Mutation screening of dynein genes in patients affected by primary ciliary diskinesia or Kartagener syndrome

Primary ciliary dyskinesia (PCD) is the most prominent genetic abnormality involving motile cilia. PCD is a rare (1:15,000), autosomal recessive, genetically heterogeneous disorder, characterized by sino-pulmonary disease and male infertility. Presence of laterality defects set up the Kartagener syndrome (KS, prevalence = 1:30,000). Ciliary ultrastructural defects are identified in about 90% of PCD patients and involve the dynein arms. Recent mutational analysis demonstrated that about 40% of PCD patients carry mutations of the dynein genes DNAI1 and DNAH5, which encode for the intermediate and high chains of dynein, respectively. The aim of our study was to perform molecular screening of 28 Southern-Italy independent patients affected by PCD (n= 8) or KS (n= 20) syndrome. We set up a protocol to amplify the 80 DNAH5 and the 20 DNAI1 exons by means 30 and 8 multiplex-PCR, respectively. Particularly, 38 different multiplex-PCR were made using 10 different annealing temperatures. So far, we have analysed 10 patients and we identified 4 new mutations in the DNAH5 gene in 3 independent patients: p.R224X, p.Q1450X, p.R1883X and c.3876_4053+158del. Mutations p.R224X, p.Q1450X and p.R1883X introduce a stop codon, producing truncated proteins. We analysed mRNA extracted from nasal brushing to evaluate effects of c.3876_4053+158del and identified 3 fragments of unequal intensity: a WT fragment, a fragment with deletion of exon 25, and a fragment with deletion of exon 25 and insertion of intron 26. We are now performing in vitro splicing assay to better characterize this splice site mutation. Moreover, we identified 4 missense variations in the DNAH5 gene in 4 patients: p.N402K, p.N549K, p.A3597S and p.F4201F. Variations p.N402K and p.N549K were found in healthy population at frequency of 2 and 0,77%, respectively. Therefore these variants may be considered polymorphisms of DNAH5 gene. Variations p.A3597S and p.F4201F were excluded in 656 and 322 chromosomes, respectively. More data are necessary to evaluate the pathogenetic role of these new variants. Furthermore, we purpose to use the new technology of ultra-high throughput DNA sequencing for a much more extensive study of all the loci that may be involved in PCD and KS.