Mutations in Radial Spoke Head Genes and Ultrastructural Cilia Defects in East-European Cohort of Primary Ciliary Dyskinesia Patients

Primary ciliary dyskinesia (PCD) is a rare (1/20,000), multisystem disease with a complex phenotype caused by the impaired motility of cilia/flagella, usually related to ultrastructural defects of these organelles. Mutations in genes encoding radial spoke head (RSPH) proteins, elements of the ciliary ultrastructure, have been recently described. However, the relative involvement of RSPH genes in PCD pathogenesis remained unknown, due to a small number of PCD families examined for mutations in these genes. The purpose of this study was to estimate the involvement of RSPH4A and RSPH9 in PCD pathogenesis among East Europeans (West Slavs), and to shed more light on ultrastructural ciliary defects caused by mutations in these genes. The coding sequences of RSPH4A and RSPH9 were screened in PCD patients from 184 families, using single strand conformational polymorphism analysis and sequencing. Two previously described (Q109X; R490X) and two new RSPH4A mutations (W356X; IVS3_2–5del), in/around exons 1 and 3, were identified; no mutations were found in RSPH9. We estimate that mutations in RSPH4A, but not in RSPH9, are responsible for 2–3% of cases in the East European PCD population (4% in PCD families without situs inversus; 11% in families preselected for microtubular defects). Analysis of the SNP-haplotype background provided insight into the ancestry of repetitively found mutations (Q109X; R490X; IVS3_2–5del), but further studies involving other PCD cohorts are required to elucidate whether these mutations are specific for Slavic people or spread among other European populations. Ultrastructural defects associated with the mutations were analyzed in the transmission electron microscope images; almost half of the ciliary cross-sections examined in patients with RSPH4A mutations had the microtubule transposition phenotype (9+0 and 8+1 pattern). While microtubule transposition was a prevalent ultrastructural defect in cilia from patients with RSPH4A mutations, similar defects were also observed in PCD patients with mutations in other genes

MT defects in ciliary cross-sections from patients with <i>RSPH4A</i> mutations.

<p>Letters C, E and P next to the 8+1 MT pattern denote position of a transposed doublet in the axoneme: central, eccentric and at the perimeter, respectively. Nd: cross-section plane and the presence of microvilli not determined, due to the low number of ciliary cross-sections.</p

Evolutionary conservation of the genomic sequence surrounding newly found sequence changes in the <i>RSPH4A</i> gene.

<p>Mutated sequences are underlined. A point denotes identity, dash – deletion; fragments of the sequences of exon 3 and 6 in <i>RSPH4A</i> are shown in bold.</p

Localization of the sequences recognized by <i>Homo sapiens</i> micro RNAs (Hsa miRNAs) within the 3′UTR of <i>RSPH9</i> sequence.

<p>The genomic sequence (last 15 bp of exon 6 and first 105 bp of 3′UTR) shown in the left column is underlined with the dotted line, with the coding sequence and UTR indicated by upper and lower case, respectively; positions +52A and +73G, mutated in some samples, are indicated (bold and heavy underline). MiRNA sequences identified through in silico search are shown below, aligned with the genomic sequence, with the complementary bases shown in uppercase. MirSVR scores (support vector regression algorithm for the prediction of the miRanda-predicted microRNA target sites; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033667#pone.0033667-Betel1" target="_blank">[38]</a>) are indicated next to each miRNA name; high scores are underlined. The A at position +52 in the 3′UTR sequence is complementary to U in four miRNAs; the A>G transition at +52 would increase complementarity between 3′UTR and the miR-127-5p. No miRNAs complementary to the sequence encompassing +73 in the 3′UTR were found.</p

SNP haplotypes in the <i>RSPH4A</i> gene.

<p>SNPs: S1: rs13213314; S2: rs41289942; S3: re117169123; S4: rs6927567; S5: rs784133; S6: 41290844; S7: rs9488991; S8: new; S9: new; S10: rs9488993; S11: rs6925922. Mutations: M1: Q109X (rs118204042); M2: W356X (new); M3: R490X (rs118204043); M4: IVS3+(2–5)del (new). Only the derived (non-ancestral) alleles are indicated in the haplotype variants; causative alleles in the “mutated” haplotypes are in bold. Counts of each haplotype in the examined PCD and non-PCD chromosomes are indicated in two rightmost columns; haplotype frequency distribution did not significantly differ between the affected and non-affected chromosomes (Fisher exact test, not shown). The single haplotype 3r, carrying one of two R490X alleles, contains a putative recombination between the mutation-carrying haplotype 3 and the frequent neutral haplotype 8. An asterisk indicates two mutation-carrying chromosomes found in the single consanguineous family. N1 = TAGG in IVS3_2–5; N2 = GATACTCACAG in 3′UTR; D1 = TAGG deletion in intron 3; D2 = GATACTCACAG deletion in 3′UTR; e – exon; i – intron; 3′ – 3′UTR.</p

PCD families with new sequence changes identified in <i>RSPH4A</i> and <i>RSPH9</i> genes.

<p><b>A</b> Causative mutations in <i>RSPH4A</i>. Segregation of the mutated alleles is consistent with the recessive mode of inheritance. No <i>situs</i> inversus was observed in any of the affected members; <b>B</b> New SNPs in <i>RSPH4A</i>; <b>C</b> New SNPs in <i>RSPH9</i>. Upper panels – pedigrees of the families; lower panels – sequencing chromatograms.</p

Transmission electron microscope analysis of the bronchial epithelium samples from PCD patients with <i>RSPH4A</i> mutations.

<p><b>A</b> Ciliary cross sections – magnification 30,000 (patient #181). <i>Left panel</i> – a typical picture of proximal ciliary cross sections (cilia are accompanied by numerous microvilli); <i>right panel</i> – ciliary cross sections taken at the distance from the cell membrane (no microvilli present); <b>B</b> Examples of MT defects in patients #181, #337 and #340 (a blown-up view). <b>C</b> Longitudinal sections of axonemes (patient #337); <i>left panel</i>: magnification 16,000; <i>right panel</i> – a blown up view of a single cilium. White, black and hashed arrows indicate 9+2, 9+0 and 8+1 MT arrangement, respectively.</p

<i>In silico</i> prediction of the effect of IVS3+2–5del in the <i>RSPH4A</i> gene.

<p>Default splice sites in IVS3 result in the proper protein sequence (donor in phase 2, acceptor in phase 0). All alternative donor sites predicted for the sequence with IVS3+2–5del mutation (located either within exon 3 or within intron 3, all in phase 1) result in a frameshift and a premature stop codon; acceptor site remains unchanged. Intronic sequences in the <i>RSPH4A</i> sequence are indicated by lowercase letters. The two most conserved positions of a consensus donor and acceptor splice site are underlined.</p

Occurrence of <i>RSPH4A</i> mutations among examined European PCD families.

<p>The 460C>T (Q154X) mutation (rs118204041), found only in four consanguineous Pakistani families <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033667#pone.0033667-Castleman1" target="_blank">[19]</a>, is not included in the Table.</p