Castleman, V.H.; (2008) The genetic and molecular analysis of primary ciliary dyskinesia. Doctoral thesis , University of London.

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Abstract

Primary Ciliary Dyskinesia (PCD) is a recessively inherited disorder caused by cilia and sperm flagella dysmotility associated with axoneme ultrastructural defects. Symptoms include recurrent respiratory tract infections, sinusitis, bronchiectasis, subfertility, and laterality defects due to defective embryonic nodal cilia. PCD is genetically heterogeneous and two genes, DNAI1 and DNAH5, account for 38% of cases. To identify new PCD genes, genome wide linkage screens were undertaken in consanguineous PCD families using homozygosity mapping: (1) five Pakistani families with missing inner and outer dynein arms (2) two Arabic families with central pair agenesis and no dextrocardia. Three disease loci were mapped on chromosome llq23.3-24.3 and 17q21.31-22 (Pakistani), and chromosome 6p21.31-21.1 (Arabic), with peak multipoint LOD scores of 3.6, 6.0 and 6.7, respectively. Comparative bioinformatic analysis identified 7 positional candidate genes which were subjected to mutational analysis. A 3 bp deletion in C6ORF206 at 6p21.31-21.1 was revealed in affected Arabic PCD individuals, resulting in a predicted in-frame loss of a C-terminal lysine residue, K268. The protein encoded by C6ORF206 was identified as homologous to the Chlamydomonas reinhardtii radial spoke head protein, RSP9. Functional work was undertaken to determine if the K268del mutation was pathogenic. RSP9 is mutated in the paralysed flagella Chlamydomonas mutant, pfl 7, and transformation of pfl 7 with wild-type RSP9 rescued motility. However transformation of pfl 7 with RSP9 R261del (equivalent to human K268del) rescued motility to a lower level, resulting in an ineffective swimming stroke. Expression of RSP9 was investigated by in situ hybridisation, and zebrafish RSP9 knock-down morphants were created to investigate its role in vertebrate ciliary function. Although expressed at the vertebrate embryonic node, knockdown of RSP9 function did not affect laterality in zebrafish, however it did cause dysfunction of the nasal cilia. This data suggests that C6ORF206/RSP9 functions as a ciliary protein in ciliated organisms and that the K268del mutation likely causes PCD without situs inversus.

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