Mutations in RSPH1 cause primary ciliary dyskinesia with a unique clinical and ciliary phenotype

Abstract

Rationale: Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder of motile cilia, but the genetic cause is not defined for all patients with PCD.

Objectives: To identify disease-causing mutations in novel genes, we performed exome sequencing, follow-up characterization, mutation scanning, and genotype-phenotype studies in patients with PCD.

Methods: Whole-exome sequencing was performed using NimbleGen capture and Illumina HiSeq sequencing. Sanger-based sequencing was used for mutation scanning, validation, and segregation analysis.

Measurements and main results: We performed exome sequencing on an affected sib-pair with normal ultrastructure in more than 85% of cilia. A homozygous splice-site mutation was detected in RSPH1 in both siblings; parents were carriers. Screening RSPH1 in 413 unrelated probands, including 325 with PCD and 88 with idiopathic bronchiectasis, revealed biallelic loss-of-function mutations in nine additional probands. Five affected siblings of probands in RSPH1 families harbored the familial mutations. The 16 individuals with RSPH1 mutations had some features of PCD; however, nasal nitric oxide levels were higher than in patients with PCD with other gene mutations (98.3 vs. 20.7 nl/min; P < 0.0003). Additionally, individuals with RSPH1 mutations had a lower prevalence (8 of 16) of neonatal respiratory distress, and later onset of daily wet cough than typical for PCD, and better lung function (FEV1), compared with 75 age- and sex-matched PCD cases (73.0 vs. 61.8, FEV1 % predicted; P = 0.043). Cilia from individuals with RSPH1 mutations had normal beat frequency (6.1 ± Hz at 25°C), but an abnormal, circular beat pattern.

Conclusions: The milder clinical disease and higher nasal nitric oxide in individuals with biallelic mutations in RSPH1 provides evidence of a unique genotype-phenotype relationship in PCD, and suggests that mutations in RSPH1 may be associated with residual ciliary function.

Figure 1.

Genomic organization, location of primary ciliary dyskinesia (PCD)-causing mutations, and transcript analysis of RSPH1. (A) Schematic showing genomic organization of RSPH1 (NM_080860.2), consisting of nine exons with 1,430 bp transcript, encoding 309 amino acids protein. Solid box designates exons “E,” horizontal lines designates introns “IVS,” and locations of 5′UTR, 3′UTR, start and stop codons are shown. Introns/exons are not drawn to scale. The positions of all four identified RSPH1 putative mutations are indicated. (B and C) Homozygous (−/−) mutation in affected (bottom), heterozygous (+/−) carrier (middle), and corresponding wild-type (WT) sequences in control (top) for c.85G>T (p.Glu29*) and c.275–2A>C (splice acceptor site) mutations, respectively. (D and E) Heterozygous (+/−) mutation in affected (bottom) and corresponding WT sequences in control (top) for c.287dupA (p.Asn96Lysfs*2) and c.407_410delAGTA (p.Lys136Metfs*6) mutations, respectively. (F) Schematic showing WT transcripts of RSPH1. (G–J) Effect of homozygous c.275–2A>C splice acceptor site mutation from affected individual (#1187 [II-1]) was interrogated using reverse-transcriptase polymerase chain reaction with 2F+9R primers (see Table E3 for primer sequences). Two major transcripts and multiple minor transcripts were observed. (G and H) Major transcript 1 and 2 led to the out-of-frame deletion of exons 4–6 and exons 4 and 5, respectively, resulting in the premature translation termination signal. Schematic of the mutant transcripts and the corresponding electropherograms with exact location of the out-of-frame deletions are shown. Additional minor mutant transcripts are depicted in Figure E3. Forward and reverse primers are designated as “F” and “R,” respectively, along with their cDNA locations, and amplicon sizes are shown in red fonts. Base sequence, amino acid sequence, and codon numbers are shown. Locations of the mutations are designate by an arrow and exon-exon junctions are shown by the vertical solid red lines. Further details with 2F+5R primer set are depicted in Figure E4.

Figure 2.

Effect of the c.275–2A>C splice site mutation on RSPH1 expression in vivo. (A) RNA was prepared from cultured human bronchial epithelial (HBE) cells from a normal subject and nasal scrape biopsies from individuals with primary ciliary dyskinesia (PCD) (#1196 [II-2], #1187 [II-1]) with homozygous splice site (c.275–2A>C) mutations, a carrier (#1373 [II-2]) with one splice site (c.275–2A>C) mutation, and an individual (#1371 [III-2]) with a splice site (c.275–2A>C) and a frameshift (c.287dupA) compound heterozygous mutations. Reverse-transcriptase polymerase chain reaction of the control sample resulted in a single, full-length product of approximately 930 bp, whereas the c.275–2A>C mutation resulted in predominantly two smaller products of approximately 650 and 750 bp, along with multiple other aberrantly spliced products (brightest fragment on ladder represents 500 bp). Heterozygous individuals generated a mixture of the full-length and aberrant products. Extra lanes between the samples and the marker lane were removed from the image, as indicated by the white line. (B) Western blot of total protein from a normal control nasal biopsy, a PCD subject (#1370 [III-1]) with a splice site (c.275–2A>C) and a frameshift (c.287dupA) compound heterozygous mutations, and cultured (normal) HBE cells. The PCD sample shows no clear signal for RSPH1. The blot was reprobed with an antibody against DNALI1 to demonstrate the presence of approximately equal numbers of cilia between samples.

Figure 3.

Expression of RSPH1 in cultured human bronchial epithelial (HBE) cells. (A) Control HBE cells were cultured at an air-liquid interface for the indicated time and total RNA was isolated (44). Reverse-transcriptase polymerase chain reaction with specific primers demonstrates that RSPH1 and DNAI1 (NM_012144.2) are not expressed in early, undifferentiated cultures, but are induced during ciliated cell differentiation. Cyclophilin A (PPIA) (NM_021130.3) expression (positive control) does not change over time. (B) Western blot of total cell lysates from undifferentiated and differentiated HBE cells and isolated ciliary axonemes (50). RSPH1 is not detected in the undifferentiated cells, but is present in the differentiated cell lysates and in the isolated ciliary axonemes. Extra lanes between the samples were removed from the image, as indicated by the black line. Two separate Western blots were probed with RSPH1 (top) and DNAI1 (bottom). (C) Cultured HBE cells were immunostained with a monoclonal antibody against RSPH1 or with control IgG. The RSPH1 antibody clearly reacts with the cilia, confirming the presence of RSPH1 in the axoneme.

Figure 4.

Electron micrographic findings. A total of 756 ciliary cross-sections were scored from 15 subjects affected with primary ciliary dyskinesia (PCD) with biallelic mutations in RSPH1, and representative images are shown. (A) Normal (9 + 2) axonemal structure including outer doublet and the central apparatus (80.1% of cilia images). (B and C) Complete absence of the central apparatus (9 + 0), or electron-dense material in central area (12.1% of cilia images). (D–F) Translocation of outer doublet to either replace central pair or into the central region (8 + 1) with extra singlet microtubule, or other abnormalities including eight outer doublets without normal central apparatus (8 + 0) (5.1% of cilia images). (G) A single microtubule in the center instead of central pair (9 + 1) (1.4% of cilia images). (H) Off-center central pair with extra singlet (1.2% of cilia images).

Figure 5.

Mutations in RSPH1 result in a circular wave form. (A) Nasal epithelial cells from a heterozygous carrier mother (#1373 [II-2]), and (B and C) two affected children (#1370 [III-1] and #1371 [III-2]) were expanded in culture (43) and allowed to differentiate at the air-liquid interface (44). High-speed videos were recorded from above the cultures and analyzed in slow motion (see Videos E6–E8). (A) The cilia on three individual cells from the carrier mother (#1373 [II-2]) beat in a mostly planar (back and forth) direction. (B and C) The cilia on three individual cells from each of the children affected with primary ciliary dyskinesia (PCD) (#1370 [III-1] and #1371 [III-2]) moved in a clockwise circle when viewed from above. To compare the ciliary activity quantitatively, a line of best fit was determined for each cilia track. The mean distance (d_mean) of the cilium from that line and the maximum distance (d_max) from the centroid along that line were calculated. The d_mean values (micrometers, ± SD) were 0.27 ± 0.26 (n = 15) for the mother (#1373 [II-2]) but much greater for children affected with PCD (#1370 [III-1], 0.53 ± 0.10 [n = 10]; #1371 [III-2], 0.36 ± 0.13 [n = 13]). The ratio of these values (r = d_mean / d_max) gives a measure of the ciliary beat shape with a lower value denoting a straighter, more planar, beat. The r values for the PCD cells (#1370 [III-1], 0.35 ± 0.068; #1371 [III-2], 0.36 ± 0.085) were significantly different than those of the carrier mother (#1373 [II-2], 0.11 ± 0.08; P < 6.4 × 10⁸), reflecting the defective ciliary activity.