DNAH5 mutations are a common cause of primary ciliary dyskinesia with outer dynein arm defects

Abstract

Rationale: Primary ciliary dyskinesia (PCD) is characterized by recurrent airway infections and randomization of left-right body asymmetry. To date, autosomal recessive mutations have only been identified in a small number of patients involving DNAI1 and DNAH5, which encode outer dynein arm components.

Methods: We screened 109 white PCD families originating from Europe and North America for presence of DNAH5 mutations by haplotype analyses and/or sequencing.

Results: Haplotype analyses excluded linkage in 26 families. In 30 PCD families, we identified 33 novel (12 nonsense, 8 frameshift, 5 splicing, and 8 missense mutations) and two known DNAH5 mutations. We observed clustering of mutations within five exons harboring 27 mutant alleles (52%) of the 52 detected mutant alleles. Interestingly, 6 (32%) of 19 PCD families with DNAH5 mutations from North America carry the novel founder mutation 10815delT. Electron microscopic analyses in 22 patients with PCD with mutations invariably detected outer dynein arm ciliary defects. High-resolution immunofluorescence imaging of respiratory epithelial cells from eight patients with DNAH5 mutations showed mislocalization of mutant DNAH5 and accumulation at the microtubule organizing centers. Mutant DNAH5 was absent throughout the ciliary axoneme in seven patients and remained detectable in the proximal ciliary axoneme in one patient carrying compound heterozygous splicing mutations at the 3'-end (IVS75-2A>T, IVS76+5G>A). In a preselected subpopulation with documented outer dynein arm defects (n = 47), DNAH5 mutations were identified in 53% of patients.

Conclusions: DNAH5 is frequently mutated in patients with PCD exhibiting outer dynein arm defects and mutations cluster in five exons.

Figure 1.

Distribution of novel and published DNAH5 mutations. Top: schematic presentation of the genomic structure of DNAH5. Exons are numbered and indicated by black boxes. Open boxes represent untranslated regions. ATG, start codons, TAA, stop codon. Intron sizes are not drawn to scale. The positions of the intronic DNAH5 mutations are indicated by vertical lines. Bottom: schematic drawing of DNAH5. The P-loop domains and the microtubule binding domain are shown. The positions of all identified exonic DNAH5 mutations are indicated by vertical lines. Mutations that were detected in several unrelated families are marked by an asterisk (⩾ 4 PCD families) and dagger (2 PCD families). Mutations that were identified in our previous study are indicated by gray boxes (19). Novel mutations are depicted in red.

Figure 2.

Mis-localization of mutant DNAH5 in respiratory epithelial cells from patients with primary ciliary dyskinesia carrying DNAH5 mutations. Confocal immunofluorescence analysis of human respiratory epithelial cells with anti-DNAH5 antibodies (red) and with antibodies against axoneme-specific acetylated α-tubulin (green) as control (21). Nuclei were stained with Hoechst 33342 (blue). (A) In respiratory epithelial cells from healthy probands, DNAH5 localizes along the entire length of the axonemes and at the microtubule organizing centers. (B) In respiratory epithelial cells from patient OP40 II1, carrying the compound heterozygous DNAH5 mutations R1716L and 4487NfsX1, mutant DNAH5 is expressed and correctly targeted to the ciliary base where it accumulates. In contrast to healthy control subjects, we observed complete absence of mutant DNAH5 from the ciliary axonemes. (C) In Patient F718 II1, who carries compound heterozygous DNAH5 mutations affecting splicing at the 3′-end of the gene, mutant DNAH5 is absent from the distal part of the ciliary axonemes but is still detectable within the proximal part.