Mutations of DNAH11 in patients with primary ciliary dyskinesia with normal ciliary ultrastructure

Abstract

Rationale: Primary ciliary dyskinesia (PCD) is an autosomal recessive, genetically heterogeneous disorder characterised by oto-sino-pulmonary disease and situs abnormalities (Kartagener syndrome) due to abnormal structure and/or function of cilia. Most patients currently recognised to have PCD have ultrastructural defects of cilia; however, some patients have clinical manifestations of PCD and low levels of nasal nitric oxide, but normal ultrastructure, including a few patients with biallelic mutations in dynein axonemal heavy chain 11 (DNAH11).

Objectives: To test further for mutant DNAH11 as a cause of PCD, DNAH11 was sequenced in patients with a PCD clinical phenotype, but no known genetic aetiology.

Methods: 82 exons and intron/exon junctions in DNAH11 were sequenced in 163 unrelated patients with a clinical phenotype of PCD, including those with normal ciliary ultrastructure (n=58), defects in outer and/or inner dynein arms (n=76), radial spoke/central pair defects (n=6), and 23 without definitive ultrastructural results, but who had situs inversus (n=17), or bronchiectasis and/or low nasal nitric oxide (n=6). Additionally, DNAH11 was sequenced in 13 subjects with isolated situs abnormalities to see if mutant DNAH11 could cause situs defects without respiratory disease.

Results: Of the 58 unrelated patients with PCD with normal ultrastructure, 13 (22%) had two (biallelic) mutations in DNAH11; and two patients without ultrastructural analysis had biallelic mutations. All mutations were novel and private. None of the patients with dynein arm or radial spoke/central pair defects, or isolated situs abnormalities, had mutations in DNAH11. Of the 35 identified mutant alleles, 24 (69%) were nonsense, insertion/deletion or loss-of-function splice-site mutations.

Conclusions: Mutations in DNAH11 are a common cause of PCD in patients without ciliary ultrastructural defects; thus, genetic analysis can be used to ascertain the diagnosis of PCD in this challenging group of patients.

Figure 1. Schematic representation of DNAH11 (not to the scale) showing AAA 1–6 domains, four P-loop, Microtubule binding domain (MTB) and Helix-1 and 2

Positions of the all the mutations are shown.

Figure 2. Representative pedigrees showing autosomal recessive mode of inheritance for DNAH11 mutations

Segregation analysis from the parents, siblings and the extended family members demonstrates that mutations were inherited in trans (A–D), and there was no bias for gender or situs status. Additional pedigrees are presented in supplemental data.

Figure 3. Effect of splice-site mutations on the DNAH11 transcript using Reverse Transcriptase-polymerase chain reaction (RT-PCR)

(A) Splice-acceptor site mutation in intron 13 (c.2275-1 G>C) in patient PCD761 led to the in-frame deletion of exon 14 that consisted of 131 amino-acid residues. (B) Splice-donor site mutation in intron 23 (c.4254+5G>T) in patient OP406-II:2 led to the in-frame deletion of exon 23 that consisted of 53 amino-acid residues. (C) Splice-acceptor site mutation in intron 26 (c.4726-1G>A) in patient OP406-II:2 led to out-of-frame deletion of exon 27, and resulted in a premature stop signal. (D) Splice-donor site mutation in intron 44 (c.7266+1G>A) in patient PCD 108 led to the in-frame deletion of exon 44 that consisted of 44 amino-acid residues. (E) Splice-donor site mutation in exon 48 (c.7914G>C) in patient OP98-II:1 led to out-of-frame deletion of exon 48, and resulted in a premature stop signal. (F) Splice-donor site mutation in intron 33 (c.5778+1G>A) in patient PCD565 led to out-of-frame deletion of exons 32–35, and resulted in a premature stop signal. The cDNA was available only from the carrier parent of the patient PCD565, which was used to check the transcript. All of the six panels with three electropherograms each shows the genomic location of the mutation (top) with a red arrow and bases underlined, mutant cDNA transcript (middle) and wild type transcript (bottom). Amino-acid residues are italicized and the protein product due to the out-of frame mutation is shown with the red fonts. Genomic base change for the mutation is shown with underline. A known single nucleotide polymorphism (SNP) was observed in OP98-II:1 and its location is shown. Further details on RT-PCR are shown in Table 3 (primer sequences shown in Supplement, Table E3).