Peter Hackman

Tibial muscular dystrophy is a titinopathy caused by mutations in TTN, the gene encoding the giant skeletal-muscle protein titin

Tibial muscular dystrophy (TMD) is an autosomal dominant late-onset distal myopathy linked to chromosome 2q31. The linked region includes the giant TTN gene, which encodes the central sarcomeric protein, titin. We have previously shown a secondary calpain-3 defect to be associated with TMD, which further underscored that titin is the candidate. We now report the first mutations in TTN to cause a human skeletal-muscle disease, TMD. In Mex6, the last exon of TTN, a unique 11-bp deletion/insertion mutation, changing four amino acid residues, completely cosegregated with all tested 81 Finnish patients with TMD in 12 unrelated families. The mutation was not found in 216 Finnish control samples. In a French family with TMD, a Leu-->Pro mutation at position 293,357 in Mex6 was discovered. Mex6 is adjacent to the known calpain-3 binding site Mex5 of M-line titin. Immunohistochemical analysis using two exon-specific antibodies directed to the M-line region of titin demonstrated the specific loss of carboxy-terminal titin epitopes in the TMD muscle samples that we studied, thus implicating a functional defect of the M-line titin in the genesis of the TMD disease phenotype.

Mitochondrial DNA Polymerase W748S Mutation: A Common Cause of Autosomal Recessive Ataxia with Ancient European Origin

Mutations in the catalytic subunit of the mitochondrial DNA polymerase gamma (POLG) have been found to be an important cause of neurological disease. Recently, we and collaborators reported a new neurodegenerative disorder with autosomal recessive ataxia in four patients homozygous for two amino acid changes in POLG: W748S in cis with E1143G. Here, we studied the frequency of this allele and found it to be among the most common genetic causes of inherited ataxia in Finland. We identified 27 patients with mitochondrial recessive ataxia syndrome (MIRAS) from 15 Finnish families, with a carrier frequency in the general population of 1 : 125. Since the mutation pair W748S+E1143G has also been described in European patients, we examined the haplotypes of 13 non-Finnish, European patients with the W748S mutation. Haplotype analysis revealed that all the chromosomes carrying these two changes, in patients from Finland, Norway, the United Kingdom, and Belgium, originate from a common ancient founder. In Finland and Norway, long, common, northern haplotypes, outside the core haplotype, could be identified. Despite having identical homozygous mutations, the Finnish patients with this adult- or juvenile-onset disease had surprisingly heterogeneous phenotypes, albeit with a characteristic set of features, including ataxia, peripheral neuropathy, dysarthria, mild cognitive impairment, involuntary movements, psychiatric symptoms, and epileptic seizures. The high carrier frequency in Finland, the high number of patients in Norway, and the ancient European founder chromosome indicate that this newly identified ataxia should be considered in the first-line differential diagnosis of progressive ataxia syndromes.

POLG mutations in neurodegenerative disorders with ataxia but no muscle involvement

Objective: To identify POLG mutations in patients with sensory ataxia and CNS features. Methods: The authors characterized clinical, laboratory, and molecular genetic features in eight patients from five European families. The authors conducted sequencing of coding exons of POLG, C10orf2 (Twinkle), and ANT1 and analyzed muscle mitochondrial DNA (mtDNA), including Southern blot analysis and long-range PCR. Results: Ataxia occurred in combination with various CNS features, including myoclonus, epilepsy, cognitive decline, nystagmus, dysarthria, thalamic and cerebellar white matter lesions on MRI, and neuronal loss in discrete gray nuclei on autopsy. Gastrointestinal dysmotility, weight loss, cardiomyopathy, and valproate-induced hepatotoxicity occurred less frequently. Two patients died without preceding signs of progressive external ophthalmoplegia. In muscle, typical findings of mitochondrial disease, such as ragged red fibers and Southern blot mtDNA abnormalities, were absent. POLG mutations were present in eight patients, including two isolated cases, and one Finnish and two unrelated Belgian families contained in total six patients. All POLG mutations were recessive, occurring in a homozygous state in seven patients and in a compound heterozygous state in one patient. The novel W748S mutation was identified in five patients from three unrelated families. Conclusions: The clinical spectrum of recessive POLG mutations is expanded by sensory ataxic neuropathy, combined with variable features of involvement of CNS and other organs. Progressive external ophthalmoplegia, myopathy, ragged red fibers, and Southern blot abnormalities of muscle mitochondrial DNA also are not mandatory features associated with POLG mutations.

Mutations affecting the cytoplasmic functions of the co-chaperone DNAJB6 cause limb-girdle muscular dystrophy

Limb-girdle muscular dystrophy type 1D (LGMD1D) was linked to chromosome 7q36 over a decade ago, but its genetic cause has remained elusive. Here we studied nine LGMD-affected families from Finland, the United States and Italy and identified four dominant missense mutations leading to p.Phe93Leu or p.Phe89Ile changes in the ubiquitously expressed co-chaperone DNAJB6. Functional testing in vivo showed that the mutations have a dominant toxic effect mediated specifically by the cytoplasmic isoform of DNAJB6. In vitro studies demonstrated that the mutations increase the half-life of DNAJB6, extending this effect to the wild-type protein, and reduce its protective anti-aggregation effect. Further, we show that DNAJB6 interacts with members of the CASA complex, including the myofibrillar myopathy–causing protein BAG3. Our data identify the genetic cause of LGMD1D, suggest that its pathogenesis is mediated by defective chaperone function and highlight how mutations in a ubiquitously expressed gene can exert effects in a tissue-, isoform- and cellular compartment–specific manner.

Titinopathies and extension of the M-line mutation phenotype beyond distal myopathy and LGMD2J

Objective: To determine the phenotype variability associated with the specific C-terminal M-line titin mutation known to cause autosomal dominant distal myopathy, tibial muscular dystrophy (TMD; MIM 600334), and limb girdle muscular dystrophy 2J (LGMD2J). Methods: Three hundred eighty-six individuals were genotyped for the Finnish founder mutation in titin (FINmaj) causing TMD/LGMD2J. Results: Two hundred seven patients were heterozygous for the mutation. Among these patients, 189 (91%) had a more common phenotype compatible with the classic description of TMD. However, 18 (9%) had unusual phenotypes such as proximal leg or posterior lower leg muscle weakness and atrophy even at onset. Four patients were confirmed homozygotes representing the LGMD2J phenotype. These homozygotes were half of the eight LGMD patients previously described in the original large consanguineous kindred. Conclusions: Large variability of phenotypic expression caused by just one mutation, the Finnish FINmaj, suggests that no certain phenotype of myopathy/dystrophy can be excluded from being caused by mutated titin. Yet unknown homozygous or compound heterozygous titin mutations without phenotype in the heterozygote carriers may be responsible for undetermined recessive MD and LGMD.

Titin mutation segregates with hereditary myopathy with early respiratory failure

In 2001, we described an autosomal dominant myopathy characterized by neuromuscular ventilatory failure in ambulant patients. Here we describe the underlying genetic basis for the disorder, and we define the neuromuscular, respiratory and radiological phenotype in a study of 31 mutation carriers followed for up to 31 years. A combination of genome-wide linkage and whole exome sequencing revealed the likely causal genetic variant in the titin (TTN) gene (g.274375T>C; p.Cys30071Arg) within a shared haplotype of 2.93 Mbp on chromosome 2. This segregated with the phenotype in 21 individuals from the original family, nine subjects in a second family with the same highly selective pattern of muscle involvement on magnetic resonance imaging and a third familial case with a similar phenotype. Comparing the mutation carriers revealed novel features not apparent in our original report. The clinical presentation included predominant distal, proximal or respiratory muscle weakness. The age of onset was highly variable, from early adulthood, and including a mild phenotype in advanced age. Muscle weakness was earlier onset and more severe in the lower extremities in nearly all patients. Seven patients also had axial muscle weakness. Respiratory function studies demonstrated a gradual deterioration over time, reflecting the progressive nature of this condition. Cardiomyopathy was not present in any of our patients despite up to 31 years of follow-up. Magnetic resonance muscle imaging was performed in 21 affected patients and revealed characteristic abnormalities with semitendinosus involvement in 20 of 21 patients studied, including 3 patients who were presymptomatic. Diagnostic muscle histopathology most frequently revealed eosinophilic inclusions (inclusion bodies) and rimmed vacuoles, but was non-specific in a minority of patients. These findings have important clinical implications. This disease should be considered in patients with adult-onset proximal or distal myopathy and early respiratory failure, even in the presence of non-specific muscle pathology. Muscle magnetic resonance imaging findings are characteristic and should be considered as an initial investigation, and if positive should prompt screening for mutations in TTN. With 363 exons, screening TTN presented a major challenge until recently. However, whole exome sequencing provides a reliable cost-effective approach, providing the gene of interest is adequately captured.

Mutations in the N-terminal Actin-Binding Domain of Filamin C Cause a Distal Myopathy

Linkage analysis of the dominant distal myopathy we previously identified in a large Australian family demonstrated one significant linkage region located on chromosome 7 and encompassing 18.6 Mbp and 151 genes. The strongest candidate gene was FLNC because filamin C, the encoded protein, is muscle-specific and associated with myofibrillar myopathy. Sequencing of FLNC cDNA identified a c.752T>C (p.Met251Thr) mutation in the N-terminal actin-binding domain (ABD); this mutation segregated with the disease and was absent in 200 controls. We identified an Italian family with the same phenotype and found a c.577G>A (p.Ala193Thr) filamin C ABD mutation that segregated with the disease. Filamin C ABD mutations have not been described, although filamin A and filamin B ABD mutations cause multiple musculoskeletal disorders. The distal myopathy phenotype and muscle pathology in the two families differ from myofibrillar myopathies caused by filamin C rod and dimerization domain mutations because of the distinct involvement of hand muscles and lack of pathological protein aggregation. Thus, like the position of FLNA and B mutations, the position of the FLNC mutation determines disease phenotype. The two filamin C ABD mutations increase actin-binding affinity in a manner similar to filamin A and filamin B ABD mutations. Cell-culture expression of the c.752T>C (p.Met251)Thr mutant filamin C ABD demonstrated reduced nuclear localization as did mutant filamin A and filamin B ABDs. Expression of both filamin C ABD mutants as full-length proteins induced increased aggregation of filamin. We conclude filamin C ABD mutations cause a recognizable distal myopathy, most likely through increased actin affinity, similar to the pathological mechanism of filamin A and filamin B ABD mutations.

Population frequency of myotonic dystrophy: higher than expected frequency of myotonic dystrophy type 2 (DM2) mutation in Finland.

Myotonic dystrophy (DM) is the most common adult-onset muscular dystrophy with an estimated prevalence of 1/8000. There are two genetically distinct types, DM1 and DM2. DM2 is generally milder with more phenotypic variability than the classic DM1. Our previous data on co-segregation of heterozygous recessive CLCN1 mutations in DM2 patients indicated a higher than expected DM2 prevalence. The aim of this study was to determine the DM2 and DM1 frequency in the general population, and to explore whether the DM2 mutation functions as a modifier in other neuromuscular diseases (NMD) to account for unexplained phenotypic variability. We genotyped 5535 Finnish individuals: 4532 normal blood donors, 606 patients with various non-myotonic NMD, 221 tibial muscular dystrophy patients and their 176 healthy relatives for the DM2 and DM1 mutations. We also genotyped an Italian idiopathic non-myotonic proximal myopathy cohort (n=93) for the DM2 mutation. In 5496 samples analyzed for DM2, we found three DM2 mutations and two premutations. In 5511 samples analyzed for DM1, we found two DM1 mutations and two premutations. In the Italian cohort, we identified one patient with a DM2 mutation. We conclude that the DM2 mutation frequency is significantly higher in the general population (1/1830; P-value=0.0326) than previously estimated. The identification of DM2 mutations in NMD patients with clinical phenotypes not previously associated with DM2 is of particular interest and is in accord with the high overall prevalence. On the basis of our results, DM2 appears more frequent than DM1, with most DM2 patients currently undiagnosed with symptoms frequently occurring in the elderly population.

Truncating mutations in C-terminal titin may cause more severe tibial muscular dystrophy (TMD).

Mutations in C-terminal titin cause autosomal dominant tibial muscular dystrophy (TMD) as reported previously. Samples from 25 new families and 25 sporadic new distal myopathy cases were screened for titin mutations. Three novel mutations were discovered in two families from Spain and two families from France. Two mutations, g.292998delT and g.293376delA lead to frameshift and premature stop codons in the second last and the last titin gene (TTN) exons, Mex5 and Mex6, respectively. The third was a nonsense mutation g.293379C>T (p.Q33396X) in Mex6. Patients with the upstream Mex5 mutation showed a more severe phenotype with earlier onset implying a genotype-phenotype correlation.

Tibial muscular dystrophy in a Belgian family

We report a Belgian family with autosomal dominant, late‐onset, distal myopathy with selective foot extensor muscle involvement of the lower legs. Linkage to the tibial muscular dystrophy (TMD) locus 2q31 was not evident at first because of incomplete disease penetrance in a 50‐year‐old asymptomatic family member. An abnormal tibialis anterior muscle biopsy established her subclinical status and linkage of the family to the TMD locus. Mutation analysis showed a disease‐specific, heterozygous point mutation in the last exon, Mex6, of the titin gene. This is the third mutation found in TMD and the second European family with TMD outside the Finnish population, suggesting that titinopathies may occur in various populations. Ann Neurol 2003