Sir, The letter from Hedberg et al. (2013) is of great interest because it addresses
an important question relating to the genetic aetiology of hereditary myopathy with
early respiratory failure (HMERF). The original report by Lange et al. (2005) indicated
that HMERF was associated with a heterozygous g.296459C>T/p.R32450W mutation in the
kinase domain of titin (TTN) (using Genebank AJ277892 and Uniprot Q8WZ42 as the reference
sequences). Since then, no further cases of HMERF caused by kinase domain mutations
in TTN have been reported. In contrast, only a year since the report of a mutation
in the 119th fibronectin-3 (FN3) domain of TTN causing HMERF (Ohlsson et al., 2012;
Pfeffer et al., 2012), numerous reports have confirmed an association between the
g.274375T>C/p.C30071R FN3 domain mutation and this disease, and seven other mutations
have been reported affecting the same domain of titin (Izumi et al., 2013; Palmio
et al., 2013; Pfeffer et al., 2013; Toro et al., 2013). Furthermore, our recent study
screening 127 patients with myofibrillar myopathy for mutations in TTN revealed seven
families with mutations in the 119th FN3 domain, but none with kinase domain mutations
(Pfeffer et al., 2013).
Ostensibly, the patients from Lange et al.’s (2005) original report were from three
separate families, although they all shared the same TTN haplotype (Lange et al.,
2005). Therefore, for all intents and purposes, the kinase domain variant has still
only been reported in a single HMERF family. Hedberg et al. (2013) now report that
patients from the same family described by Lange et al. (2005) not only carry the
kinase domain mutation (g.296459C>T/p.R32450W), but also have a g.284762C>T mutation
in the 119th fibronectin-3 domain, predicted to cause p.P30091L, which is known to
cause HMERF on its own (Palmio et al., 2013; Pfeffer et al., 2013). This finding should
be confirmed by the authors of the study by Lange et al. (2005), but assuming that
Hedberg et al.’s (2013) report applies to all family members, these data suggest that
the g.284762C>T/p.P30091L FN3 domain variant is the true cause of HMERF in the original
study of Lange et al. (2005). The segregation of both the FN3 and kinase variants
with the disease in these patients is likely because of the shared TTN haplotype between
the patients.
It should be noted that Lange et al. (2005) presented functional data in support of
the kinase variant’s pathogenicity by creating a recombinant TTN kinase construct
using previously published methods (Mayans et al., 1998). The catalytic activity of
the mutated construct was similar to wild-type, although binding with NBR1 was reduced.
This altered NBR1 binding was postulated to be the disease-causing mechanism for this
variant. However, the construct included only 324 amino acids from this giant protein,
whose inferred complete model includes up to 34 350 amino acids (Uniprot Q8WZ42).
We therefore do not know whether this kinase domain variant affects the full-length
protein, nor whether the altered NBR1 binding of this particular domain is, in itself,
pathogenic.
To determine whether kinase domain variants contribute to the pathogenesis, we sequenced
the entire kinase domain (according to Uniprot Q8WZ42, including positions p.32115–32496)
in 33 HMERF patients from eight families with the g.274375T>C/p.C30071R mutation in
the 119th FN3 domain of TTN (Pfeffer et al., 2012, 2013). Our hypothesis was that
amino acid sequence-altering variants (previously reported, or novel) would be more
prevalent in patients with HMERF than control subjects, and/or that kinase variants
would be present in HMERF patients with more severe phenotype. Our control group consisted
of 343 disease controls from 261 pedigrees (70% of whom originate from the UK) who
have had their exomes sequenced at our centre.
PCR was performed with Immolase™ (Bioline) according to manufacturer’s protocol, using
∼50 ng of DNA, 0.25 mM of each oligonucleotide and 2 mM MgCl2, for 30 cycles with
annealing temperature of 60°C. Sequencing was performed using BigDye® (Applied Biosystems)
according to the manufacturer’s protocol with an ABI 3130XL sequencer. Primer sequences
are available on request.
One HMERF patient (Patient A-III:7 from Pfeffer et al., 2012) with the g.274375T>C/p.C30071R
mutation had a protein-altering variant, g.295853G>A predicted to cause p.V32248I
in the kinase domain (listed in dbSNP as rs34924609—note this is not the same kinase
variant as the report from Lange et al., 2005). No sequence-altering variants were
found in the other 32 patients. Given that all 33 share the same TTN haplotype (Pfeffer
et al., 2013), the g.295853G>A/p.V32248I kinase variant in Patient A-III:7 is likely
to be in trans with the FN3 domain mutation. This patient did not have distinguishing
phenotypic features compared with other family members: she had proximal-predominant
skeletal muscle weakness, respiratory muscle weakness with abnormal pulmonary function
testing, and disease onset occurred at age 55 (onset age ranged from 33 to 71 years
in this family).
Comparison of sequence variants in our control group of 343 individuals identified
a sibling pair with the g.296459C>T/p.R32450W mutation in the kinase domain (i.e.
the variant associated with HMERF in Lange et al., 2005). These patients have a confirmed
diagnosis of an autosomal recessive limb girdle muscular dystrophy as a result of
compound heterozygous CAPN3 mutations, and have muscle pathology findings that are
not compatible with HMERF. The clinical picture was of juvenile onset proximal myopathy
without respiratory muscle weakness, and thus also distinct from HMERF’s described
clinical spectrum. Furthermore, sequencing of two unaffected family members from the
same family revealed the presence of the TTN g.296459C>T/p.R32450W kinase variant
in one of them. This is consistent with the kinase variant being a neutral polymorphism,
and not the cause of disease in this family.
We agree with Hedberg et al. (2013) that mutations in the 119th fibronectin-3 domain
of TTN cause HMERF, and that the p.R32450W kinase domain variant is not sufficient
to cause the disease. The data presented here, from both patients and controls, provide
no evidence to support the role of kinase domain mutations in HMERF, in keeping with
public data listing the g.296459C>T/p.R32450W variant in the kinase domain of TTN
as a single nucleotide polymorphism (rs140319117). Although currently also listed
as pathogenic on the Human Gene Mutation Database (HGMD, CM057411), we suggest that
g.296459C>T/p.R32450W should be removed from HGMD to avoid erroneous genetic counselling
advice in the future.