Combined exome and whole-genome sequencing identifies mutations in ARMC4 as a cause of primary ciliary dyskinesia with defects in the outer dynein arm

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous ciliopathy disorder affecting cilia and sperm motility. A range of ultrastructural defects of the axoneme underlie the disease, which is characterised by chronic respiratory symptoms and obstructive lung disease, infertility and body axis laterality defects. We applied a next-generation sequencing approach to identify the gene responsible for this phenotype in two consanguineous families

Primary ciliary dyskinesia with normal ultrastructure:three-dimensional tomography detects absence of DNAH11

In primary ciliary dyskinesia (PCD), motile ciliary dysfunction arises from ciliary defects usually confirmed by transmission electron microscopy (TEM). In 30% of patients, such as those with DNAH11 mutations, apparently normal ultrastructure makes diagnosis difficult. Genetic analysis supports diagnosis, but may not identify definitive causal variants. Electron tomography, an extension of TEM, produces three-dimensional ultrastructural ciliary models with superior resolution to TEM. Our hypothesis is that tomography using existing patient samples will enable visualisation of DNAH11-associated ultrastructural defects. Dual axis tomograms from araldite-embedded nasal cilia were collected in 13 PCD patients with normal ultrastructure (DNAH11 n=7, HYDIN n=2, CCDC65 n=3 and DRC1 n=1) and six healthy controls, then analysed using IMOD and Chimera software. DNAH11 protein is localised to the proximal ciliary region. Within this region, electron tomography indicated a deficiency of >25% of proximal outer dynein arm volume in all patients with DNAH11 mutations (n=7) compared to other patients with PCD and normal ultrastructure (n=6) and healthy controls (n=6). DNAH11 mutations cause a shared abnormality in ciliary ultrastructure previously undetectable by TEM. Advantageously, electron tomography can be used on existing diagnostic samples and establishes a structural abnormality where ultrastructural studies were previously normal

Exome sequencing and genotyping identify a rare variant in NLRP7 gene associated with ulcerative colitis.

Background and Aims Although genome-wide association studies [GWAS] in inflammatory bowel disease [IBD] have identified a large number of common disease susceptibility alleles for both Crohn’s disease [CD] and ulcerative colitis [UC], a substantial fraction of IBD heritability remains unexplained, suggesting that rare coding genetic variants may also have a role in pathogenesis. We used high-throughput sequencing in families with multiple cases of IBD, followed by genotyping of cases and controls, to investigate whether rare protein-altering genetic variants are associated with susceptibility to IBD. Methods Whole-exome sequencing was carried out in 10 families in whom three or more individuals were affected with IBD. A stepwise filtering approach was applied to exome variants, to identify potential causal variants. Follow-up genotyping was performed in 6025 IBD cases [2948 CD; 3077 UC] and 7238 controls. Results Our exome variant analysis revealed coding variants in the NLRP7 gene that were present in affected individuals in two distinct families. Genotyping of the two variants, p.S361L and p.R801H, in IBD cases and controls showed that the p.S361L variant was significantly associated with an increased risk of ulcerative colitis [odds ratio 4.79, p = 0.0039] and IBD [odds ratio 3.17, p = 0.037]. A combined analysis of both variants showed suggestive association with an increased risk of IBD [odds ratio 2.77, p = 0.018]. Conclusions The results suggest that NLRP7 signalling and inflammasome formation may be a significant component in the pathogenesis of IBD

Risk factors for situs defects and congenital heart disease in primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is associated with abnormal organ positioning (situs) and congenital heart disease (CHD). This study investigated genotype–phenotype associations in PCD to facilitate risk predictions for cardiac and laterality defects. This retrospective cohort study of 389 UK patients with PCD found 51% had abnormal situs and 25% had CHD and/or laterality defects other than situs inversus totalis. Patients with biallelic mutations in a subset of nine PCD genes had normal situs. Patients with consanguineous parents had higher odds of situs abnormalities than patients with non-consanguineous parents. Patients with abnormal situs had higher odds of CHD and/or laterality defects

Targeted NGS gene panel identifies mutations in RSPH1 causing primary ciliary dyskinesia and a common mechanism for ciliary central pair agenesis due to radial spoke defects.

Primary ciliary dyskinesia (PCD) is an inherited chronic respiratory obstructive disease with randomized body laterality and infertility, resulting from cilia and sperm dysmotility. PCD is characterized by clinical variability and extensive genetic heterogeneity, associated with different cilia ultrastructural defects and mutations identified in >20 genes. Next generation sequencing (NGS) technologies therefore present a promising approach for genetic diagnosis which is not yet in routine use. We developed a targeted panel-based NGS pipeline to identify mutations by sequencing of selected candidate genes in 70 genetically undefined PCD patients. This detected loss-of-function RSPH1 mutations in four individuals with isolated central pair (CP) agenesis and normal body laterality, from two unrelated families. Ultrastructural analysis in RSPH1-mutated cilia revealed transposition of peripheral outer microtubules into the 'empty' CP space, accompanied by a distinctive intermittent loss of the central pair microtubules. We find that mutations in RSPH1, RSPH4A and RSPH9, which all encode homologs of components of the 'head' structure of ciliary radial spoke complexes identified in Chlamydomonas, cause clinical phenotypes that appear to be indistinguishable except at the gene level. By high-resolution immunofluorescence we identified a loss of RSPH4A and RSPH9 along with RSPH1 from RSPH1-mutated cilia, suggesting RSPH1 mutations may result in loss of the entire spoke head structure. CP loss is seen in up to 28% of PCD cases, in whom laterality determination specified by CP-less embryonic node cilia remains undisturbed. We propose this defect could arise from instability or agenesis of the ciliary central microtubules due to loss of their normal radial spoke head tethering

Recessive <i>HYDIN</i> mutations cause primary ciliary dyskinesia without randomization of left-right body asymmetry

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder characterized by defective cilia and flagella motility. Chronic destructive-airway disease is caused by abnormal respiratory-tract mucociliary clearance. Abnormal propulsion of sperm flagella contributes to male infertility. Genetic defects in most individuals affected by PCD cause randomization of left-right body asymmetry; approximately half show situs inversus or situs ambiguous. Almost 70 years after the hy3 mouse possessing Hydin mutations was described as a recessive hydrocephalus model, we report HYDIN mutations in PCD- affected persons without hydrocephalus. By homozygosity mapping, we identified a PCD-associated locus, chromosomal region 16q21- q23, which contains HYDIN. However, a nearly identical 360 kb paralogous segment (HYDIN2) in chromosomal region 1q21.1 complicated mutational analysis. In three affected German siblings linked to HYDIN, we identified homozygous c.3985G>T mutations that affect an evolutionary conserved splice acceptor site and that subsequently cause aberrantly spliced transcripts predicting premature protein termination in respiratory cells. Parallel whole-exome sequencing identified a homozygous nonsense HYDIN mutation, c.922A>T (p.Lys307( *)), in six individuals from three Faroe Island PCD-affected families that all carried an 8.8 Mb shared haplotype across HYDIN, indicating an ancestral founder mutation in this isolated population. We demonstrate by electron microscopy tomography that, consistent with the effects of loss-of-function mutations, HYDIN mutant respiratory cilia lack the C2b projection of the central pair (CP) apparatus; similar findings were reported in Hydin-deficient Chlamydomonas and mice. High-speed videomicroscopy demonstrated markedly reduced beating amplitudes of respiratory cilia and stiff sperm flagella. Like the hy3 mouse model, all nine PCD-affected persons had normal body composition because nodal cilia function is apparently not dependent on the function of the CP apparatus

X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3

By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2–DNAAF4–HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins

Mutations in CCDC 39 and CCDC 40 are the Major Cause of Primary Ciliary Dyskinesia with Axonemal Disorganization and Absent Inner Dynein Arms

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder caused by cilia and sperm dysmotility. About 12% of cases show perturbed 9+2 microtubule cilia structure and inner dynein arm (IDA) loss, historically termed ‘radial spoke defect’. We sequenced CCDC39 and CCDC40 in 54 ‘radial spoke defect’ families, as these are the two genes identified so far to cause this defect. We discovered biallelic mutations in a remarkable 69% (37/54) of families, including identification of 25 (19 novel) mutant alleles (12 in CCDC39 and 13 in CCDC40). All the mutations were nonsense, splice and frameshift predicting early protein truncation, which suggests this defect is caused by ‘null’ alleles conferring complete protein loss. Most families (73%; 27/37) had homozygous mutations, including families from outbred populations. A major putative hotspot mutation was identified, CCDC40 c.248delC, as well as several other possible hotspot mutations. Together, these findings highlight the key role of CCDC39 and CCDC40 in PCD with axonemal disorganisation and IDA loss, and these genes represent major candidates for genetic testing in families affected by this ciliary phenotype. We show that radial spoke structures are largely intact in these patients and propose this ciliary ultrastructural abnormality be referred to as ‘IDA and nexin-dynein regulatory complex (N-DRC) defect’, rather than ‘radial spoke defect’