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author:("UDD, biarnes")
1.  Recessive TTN truncating mutations define novel forms of core myopathy with heart disease 
Human Molecular Genetics  2013;23(4):980-991.
Core myopathies (CM), the main non-dystrophic myopathies in childhood, remain genetically unexplained in many cases. Heart disease is not considered part of the typical CM spectrum. No congenital heart defect has been reported, and childhood-onset cardiomyopathy has been documented in only two CM families with homozygous mutations of the TTN gene. TTN encodes titin, a giant protein of striated muscles. Recently, heterozygous TTN truncating mutations have also been reported as a major cause of dominant dilated cardiomyopathy. However, relatively few TTN mutations and phenotypes are known, and titin pathophysiological role in cardiac and skeletal muscle conditions is incompletely understood. We analyzed a series of 23 families with congenital CM and primary heart disease using TTN M-line-targeted sequencing followed in selected patients by whole-exome sequencing and functional studies. We identified seven novel homozygous or compound heterozygous TTN mutations (five in the M-line, five truncating) in 17% patients. Heterozygous parents were healthy. Phenotype analysis identified four novel titinopathies, including cardiac septal defects, left ventricular non-compaction, Emery–Dreifuss muscular dystrophy or arthrogryposis. Additionally, in vitro studies documented the first-reported absence of a functional titin kinase domain in humans, leading to a severe antenatal phenotype. We establish that CM are associated with a large range of heart conditions of which TTN mutations are a major cause, thereby expanding the TTN mutational and phenotypic spectrum. Additionally, our results suggest titin kinase implication in cardiac morphogenesis and demonstrate that heterozygous TTN truncating mutations may not manifest unless associated with a second mutation, reassessing the paradigm of their dominant expression.
doi:10.1093/hmg/ddt494
PMCID: PMC3954110  PMID: 24105469
2.  Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2 
Acta neuropathologica  2010;119(4):465-479.
Aberrant transcription and mRNA processing of multiple genes due to RNA-mediated toxic gain-of-function has been suggested to cause the complex phenotype in myotonic dystrophies type 1 and 2 (DM1 and DM2). However, the molecular basis of muscle weakness and wasting and the different pattern of muscle involvement in DM1 and DM2 are not well understood. We have analyzed the mRNA expression of genes encoding muscle-specific proteins and transcription factors by microarray profiling and studied selected genes for abnormal splicing. A subset of the abnormally regulated genes was further analyzed at the protein level. TNNT3 and LDB3 showed abnormal splicing with significant differences in proportions between DM2 and DM1. The differential abnormal splicing patterns for TNNT3 and LDB3 appeared more pronounced in DM2 relative to DM1 and are among the first molecular differences reported between the two diseases. In addition to these specific differences, the majority of the analyzed genes showed an overall increased expression at the mRNA level. In particular, there was a more global abnormality of all different myosin isoforms in both DM1 and DM2 with increased transcript levels and a differential pattern of protein expression. Atrophic fibers in DM2 patients expressed only the fast myosin isoform, while in DM1 patients they co-expressed fast and slow isoforms. However, there was no increase of total myosin protein levels, suggesting that aberrant protein translation and/or turnover may also be involved.
doi:10.1007/s00401-010-0637-6
PMCID: PMC4199327  PMID: 20066428
Myotonic dystrophy type 1 (DM1); Myotonic dystrophy type 2 (DM2); Skeletal muscle; Aberrant splicing; Microarray expression profiling
3.  ALTERED MEF2 ISOFORMS IN MYOTONIC DYSTROPHY AND OTHER NEUROMUSCULAR DISORDERS 
Muscle & nerve  2010;42(6):856-863.
Because of their central role in muscle development and maintenance, MEF2 family members represent excellent candidate effectors of the muscle pathology in myotonic dystrophy (DM). We investigated the expression and alternative splicing of all four MEF2 genes in muscle from neuromuscular disorder (NMD) patients, including DM1 and DM2. We observed MEF2A and MEF2C overexpression in all NMD muscle, including 12 MEF2-interacting genes. Exon 4 and 5 usage in MEF2A and MEF2C was different between DM and normal muscle, with DM showing the embryonic isoform. Similar splicing differences were observed in other NMD muscle. For MEF2C, missplicing was more pronounced in DM than in other dystrophies. Our data confirm dysregulation of MEF2A and MEF2C expression and splicing in several NMD, including DM. Our findings demonstrate that aberrant splicing in NMD is independent from expression of mutant repeats, and suggests that some aberrant splicing, even in DM, may be compensatory rather than primary.
doi:10.1002/mus.21789
PMCID: PMC4136472  PMID: 21104860
dysregulation; MADS-domain transcription enhancer factor 2; MEF2; myotonic dystrophy; splicing
4.  High frequency of co-segregating CLCN1 mutations among myotonic dystrophy type 2 patients from Finland and Germany 
Journal of neurology  2008;255(11):1731-1736.
Based on previous reports the frequency of co-segregating recessive chloride channel (CLCN1) mutations in families with myotonic dystrophy type 2 (DM2) was suspected to be increased. We have studied the frequency of CLCN1 mutations in two separate patient and control cohorts from Germany and Finland, and for comparison in a German myotonic dystrophy type 1 (DM1) patient cohort. The frequency of heterozygous recessive chloride channel (CLCN1) mutations is disproportionally higher (5%) in currently diagnosed DM2 patients compared to 1.6% in the control population (p = 0.037), while the frequency in DM1 patients was the same as in the controls. Because the two genes segregate independently, the prevalence of CLCN1 mutations in the total DM2 patient population is, by definition, the same as in the control population. Our findings are, however, not based on the total DM2 population but on the currently diagnosed DM2 patients and indicate a selection bias in molecular diagnostic referrals. DM2 patients with co-segregating CLCN1 mutation have an increased likelihood to be referred for molecular diagnostic testing compared to DM2 patients without co-segregating CLCN1 mutation.
doi:10.1007/s00415-008-0010-z
PMCID: PMC4079033  PMID: 18807109
myotonic dystrophy; co-segregation; CLCN1; genetic modifier; phenotype variation
5.  Altered expression and splicing of Ca2+ metabolism genes in myotonic dystrophies DM1 and DM2 
Neuropathology and applied neurobiology  2013;39(4):10.1111/j.1365-2990.2012.01289.x.
Aims
Myotonic dystrophy types 1 and 2 (DM1 and DM2) are multisystem disorders caused by similar repeat expansion mutations, with similar yet distinct clinical features. Aberrant splicing of multiple effector genes, as well as dysregulation of transcription and translation, have been suggested to underlie different aspects of the complex phenotypes in DM1 and DM2. Ca2+ plays a central role in both muscle contraction and control of gene expression, and recent expression profiling studies have indicated major perturbations of the Ca2+ signaling pathways in DM. Here we have further investigated the expression of genes and proteins involved in Ca2+ metabolism in DM patients, including Ca2+ channels and Ca2+ binding proteins.
Methods
We used patient muscle biopsies to analyze mRNA expression and splicing of genes by microarray expression profiling and RT-PCR. We studied protein expression by immunohistochemistry and immunoblotting.
Results
Most of the genes studied showed mRNA up-regulation in expression profiling. When analyzed by immunohistochemistry the Ca2+ release channel ryanodine receptor was reduced in DM1 and DM2, as was calsequestrin 2, a sarcoplasmic reticulum lumen Ca2+ storage protein. Abnormal splicing of ATP2A1 was more pronounced in DM2 than DM1.
Conclusions
We observed abnormal mRNA and protein expression in DM affecting several proteins involved in Ca2+ metabolism, with some differences between DM1 and DM2. Our protein expression studies are suggestive of a post-transcriptional defect(s) in the myotonic dystrophies.
doi:10.1111/j.1365-2990.2012.01289.x
PMCID: PMC3882430  PMID: 22758909
Myotonic dystrophy type 1 (DM1); myotonic dystrophy type 2 (DM2); skeletal muscle; calcium metabolism
6.  Gene Expression Profiling in Tibial Muscular Dystrophy Reveals Unfolded Protein Response and Altered Autophagy 
PLoS ONE  2014;9(3):e90819.
Tibial muscular dystrophy (TMD) is a late onset, autosomal dominant distal myopathy that results from mutations in the two last domains of titin. The cascade of molecular events leading from the causative Titin mutations to the preterm death of muscle cells in TMD is largely unknown. In this study we examined the mRNA and protein changes associated with the myopathology of TMD. To identify these components we performed gene expression profiling using muscle biopsies from TMD patients and healthy controls. The profiling results were confirmed through quantitative real-time PCR and protein level analysis. One of the pathways identified was activation of endoplasmic reticulum (ER) stress response. ER stress activates the unfolded protein response (UPR) pathway. UPR activation was supported by elevation of the marker genes HSPA5, ERN1 and the UPR specific XBP1 splice form. However, UPR activation appears to be insufficient to correct the protein abnormalities causing its activation because degenerative TMD muscle fibres show an increase in ubiquitinated protein inclusions. Abnormalities of VCP-associated degradation pathways are also suggested by the presence of proteolytic VCP fragments in western blotting, and VCP's accumulation within rimmed vacuoles in TMD muscle fibres together with p62 and LC3B positive autophagosomes. Thus, pathways controlling turnover and degradation, including autophagy, are distorted and lead to degeneration and loss of muscle fibres.
doi:10.1371/journal.pone.0090819
PMCID: PMC3949689  PMID: 24618559
7.  Borderlines between Sarcopenia and Mild Late-Onset Muscle Disease 
Numerous natural or disease-related alterations occur in different tissues of the body with advancing age. Sarcopenia is defined as age-related decrease of muscle mass and strength beginning in mid-adulthood and accelerating in people older than 60 years. Pathophysiology of sarcopenia involves both neural and muscle dependent mechanisms and is enhanced by multiple factors. Aged muscles show loss in fiber number, fiber atrophy, and gradual increase in the number of ragged red fibers and cytochrome c oxidase-negative fibers. Generalized loss of muscle tissue and increased amount of intramuscular fat are seen on muscle imaging. However, the degree of these changes varies greatly between individuals, and the distinction between normal age-related weakening of muscle strength and clinically significant muscle disease is not always obvious. Because some of the genetic myopathies can present at a very old age and be mild in severity, the correct diagnosis is easily missed. We highlight this difficult borderline zone between sarcopenia and muscle disease by two examples: LGMD1D and myotonic dystrophy type 2. Muscle magnetic resonance imaging (MRI) is a useful tool to help differentiate myopathies from sarcopenia and to reach the correct diagnosis also in the elderly.
doi:10.3389/fnagi.2014.00267
PMCID: PMC4179539  PMID: 25324776
sarcopenia; myopathy; late-onset; genetic; muscle imaging
8.  Autosomal dominant late-onset spinal motor neuronopathy is linked to a new locus on chromosome 22q11.2-q13.2 
European Journal of Human Genetics  2012;20(11):1193-1196.
Spinal muscular atrophies (SMAs) are hereditary disorders characterized by degeneration of lower motor neurons. Different SMA types are clinically and genetically heterogeneous and many of them show significant phenotypic overlap. We recently described the clinical phenotype of a new disease in two Finnish families with a unique autosomal dominant late-onset lower motor neuronopathy. The studied families did not show linkage to any known locus of hereditary motor neuron disease and thus seemed to represent a new disease entity. For this study, we recruited two more family members and performed a more thorough genome-wide scan. We obtained significant linkage on chromosome 22q, maximum LOD score being 3.43 at marker D22S315. The linked area is defined by flanking markers D22S686 and D22S276, comprising 18.9 Mb. The region harbours 402 genes, none of which is previously known to be associated with SMAs. This study confirms that the disease in these two families is a genetically distinct entity and also provides evidence for a founder mutation segregating in both pedigrees.
doi:10.1038/ejhg.2012.76
PMCID: PMC3477865  PMID: 22535186
motor neuron disease; spinal muscular atrophy; linkage analysis
9.  Novel FLNC mutation in a patient with myofibrillar myopathy in combination with late-onset cerebellar ataxia 
Muscle & nerve  2012;46(2):275-282.
Introduction
Mutations in the gene that encodes filamin C, FLNC, represent a rare cause of a distinct type of myofibrillar myopathy (MFM).
Methods
We investigated an Italian patient by means of muscle biopsy, muscle and brain imaging and molecular analysis of MFM genes.
Results
The patient harbored a novel 7256C>T, p.Thr2419Met mutation in exon 44 of FLNC. Clinical, pathological and muscle MRI findings were similar to the previously described filaminopathy cases. This patient had, in addition, cerebellar ataxia with atrophy of cerebellum and vermis evident on brain MRI scan. Extensive screening failed to establish a cause of cerebellar atrophy.
Discussion
We report an Italian filaminopathy patient, with a novel mutation in a highly conserved region. This case raises the possibility that the disease spectrum caused by FLNC may include cerebellar dysfunction.
doi:10.1002/mus.23349
PMCID: PMC3400116  PMID: 22806379
filaminopathy; FLNC; myofibrillar myopathy; cerebellar ataxia; muscle MRI
10.  Titin mutation segregates with hereditary myopathy with early respiratory failure 
Brain  2012;135(6):1695-1713.
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.
doi:10.1093/brain/aws102
PMCID: PMC3359754  PMID: 22577215
hereditary myopathy with early respiratory failure; cytoplasmic body; titin; exome sequencing; distal myopathy
11.  New immunohistochemical method for improved myotonia and chloride channel mutation diagnostics 
Neurology  2012;79(22):2194-2200.
Objective:
The objective of this study was to validate the immunohistochemical assay for the diagnosis of nondystrophic myotonia and to provide full clarification of clinical disease to patients in whom basic genetic testing has failed to do so.
Methods:
An immunohistochemical assay of sarcolemmal chloride channel abundance using 2 different ClC1-specific antibodies.
Results:
This method led to the identification of new mutations, to the reclassification of W118G in CLCN1 as a moderately pathogenic mutation, and to confirmation of recessive (Becker) myotonia congenita in cases when only one recessive CLCN1 mutation had been identified by genetic testing.
Conclusions:
We have developed a robust immunohistochemical assay that can detect loss of sarcolemmal ClC-1 protein on muscle sections. This in combination with gene sequencing is a powerful approach to achieving a final diagnosis of nondystrophic myotonia.
doi:10.1212/WNL.0b013e31827595e2
PMCID: PMC3570820  PMID: 23152584
12.  Mutations affecting the cytoplasmic functions of the co-chaperone DNAJB6 cause limb-girdle muscular dystrophy 
Nature genetics  2012;44(4):450-S2.
Limb-girdle muscular dystrophy type 1D (LGMD1D) was linked to 7q36 over a decade ago1, but its genetic cause has remained elusive. We have studied nine LGMD families from Finland, the U.S., 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 provide the genetic cause of LGMD1D, suggest that the pathogenesis is mediated by defective chaperone function, and highlight how mutations expressed ubiquitously can exert their effect in a tissue-, cellular compartment-, and isoform-specific manner.
doi:10.1038/ng.1103
PMCID: PMC3315599  PMID: 22366786
13.  TARDBP mutations are not a frequent cause of ALS in Finnish patients 
Acta Myologica  2012;31(2):134-138.
In previous studies 1-3 % of ALS patients have TARDBP mutations as the cause of the disease. TARDBP mutations have been reported in ALS patients in different populations but so far there are no studies on the frequency of TARDBP mutations in Finnish ALS patients. A cohort of 50 Finnish patients, 44 SALS and 6 FALS patients, were included in the study. Genomic DNA was extracted from venous blood or muscle tissue and a mutation analysis of TARDBP was performed. No definitely pathogenic mutations could be identified in TARDBP in our patient cohort. However, two previously unknown variations were found: one silent mutation in exon 2 and one relatively deep intronic single nucleotide insertion in intron 5. In addition, two previously known non-pathogenic polymorphisms in intron 5 were detected. The size of our cohort is obviously not large enough to conclusively exclude TARDBP mutations as a very rare cause of ALS in Finland. However, based on our results TARDBP mutations do not appear to be a frequent cause of familial or sporadic ALS in Finland.
PMCID: PMC3476858  PMID: 23097605
Amyotrophic lateral sclerosis; mutation screening; TARDBP
14.  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.
doi:10.1038/ejhg.2011.23
PMCID: PMC3137497  PMID: 21364698
myotonic dystrophy; mutation frequency; prevalence; population
16.  ZNF9 Activation of IRES-Mediated Translation of the Human ODC mRNA Is Decreased in Myotonic Dystrophy Type 2 
PLoS ONE  2010;5(2):e9301.
Myotonic dystrophy types 1 and 2 (DM1 and DM2) are forms of muscular dystrophy that share similar clinical and molecular manifestations, such as myotonia, muscle weakness, cardiac anomalies, cataracts, and the presence of defined RNA-containing foci in muscle nuclei. DM2 is caused by an expansion of the tetranucleotide CCTG repeat within the first intron of ZNF9, although the mechanism by which the expanded nucleotide repeat causes the debilitating symptoms of DM2 is unclear. Conflicting studies have led to two models for the mechanisms leading to the problems associated with DM2. First, a gain-of-function disease model hypothesizes that the repeat expansions in the transcribed RNA do not directly affect ZNF9 function. Instead repeat-containing RNAs are thought to sequester proteins in the nucleus, causing misregulation of normal cellular processes. In the alternative model, the repeat expansions impair ZNF9 function and lead to a decrease in the level of translation. Here we examine the normal in vivo function of ZNF9. We report that ZNF9 associates with actively translating ribosomes and functions as an activator of cap-independent translation of the human ODC mRNA. This activity is mediated by direct binding of ZNF9 to the internal ribosome entry site sequence (IRES) within the 5′UTR of ODC mRNA. ZNF9 can activate IRES-mediated translation of ODC within primary human myoblasts, and this activity is reduced in myoblasts derived from a DM2 patient. These data identify ZNF9 as a regulator of cap-independent translation and indicate that ZNF9 activity may contribute mechanistically to the myotonic dystrophy type 2 phenotype.
doi:10.1371/journal.pone.0009301
PMCID: PMC2823779  PMID: 20174632
17.  Myotonic Dystrophy Type 2 Found in Two of Sixty-Three Persons Diagnosed as Having Fibromyalgia 
Arthritis and rheumatism  2008;58(11):3627-3631.
Because of its high prevalence, fibromyalgia (FM) is a major general health issue. Myotonic dystrophy type 2 (DM2) is a recently described autosomal-dominant multisystem disorder. Besides variable proximal muscle weakness, myotonia, and precocious cataracts, muscle pain and stiffness are prominent presenting features of DM2. After noting that several of our mutation-positive DM2 patients had a previous diagnosis of FM, suggesting that DM2 may be misdiagnosed as FM, we invited 90 randomly selected patients diagnosed as having FM to undergo genetic testing for DM2. Of the 63 patients who agreed to participate, 2 (3.2%) tested positive for the DM2 mutation. Their cases are described herein. DM2 was not found in any of 200 asymptomatic controls. We therefore suggest that the presence of DM2 should be investigated in a large sample of subjects diagnosed as having FM, and clinicians should be aware of overlap in the clinical presentation of these 2 distinct disorders.
doi:10.1002/art.24037
PMCID: PMC2585600  PMID: 18975316

Results 1-17 (17)