YI6 / 158D – Mutations in DNAH5 and DNAI1 hot-spot exons in Czech pediatric patients with primary ciliary dyskinesia.

JDjakow1, TSvobodova1, JUhlik2, OCinek1, PPohunek1.

1University Hospital Motol and 2nd Faculty of Medicine Department of Pediatrics – Prague, Czech Republic

22nd Faculty of Medicine Department of Histology and Embryology – Prague, Czech Republic

Introduction

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease characterised by a congenital dysfunction of ciliary motility. The aim of this study was to test for mutations in the regions with a high occurence of mutations in the DNAH5 and DNAI1 genes in Czech paediatric patients with the diagnosis of PCD.

Methods

Phenotype data and informed consents were obtained from patients with PCD whose diagnosis was based on typical clinical findings and either electron microscopy or repeated high-speed videomicroscopy or both. The DNA of the patients was extracted from saliva or blood. As the genes are considerably long, selected hot-spot exons and their flanking regionswere bidirectionally sequenced in DNAH5 (exons: 33, 34, 36, 50, 63, 76 and 77) and in DNAI1 (exons: 1, 16, 17, 18 and 19). Two independent evaluators reviewed the results.

Results

Out of 24 tested patients with the PCD, three carried a heterozygous mutation in the tested exons of the DNAH5 gene; one patient was a compound heterozygote carrying mutations in DNAH5 exon 50 and 63; and aheterozygous mutation in the tested region of DNAI1 was found in one patient. Two of the mutations found in DNAH5 gene are novel missence mutations affecting highly conserved sites of the gene. They were not found in healthy controls. All the patients with mutations in DNAH5 gene had immotile cilia and outer dynein arms defects. Three patients had situs viscerum inversus. Previously described splice-site mutation in DNAI1 intron 1 was found in a patient with typical clinical presentation and situs viscerum inversus. The cilia of the patient were characterised by residual movement with slow mean frequency of 2 Hz. Electron microscopy detected no structural abnormality.

Conclusion

Sequencing of the hot-spot exons has been proposed as the first-step of genetic testing in the diagnostic protocol as it should be capable of identifying at least one mutated allele in most patients. However, our data show that the locations of the mutations may be population-specific as the selection of hot-spot exons performs suboptimally in our dataset. The strategy towards the genetic confirmation of PCD may therefore have to be reconsidered, be it either adding further exons, whole gene sequencing, or RNA analysis. Also in patients with one detected mutation these methods should be used for detection of the second mutation.

Acknowledgement

Project has been supported by the Charles University Grant Agency, Project No. 48409.