men. We studied two male heterozygotes for the SPAG16 mutation, both of which were fertile. Freezing-boiling of isolated sperm from both affected males resulted in the loss of the SPAG16L protein, SPAG6, another central apparatus protein that interacts with SPAG16L, and the 28-kDa fragment of SPAG17, which associates with SPAG6. These proteins were also lost after freezing-boiling cycles of sperm extracts from mice that were heterozygous for an inactivating mutation (exons 2 and 3) in Spag16. Our findings suggest that a heterozygous mutation that affects both SPAG16L and SPAG16S does not cause male infertility in man, but is associated with reduced stability of the interacting proteins of the central apparatus in response to a thermal challenge, a phenotype shared by the sperm of mice heterozygous for a mutation that affects SPAG16L.

A common variant in combination with a nonsense mutation in a member of the thioredoxin family causes primary ciliary dyskinesia.

Thioredoxins belong to a large family of enzymatic proteins that function as general protein disulfide reductases, therefore participating in several cellular processes via redox-mediated reactions. So far, none of the 18 members of this family has been involved in human pathology. Here we identified TXNDC3, which encodes a thioredoxin-nucleoside diphosphate kinase, as a gene implicated in primary ciliary dyskinesia (PCD), a genetic condition characterized by chronic respiratory tract infections, left-right asymmetry randomization, and male infertility. We show that the disease, which segregates as a recessive trait, results from the unusual combination of the following two transallelic defects: a nonsense mutation and a common intronic variant found in 1% of control chromosomes. This variant affects the ratio of two physiological TXNDC3 transcripts: the full-length isoform and a novel isoform, TXNDC3d7, carrying an in-frame deletion of exon 7. In vivo and in vitro expression data unveiled the physiological importance of TXNDC3d7 (whose expression was reduced in the patient) and the corresponding protein that was shown to bind microtubules. PCD is known to result from defects of the axoneme, an organelle common to respiratory cilia, embryonic nodal cilia, and sperm flagella, containing dynein arms, with, to date, the implication of genes encoding dynein proteins. Our findings, which identify a another class of molecules involved in PCD, disclose the key role of TXNDC3 in ciliary function; they also point to an unusual mechanism underlying a Mendelian disorder, which is an SNP-induced modification of the ratio of two physiological isoforms generated by alternative splicing.

Mutations of DNAI1 in primary ciliary dyskinesia: evidence of founder effect in a common mutation.

RATIONALE: Primary ciliary dyskinesia (PCD) is a rare, usually autosomal recessive, genetic disorder characterized by ciliary dysfunction, sino-pulmonary disease, and situs inversus. Disease-causing mutations have been reported in DNAI1 and DNAH5 encoding outer dynein arm (ODA) proteins of cilia. OBJECTIVES: We analyzed DNAI1 to identify disease-causing mutations in PCD and to determine if the previously reported IVS1+2_3insT (219+3insT) mutation represents a "founder" or "hot spot" mutation. METHODS: Patients with PCD from 179 unrelated families were studied. Exclusion mapping showed no linkage to DNAI1 for 13 families; the entire coding region was sequenced in a patient from the remaining 166 families. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on nasal epithelial RNA in 14 families. RESULTS: Mutations in DNAI1 including 12 novel mutations were identified in 16 of 179 (9%) families; 14 harbored biallelic mutations. Deep intronic splice mutations were not identified by reverse transcriptase-polymerase chain reaction. The prevalence of mutations in families with defined ODA defect was 13%; no mutations were found in patients without a defined ODA defect. The previously reported IVS1+2_3insT mutation accounted for 57% (17/30) of mutant alleles, and marker analysis indicates a common founder for this mutation. Seven mutations occurred in three exons (13, 16, and 17); taken together with previous reports, these three exons are emerging as mutation clusters harboring 29% (12/42) of mutant alleles. CONCLUSIONS: A total of 10% of patients with PCD are estimated to harbor mutations in DNAI1; most occur as a common founder IVS1+2_3insT or in exons 13, 16, and 17. This information is useful for establishing a clinical molecular genetic test for PCD.

Isolation and expression of the human hPF20 gene orthologous to Chlamydomonas PF20: evaluation as a candidate for axonemal defects of respiratory cilia and sperm flagella.

Primary ciliary dyskinesia (PCD) is a heterogeneous congenital disorder characterized by bronchiectasis and chronic sinusitis, sometimes associated with situs inversus (i.e., Kartagener's syndrome) and male infertility. At the cell level, the disease phenotype includes various axonemal abnormalities of respiratory cilia and sperm flagella. We have previously isolated DNAI1, the first gene involved in these diseases in patients lacking outer dynein arms. In this study, designed to find additional genes for other axonemal defects, we report the isolation of a novel human gene, hPF20, which is orthologous to Chlamydomonas pf20. The hPF20 gene is expressed as two major transcripts: one is expressed in testis only, whereas the second is weakly expressed in many other tissues. As flagella of Chlamydomonas strains carrying pf20 mutations lack the axonemal central complexes, we tested the involvement of the hPF20 gene in the disease phenotype of five patients in whom cilia or flagella display abnormal central complexes. Five intragenic polymorphisms were identified and used to exclude hPF20 in two consanguineous patients, while no mutation was found in the remaining patients. However, given the genetic heterogeneity of PCD, we consider that this gene remains a good candidate to be investigated in patients with abnormal central complexes.