The heterozygous g.274375T>C substitution in TTN
is highly likely to be the cause of hereditary myopathy with early respiratory failure in these families. It is a novel variant predicted to alter a highly conserved small/polar cysteine amino acid residue for a larger/basic arginine, which is likely to induce conformational change in A-band titin, and thus have functional consequences. The same variant segregates with the phenotype in three ostensibly unrelated families (a total of 31 affected and nine unaffected individuals) with the same rare disorder, and is not present in 364 control chromosomes. Other potentially pathogenic variants within the chromosomal region linked to the disease were excluded by direct sequencing. We also excluded the possibility of another mutation in TTN
by sequencing the entire coding region of this gene. The exome sequencing had >10-fold coverage for all but 19 coding regions of TTN
, and these remaining regions were excluded by Sanger sequencing. After further analysis of single nucleotide variants surrounding the putative mutation, the size of the shared haplotype in these three families was 2.93 Mbp, which contains only 10 protein-coding genes. The only sarcomeric protein in this region is TTN
, and a muscle protein is the presumed cause of this disease due to the disturbed myofibrillar architecture on electron microscopy. As expected for a missense mutation, the size and abundance of the titin protein was normal in affected muscle, but the titin-binding partner calpain 3 was reduced, further implicating titin in the pathogenesis of the disorder. Finally, by carrying out a comprehensive study of the clinical phenotype in mutation carriers, we show that the clinical spectrum of disease in these families overlaps with that described in other families with mutations in the kinase domain of TTN
), providing further evidence that TTN
is the disease gene in the families described here.
Hereditary myopathy with early respiratory failure is a rare disease, but as we have demonstrated, there is enormous phenotypic variability within these families. The phenotype was mild or severe, with onset in early to late adulthood, and the predominant muscles affected were proximal, distal, axial, respiratory or, most frequently, a combination of these. Ultimately, nearly all patients eventually developed significant proximal and distal weakness, perhaps suggesting that the various initial presentations may converge into a common late-stage phenotype, as has been demonstrated in dysferlin myopathy (Paradas et al., 2010
). Given these features of the condition and the difficulty in recognizing symptoms of respiratory failure due to muscle weakness (two of our patients were initially misdiagnosed as having obstructive sleep apnoea), it is quite possible that this disease is under-recognized, and the very small number of reported cases in the world literature () may therefore under-represent the true prevalence of this disorder. This is compounded by the fact that muscle pathology can be non-specific (as was the case for 3/15 patients in our series), and even if detected, the presence of cytoplasmic bodies are a non-specific finding seen in other well-defined muscle diseases (Caron et al., 1999
). Another common feature is Z-disc abnormality (Edstrom et al., 1990
; Abe et al., 1993
; Chinnery et al., 2001
) and the accumulation of myofibrillar protein within inclusions, vacuoles and/or cytoplasmic bodies (Bertini et al., 1990
; Baeta et al., 1996
; Caron et al., 1999
; Chinnery et al., 2001
), which could result in cases of this disease being classified as myofibrillar myopathy [e.g. myopathy due to desmin, myotilin or filamin mutations may also present with early respiratory failure (Ferrer and Olive, 2008
)]. Cytoplasmic bodies in hereditary myopathy with early respiratory failure have been found to contain desmin (Bertini et al., 1990
; Baeta et al., 1996
), dystrophin (Caron et al., 1999
) and β-amyloid (Chinnery et al., 2001
), which are also over-expressed in the muscle from patients with myofibrillar myopathy (Selcen, 2011
). On account of these clinical and pathological similarities, as well as the similar genetic aetiology (since myofibrillar myopathies are caused by mutations in proteins associated with the Z-disc), it is reasonable to consider this disorder in the pathological differential diagnosis of myofibrillar myopathy, even though the hyaline and dark cytoplasmic abnormalities on trichrome stain are lacking in this disease.
Previously reported cases of cytoplasmic body myopathy with early respiratory failure
By studying the largest series of patients described to date, our observations cast light on the optimal means to diagnose and manage patients with this disorder, providing a guide to the natural history. Selective fat infiltration of semitendinosus on muscle MRI was present in all but one of the patients in this series, and was also present in all presymptomatic mutation carriers before symptom onset. MRI abnormalities of semitendinosus and peroneus longus (the two most frequently affected muscles in our series) are also observed in myofibrillar myopathies, particularly desminopathy, αB-crystallinopathy and myotilinopathy (refer to diagnostic algorithm in of Wattjes et al., 2010
). These shared patterns on MRI are not likely to be coincidental: it has already been demonstrated that phenotypes resembling ‘titinopathy’ can be caused by mutations in αB-crystallin and that the mechanism appears to be due to altered interaction of αB-crystallin with titin protein (Inagaki et al., 2006
; Zhu et al., 2009
). Our series has provided MRI data on 21 patients and firmly defines the MRI features of hereditary myopathy with early respiratory failure, showing for the first time that thigh MRI is a sensitive predictor of mutation status. Future MRI algorithms should include hereditary myopathy with early respiratory failure in the category of diseases that have predominant semitendinosus and peroneal muscle involvement. This would improve recognition and appropriate genetic testing for this condition. After diagnosis, the early recognition of asymptomatic respiratory failure and its treatment with nocturnal ventilation is likely to have improved the quality and length of life, with several individuals remaining semi-independent at home up to 31 years after the initial diagnosis.
The reported cases of this disorder in the world literature are summarized in , with the majority not defined genetically. For those cases with onset in adulthood, it seems plausible that these might also be due to TTN mutations, given the overlap of clinical and pathological findings. One of the reported adult-onset cases had cardiomyopathy, which was not demonstrated in any of our patients. Nonetheless, titin is also expressed in cardiac muscle and it is possible a different mutation in this gene could be responsible. The massive size of titin has rendered complete screening of this gene near-impossible in the past, but exome sequencing is a cost-effective way forward. However, our findings show that this should not be considered an ‘off the shelf’ routine clinical test at present. Our initial exome sequencing failed to identify the causative mutation in Family A because only a small proportion of titin was sequenced following 38-Mb capture. Only by further re-sequencing using a 62-Mb capture system did we obtain adequate coverage to detect the causal variant in this family.
The spectrum of disease due to TTN
mutations is already broad, and a schematic of the regional distribution of mutations causing the various phenotypes is summarized in . Mutations of the M-band region (at the C-terminal limit of the protein) cause tibial muscular dystrophy in heterozygous state (Hackman et al., 2002
), which bears some similarity to hereditary myopathy with early respiratory failure due to its onset in adulthood, and preferential involvement of ankle dorsiflexion. Mutations that cause premature stop codons have been suggested to cause a more severe phenotype of this disorder in some reported families (Hackman et al., 2008
). M-band mutations in homozygous state cause a severe, early onset skeletal myopathy, LGMD type 2J (Udd et al., 2005
). Homozygous deletions in the M-band are causative of early onset cardiomyopathy and skeletal myopathy (Carmignac et al., 2007
). A missense mutation in the kinase region of TTN
(proximal to the M-band) has been demonstrated to be causative of hereditary myopathy with early respiratory failure in three families (Lange, 2005
). Interestingly, the mutation described in the current report is a missense mutation proximal to the kinase region, in the A-band of titin. This suggests that the mechanism is not directly related to altered kinase activity of titin, and further study will be required to determine the cause of early respiratory muscle involvement, which appears to be mutation-specific at present. Given the myofibrillar pathology and inclusion bodies, the mutation may cause hyperaggregation of titin and/or its binding partners in the A-band or interference with the calpain 3 proteolytic system.
Figure 11 Schematic diagram of the various domains of TTN (not drawn to scale), represented from N-terminus to C-terminus, and described based on their location within the sarcomere. There is some domain specificity for the observed phenotypes. Cardiomyopathy occurs (more ...)
Protein studies from muscle in our patients did not reveal abnormalities in the most C-terminal part of titin with the antibodies tested. However, detailed pathological studies of eight muscle biopsies and three separate skeletal muscle tissues from a single patient (post-mortem material) demonstrated decreased calpain 3 expression. Calpain 3 is a muscle-specific calcium-dependent protease responsible for LGMD2A. Secondary reduction of calpain 3 on muscle biopsies has been reported in LGMD2B (Anderson et al., 2000
) and LGMD2I (Yamamoto et al., 2008
), without any apparent correlation between the levels of calpain 3 and the stage of muscle pathology (Charlton et al., 2009
). However, in the post-mortem tissue studied here, the greatly reduced levels of calpain 3 in the diaphragm correlated with the severity of the myopathy. To our knowledge, this is the first report of comparative calpain 3 analysis in multiple muscle groups from the same patient and is consistent with the role of titin as a regulatory factor controlling calpain 3 autolysis (Ono et al., 2006
). Although preliminary, these observations suggest that secondary loss of calpain 3 may exacerbate the disease.
In conclusion, we present a detailed clinical description of the largest reported series to date of patients with hereditary myopathy with early respiratory failure. This condition has variable clinical presentation, progressive respiratory muscle weakness, occasionally highly non-specific muscle pathology and as such is likely under-recognized. We show that patients with this condition may be identified when they are presymptomatic with muscle MRI, revealing semitendinosus and peroneus longus as the first affected muscles. In more advanced cases, semitendinosus, peroneus longus and obturator externus are the most commonly affected muscles on MRI. Using microsatellite linkage analysis and whole exome sequencing, we have identified the g.274375T>C mutation in TTN, which lies within a 2.93-Mb haplotype shared between these three families, and excluded the possibility of another TTN gene mutation by sequencing all coding regions of the gene. Hereditary myopathy with early respiratory failure can be due to mutations in the A-band (as in this report) and the kinase domain, and further study will be required to discover the full repertoire of TTN lesions capable of causing this disease.