Novel mutations in RSPH4A and TTN genes lead to primary ciliary dyskinesia-hereditary myopathy with early respiratory failure overlap syndrome

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous inherited disorder arising from dysmotility of motile cilia and sperm. This is associated with a variety of ultrastructural defects of the cilia and sperm axoneme that affect movement, leading to clinical consequences on respiratory-tract mucociliary clearance and lung function, fertility, and left-right body-axis determination. We performed whole-genome SNP-based linkage analysis in seven consanguineous families with PCD and central-microtubular-pair abnormalities. This identified two loci, in two families with intermittent absence of the central-pair structure (chromosome 6p21.1, Zmax 6.7) and in five families with complete absence of the central pair (chromosome 6q22.1, Zmax 7.0). Mutations were subsequently identified in two positional candidate genes, RSPH9 on chromosome 6p21.1 and RSPH4A on chromosome 6q22.1. Haplotype analysis identified a common ancestral founder effect RSPH4A mutation present in UK-Pakistani pedigrees. Both RSPH9 and RSPH4A encode protein components of the axonemal radial spoke head. In situ hybridization of murine Rsph9 shows gene expression restricted to regions containing motile cilia. Investigation of the effect of knockdown or mutations of RSPH9 orthologs in zebrafish and Chlamydomonas indicate that radial spoke head proteins are important in maintaining normal movement in motile, "9+2"-structure cilia and flagella. This effect is rescued by reintroduction of gene expression for restoration of a normal beat pattern in zebrafish. Disturbance in function of these genes was not associated with defects in left-right axis determination in humans or zebrafish.

Hereditary myopathy with early respiratory failure and extensive myofibrillar lesions has been described in sporadic and familial cases and linked to various chromosomal regions. The mutated gene is unknown in most cases. We studied eight individuals, from three apparently unrelated families, with clinical and pathological features of hereditary myopathy with early respiratory failure. The investigations included clinical examination, muscle histopathology and genetic analysis by whole exome sequencing and single nucleotide polymorphism arrays. All patients had adult onset muscle weakness in the pelvic girdle, neck flexors, respiratory and trunk muscles, and the majority had prominent calf hypertrophy. Examination of pulmonary function showed decreased vital capacity. No signs of cardiac muscle involvement were found. Muscle histopathological features included marked muscle fibre size variation, fibre splitting, numerous internal nuclei and fatty infiltration. Frequent groups of fibres showed eosinophilic inclusions and deposits. At the ultrastructural level, there were extensive myofibrillar lesions with marked Z-disc alterations. Whole exome sequencing in four individuals from one family revealed a missense mutation, g.274375T>C; p.Cys30071Arg, in the titin gene (TTN). The mutation, which changes a highly conserved residue in the myosin binding A-band titin, was demonstrated to segregate with the disease in all three families. High density single nucleotide polymorphism arrays covering the entire genome demonstrated sharing of a 6.99 Mb haplotype, located in chromosome region 2q31 including TTN, indicating common ancestry. Our results demonstrate a novel and the first disease-causing mutation in A-band titin associated with hereditary myopathy with early respiratory failure. The typical histopathological features with prominent myofibrillar lesions and inclusions in muscle and respiratory failure early in the clinical course should be incentives for analysis of TTN mutations.