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

Abstract

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.

Immunofluorescence analysis and diagnosis of primary ciliary dyskinesia with radial spoke defects

“…PCD due to defects in RS function are very difficult to diagnose, because ultrastructural analyses of cross-sections studied by TEM can be normal (33)(34)(35)(36)(37)45), and some RS complexes can still be observed in TEM sections of patients with RSPH1 and RSPH4A mutations (35,36,46). Furthermore, ciliary beating abnormalities are very subtle and only detectable by experienced HSVM examiners (33,(35)(36)(37)45).…”

“…Furthermore, ciliary beating abnormalities are very subtle and only detectable by experienced HSVM examiners (33,(35)(36)(37)45). Here, we searched for mutations in RSPH9, RSPH4A, and RSPH1 in individuals with classical PCD symptoms with situs solitus, frequently normal TEM findings, and subtle changes in the ciliary beating pattern, as evaluated by HSVM (Table 1).…”

“…C, rs151107532; mutation of the obligatory canonical splice site; c.433C.T, p.Gln145* in RSPH1) have been reported previously (35)(36)(37)41). The mutation c.275-2A.C [p.?]…”

Genome-Wide Sequencing for Prenatal Detection of Fetal Single-Gene Disorders

“…After purification, only the purified captured fragments are sequenced, allowing increased sequencing depth and thereby accuracy at a lower cost. This is the basis for the development of diagnostic disease-focused multigene panels Brett et al 2014;Lepri et al 2014;Onoufriadis et al 2014;Xue et al 2014), containing tens to up to hundreds of genes, as well as for WES (Lemke et al 2012;Korf 2013;Xue et al 2014). For WES, the baits are designed to capture the coding exons of nearly all genes, which represent 1% -2% of the total genomic DNA but contain 85% of known disease causing mutations (Bick and Dimmock 2011;Gilissen et al 2012;Gonzaga-Jauregui et al 2012;Korf and Rehm 2013;Xue et al 2014).…”

“…Rather than a lengthy stepwise approach with sequential mutation analysis of the most obvious candidate genes first, followed by analysis of less-likely candidate genes, disease-focused NGS panels permit analysis of up to several hundreds of genes at once for defined categories of phenotypes, such as congenital heart defects, skeletal dysplasias, mitochondrial disorders, disorders of glycosylation, ciliopathies, cardiac defects, epilepsies, Noonan syndrome, etc. Korf 2013;Valencia et al 2013;Brett et al 2014;Chen et al 2014;Lepri et al 2014;Onoufriadis et al 2014;Xue et al 2014).…”