New DNAH11 mutations in primary ciliary dyskinesia with normal axonemal ultrastructure

To the Editors:

In primary ciliary dyskinesia (PCD; Mendelian Inheritance in Man database #242650), a rare genetic disorder, the dysfunctional motility of cilia and impaired mucociliary clearance result in a myriad of clinical manifestations including recurrent infections of the respiratory tract, eventually causing lung damage, such as bronchiectasis, laterality defects and male infertility 1. The heterogeneous clinical presentation of PCD and the limitations of transmission electron microscopy (TEM) to assess ultrastructural defects within the cilium may delay the diagnosis 2. Ciliary beat frequency and pattern analyses and nasal nitric oxide (nNO) measurements are helpful 1, 3, 4, but only ciliogenesis in cultured cells has been reported as decisive for the diagnosis of the atypical PCD phenotype with normal axonemal ultrastructure 1, 5. Under these conditions, the identification of disease-causing mutations could overcome the current diagnostic limitations of TEM and improve our understanding of the biology and function of the cilium 2, 6, 7. For these reasons, we have assessed whether an analysis of the DNAH11 gene, in which some nonsense mutations have been reported to be associated with a normal axonemal ultrastructure but with an abnormal nonflexible beating pattern, reduced cilium bending capacity and a hyperkinetic beat 7, could be used to identify new mutations in three atypical PCD patients and thus be used in the diagnostic work-up of these most difficult cases.

We observed three patients (A and B, sister and brother; and C, male), at the ages of 15 yrs and 5 months, 9 yrs and 4 months, and 8 yrs, respectively. The two siblings presented situs inversus. Both patients had chronic respiratory symptoms which required repeated treatment with antibiotics. Patient A also had neonatal purulent rhinitis with frequent relapses, chronic rhinosinusitis and otitis that required adenoidectomy and transtympanic drainage at the age of 6 yrs. Patient B also presented recurrent wheezy bronchitis and pneumonia. Patient C had neonatal respiratory distress unrelated to any evident cause, situs inversus, partial inter-atrial defect, recurrent otitis and pneumonia from the age of 18 months.

Moreover, patient A presented bronchiectasis and atelectasis of the left middle lobe associated with fibrotic areas at chest high-resolution computed tomography and pan-sinusitis at computed tomography scan. Pan-sinusitis and bronchioloectasis of the left middle lobe associated with fibrotic areas were also found in patient B, while a fibrotic area in the left middle lobe, maxillary and sphenoid sinusitis with agenesis of frontal sinuses were found in patient C.

In all three patients TEM evaluation showed few nonspecific abnormalities such as swollen cilia (7.7–13.6%) and compound cilia (0.8–1.5%), which are compatible with mild inflammation. None of the patients showed alterations of the central pair and dynein arm deficiencies 5–7. Ciliary motion analysis showed an abnormal nonflexible beating pattern with a reduced cilium bending capacity in 49.9–76.5% of microscopic fields and a hyperkinetic beat of cilia up to 16 Hz (mean±sd normal values in our laboratory: 12.1±1.8 Hz) with immotile cilia in 4–32% of microscopic fields, whereas a very low beat frequency resulted in the remaining fields. In all three patients functional ciliary evaluation after ciliogenesis in culture allowed us make a final diagnosis of PCD in relation to complete absence of either migration or rotation of spheroids 5. nNO was low in all three patients (33.7, 26.9 and 19.8 ppb). Therefore, the diagnosis of PCD in these children relied on clinical features together with typical ciliary beat frequency and pattern phenotypes 1, 3 and on ciliary functional analysis after ciliogenesis in culture 5, as well as on the low level of nNO 4, 8.

Moreover, in all three patients, we identified novel DNAH11 compound heterozygous mutations, which were not found in the control population of 200 chromosomes. In particular, the two siblings carried the same compound heterozygous mutations in the DNAH11 gene (fig. 1a⇓): a splice-site mutation (c.883-1G>A) located in the acceptor splice site of exon 5 and a nonsense mutation (c.4145G>A, p.W1382X) located in exon 23. An analysis of parental samples revealed that the unaffected mother had transmitted the c.883-1G>A mutation while the unaffected father had transmitted the p.W1382X mutation.

The third patient carried compound heterozygous mutations in the DNAH11 gene (fig. 1b⇑): a mis-sense mutation (c.8135A>G, p.H2712R) located in exon 50 and a mis-sense mutation (c.10284G>A, p.G3429R) located in exon 64. An analysis of parental samples revealed that the unaffected mother had transmitted the p.H2712R mutation while the unaffected father had transmitted the p.G3429R mutation.

Obviously, genetic analysis represents a particularly important diagnostic instrument in atypical cases. A homozygous nonsense mutation in dynein heavy chain DNAH11 has previously been associated with immotile cilia despite normal axonemal structure in a subject with situs inversus 9. Due to the concomitant presence of cystic fibrosis in this patient, some doubts were raised concerning the involvement of DNAH11 in this atypical case of PCD. However, DNAH11 mutations were subsequently confirmed in a German family that includes five individuals with PCD, and another with Kartagener syndrome, but with a normal ultrastructure of the respiratory cilia 7. All these six individuals harboured compound heterozygous mutations in DNAH11.

Our results provide additional evidence of the possible role of new mutations in three patients with atypical PCD; and we can confirm that involvement of DNAH11, first observed in an inversus viscerum mouse model 10, is important for organ lateralisation since all our patients presented situs inversus.

Dynein heavy chains consist of an N-terminal stem which interacts with other dynein components and the head or motor domain. The motor contains six tandemly-linked AAA domains in the head, which form a ring. A stalk-like structure protrudes between AAA4 and AAA5 and terminates in a microtubule-binding site 11.

The splice acceptor site c.883-1G>A mutation found in the first family occurs in intron 4. In order to predict whether the splice mutations will lead to an abnormal splicing, we performed in silico analysis using Splice Site Prediction by Neural Network 12. The analysis showed that the mutation may cause exon skipping, activation of different potential cryptic splice-sites and integration of part of intron 4 within the mRNA. The predicted aberrant mRNA could result in a frameshift and a premature stop codon which may lead to the synthesis of a truncated protein or to nonsense-mediated mRNA decay. To test how effectively this mutation affects the splicing, we should have performed cDNA analysis, but the respiratory epithelial cells obtained by nasal brush biopsies to isolate the RNA were not available.

The nonsense mutation p.W1382X located in exon 23 affects the N-terminal stem (1–1861 amino acids) of the protein. Therefore, the two mutations reported should truncate the entire C-terminal domain of the DNAH11 protein and if degraded by the nonsense-mediated mRNA decay pathway, they should lead to downregulate gene expression after the DNAH11 gene has been transcribed.

The mutations found in the second family are both mis-sense mutations and affect two different domains of the protein. The p.H2712R mutation affects the AAA3 domain (2479–2726 aa), while the p.G3429R mutation affects the region between the stalk-like structure (3079–3410 aa) and the AAA5 domain (3466–3693 aa).

In silico, we did an analysis using the PolyPhen program 13 to predict whether a nonsynonymous single-nucleotide substitution is expected to have a deleterious effect. PolyPhen considered it likely that both p.H2712R and p.G3429R would be damaging (PSIC score difference 3.352 and 2.739, respectively), suggesting a deleterious effect on protein conformation and function. These new variants can be categorised as pathogenic mutations and their severity is indicated by the severe phenotype of PCD in these patients.

All of the amino acid substitutions fall into highly conserved regions among different species and are not reported in the HGMD professional database 14.

The identification of the mutation in parents provides an opportunity for genetic counselling; and carrier testing for at-risk family members is an important tool for already-born children as well as for prenatal testing in the case of successive pregnancies.

In conclusion, our newly described mutations confirm and further expand the notion that mutations of DNAH11 are a possible cause of PCD in patients with normal axoneme ultrastructure. Thus, genetic evaluation of this region could be used in the diagnostic work-up in difficult cases and to improve the understanding of the basic biology and function of the cilium.

Support statement

This work was supported by grants from the Unione Industriale Pisana.

Statement of interest

None declared.