Differential isoform expression and selective muscle involvement in muscular dystrophies.

Despite the expression of the mutated gene in all muscles, selective muscles are involved in genetic muscular dystrophies. Different muscular dystrophies show characteristic patterns of fatty degenerative changes by muscle imaging, even to the extent that the patterns have been used for diagnostic purposes. However, the underlying molecular mechanisms explaining the selective involvement of muscles are not known. To test the hypothesis that different muscles may express variable amounts of different isoforms of muscle genes, we applied a custom-designed exon microarray containing probes for 57 muscle-specific genes to assay the transcriptional profiles in sets of human adult lower limb skeletal muscles. Quantitative real-time PCR and whole transcriptome sequencing were used to further analyze the results. Our results demonstrate significant variations in isoform and gene expression levels in anatomically different muscles. Comparison of the known patterns of selective involvement of certain muscles in two autosomal dominant titinopathies and one autosomal dominant myosinopathy, with the isoform and gene expression results, shows a correlation between the specific muscles involved and significant differences in the level of expression of the affected gene and exons in these same muscles compared with some other selected muscles. Our results suggest that differential expression levels of muscle genes and isoforms are one determinant in the selectivity of muscle involvement in muscular dystrophies.

Abnormal splicing of NEDD4 in myotonic dystrophy type 2: possible link to statin adverse reactions.

Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG)n repeat expansion in intron 1 of CNBP. Transcription of the repeats causes a toxic RNA gain of function involving their accumulation in ribonuclear foci. This leads to sequestration of splicing factors and alters pre-mRNA splicing in a range of downstream effector genes, which is thought to contribute to the diverse DM2 clinical features. Hyperlipidemia is frequent in DM2 patients, but the treatment is problematic because of an increased risk of statin-induced adverse reactions. Hypothesizing that shared pathways lead to the increased risk, we compared the skeletal muscle expression profiles of DM2 patients and controls with patients with hyperlipidemia on statin therapy. Neural precursor cell expressed, developmentally downregulated-4 (NEDD4), an ubiquitin ligase, was one of the dysregulated genes identified in DM2 patients and patients with statin-treated hyperlipidemia. In DM2 muscle, NEDD4 mRNA was abnormally spliced, leading to aberrant NEDD4 proteins. NEDD4 was down-regulated in persons taking statins, and simvastatin treatment of C2C12 cells suppressed NEDD4 transcription. Phosphatase and tensin homologue (PTEN), an established NEDD4 target, was increased and accumulated in highly atrophic DM2 muscle fibers. PTEN ubiquitination was reduced in DM2 myofibers, suggesting that the NEDD4-PTEN pathway is dysregulated in DM2 skeletal muscle. Thus, this pathway may contribute to the increased risk of statin-adverse reactions in patients with DM2.

Atypical phenotypes in titinopathies explained by second titin mutations.

OBJECTIVE: Several patients with previously reported titin gene (TTN) mutations causing tibial muscular dystrophy (TMD) have more complex, severe, or unusual phenotypes. This study aimed to clarify the molecular cause of the variant phenotypes in 8 patients of 7 European families. METHODS: Clinical, histopathological, and muscle imaging data of patients and family members were reanalyzed. The titin protein was analyzed by Western blotting and TTN gene by reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. RESULTS: Western blotting showed more pronounced C-terminal titin abnormality than expected for heterozygous probands, suggesting the existence of additional TTN mutations. RT-PCR indicated unequal mRNA expression of the TTN alleles in biopsies of 6 patients, 3 with an limb-girdle muscular dystrophy type 2J (LGMD2J) phenotype. Novel frameshift mutations were identified in 5 patients. A novel A-band titin mutation, c.92167C>T (p.P30723S), was found in 1 patient, and 1 Portuguese patient with a severe TMD phenotype proved to be homozygous for the previously reported Iberian TMD mutation. INTERPRETATION: The unequal expression levels of TTN transcripts in 5 probands suggested severely reduced expression of the frameshift mutated allele, probably through nonsense-mediated decay, explaining the more severe phenotypes. The Iberian TMD mutation may cause a more severe TMD rather than LGMD2J when homozygous. The Finnish patient compound heterozygous for the FINmaj TMD mutation and the novel A-band titin missense mutation showed a phenotype completely different from previously described titinopathies. Our results further expand the complexity of muscular dystrophies caused by TTN mutations and suggest that the coexistence of second mutations may constitute a more common general mechanism explaining phenotype variability.

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.

Mutant (CCTG)n expansion causes abnormal expression of zinc finger protein 9 (ZNF9) in myotonic dystrophy type 2.

The mutation that underlies myotonic dystrophy type 2 (DM2) is a (CCTG)n expansion in intron 1 of zinc finger protein 9 (ZNF9). It has been suggested that ZNF9 is of no consequence for disease pathogenesis. We determined the expression levels of ZNF9 during muscle cell differentiation and in DM2 muscle by microarray profiling, real-time RT-PCR, splice variant analysis, immunofluorescence, and Western blotting. Our results show that in differentiating myoblasts, ZNF9 protein was localized primarily to the nucleus, whereas in mature muscle fibers, it was cytoplasmic and organized in sarcomeric striations at the Z-disk. In patients with DM2, ZNF9 was abnormally expressed. First, there was an overall reduction in both the mRNA and protein levels. Second, the subcellular localization of the ZNF9 protein was somewhat less cytoplasmic and more membrane-bound. Third, our splice variant analysis revealed retention of intron 3 in an aberrant isoform, and fourth quantitative allele-specific expression analysis showed the persistence of intron 1 sequences from the abnormal allele, further suggesting that the mutant allele is incompletely spliced. Thus, the decrease in total expression appears to be due to impaired splicing of the mutant transcript. Our data indicate that ZNF9 expression in DM2 patients is altered at multiple levels. Although toxic RNA effects likely explain overlapping phenotypic manifestations between DM1 and DM2, abnormal ZNF9 levels in DM2 may account for the differences in DM1.

Human disease caused by loss of fast IIa myosin heavy chain due to recessive MYH2 mutations.

Striated muscle myosin heavy chain is a molecular motor protein that converts chemical energy into mechanical force. It is a major determinant of the physiological properties of each of the three muscle fibre types that make up the skeletal muscles. Heterozygous dominant missense mutations in myosin heavy chain genes cause various types of cardiomyopathy and skeletal myopathy, but the effects of myosin heavy chain null mutations in humans have not previously been reported. We have identified the first patients lacking fast type 2A muscle fibres, caused by total absence of fast myosin heavy chain IIa protein due to truncating mutations of the corresponding gene MYH2. Five adult patients, two males and three females, from three unrelated families in UK and Finland were clinically assessed and muscle biopsy was performed in one patient from each family. MYH2 was sequenced and the expression of the corresponding transcripts and protein was analysed in muscle tissue. The patients had early-onset symptoms characterized by mild generalized muscle weakness, extraocular muscle involvement and relatively favourable prognosis. Muscle biopsy revealed myopathic changes including variability of fibre size, internalized nuclei, and increased interstitial connective and adipose tissue. No muscle fibres expressing type IIa myosin heavy chain were identified and the MYH2 transcripts were markedly reduced. All patients were compound heterozygous for truncating mutations in MYH2. The parents were unaffected, consistent with recessive mutations. Our findings show that null mutations in the fast myosin heavy chain IIa gene cause early onset myopathy and demonstrate that this isoform is necessary for normal muscle development and function. The relatively mild phenotype is interesting in relation to the more severe phenotypes generally seen in relation to recessive null mutations in sarcomeric proteins.

Novel myosin heavy chain immunohistochemical double staining developed for the routine diagnostic separation of I, IIA and IIX fibers.

The different histochemical ATPase properties of myosins separating the muscle fiber types have been utilized in diagnostic muscle biopsy routine for more than four decades. The ATPase staining method is rather laborious and has several disadvantages, such as weakening of staining over time and non-specific staining of capillaries, making the distinction of extremely atrophic muscle fibers difficult. We have developed a reliable and advanced immunohistochemical myosin double staining method for the identification of fiber types, including highly atrophic fibers in routine diagnostics. With this double staining method, we are able to distinguish among type I (ATPase type 1), IIA (ATPase type 2A), IIX (ATPase type 2B) and remodeled ATPase type 2C fibers expressing both fast and slow myosins using a one slide technique. Immunohistochemical double staining of myosin heavy chain isoforms can be used as an alternative for the conventional ATPase staining method in routine histopathology. The method provides even more detailed information of fast fiber subtypes and highly atrophic fibers on one single slide.

Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2.

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.

The enigma of 7q36 linked autosomal dominant limb girdle muscular dystrophy.

INTRODUCTION: Two families with autosomal dominant limb girdle muscular dystrophy (LGMD) have previously been linked to a locus on chromosome 7q36 10 years ago. The locus has been termed both LGMD1D and 1E, but because of lack of additional families to narrow down the linked region of interest, this disease has remained elusive. METHODS: A large Finnish family was clinically and genetically investigated. Laboratory parameters were determined, including creatine kinase (CK) value, neurographic and electromyography studies, cardiac and respiratory function examinations, muscle biopsies and muscle imaging by CT or MRI. RESULTS: Patients had onset of muscle weakness in the pelvic girdle between the fourth and sixth decades with an autosomal dominant pattern of inheritance. CK values were slightly elevated and electromyography was myopathic only. Muscle biopsies showed myopathic and/or dystrophic features with very minor rimmed vacuolation and protein aggregation findings. Molecular genetic analysis indicates linkage of the disease to the locus on chromosome 7q36 completely overlapping with the previously reported locus LGMD1D/E. DISCUSSION: Advancement towards the causative gene defect in the 7q36 linked disease needs new additional families to narrow the region of interest. The phenotype in the previously linked families has not been reported in full detail, which may be one reason for the shortage of additional families. A comprehensive clinical and morphological phenotype of chromosome 7q36 linked autosomal dominant LGMD with a restricted and updated 6.4 Mb sized haplotype is reported here.

Cytoplasmic LRIG2 expression is associated with poor oligodendroglioma patient survival.

The three leucine-rich repeats and immunoglobulin-like domains (LRIG) genes encode integral membrane proteins. Of these, LRIG1 negatively regulates growth factor signaling and is implicated as a tumor suppressor in certain malignancies. In astrocytic tumors, the subcellular distribution of LRIG proteins is associated with specific clinicopathological features and patient survival. The role of LRIG proteins in oligodendroglioma has not previously been studied. Here we used immunohistochemistry to analyze the expression of the LRIG proteins in 63 oligodendroglial tumors, and evaluated possible associations between LRIG protein expression and clinicopathological parameters. Notably, cytoplasmic LRIG2 expression was found to be an independent prognostic factor associated with poor oligodendroglioma patient survival. This is the first report of an LRIG protein showing a negative effect on survival, suggesting that LRIG2 might have a function different from that of LRIG1, and possibly contributing to the etiology of oligodendroglioma.

Distinct Muscle Biopsy Findings in Genetically Defined Adult-Onset Motor Neuron Disorders.

The objective of this study was to characterize and compare muscle histopathological findings in 3 different genetic motor neuron disorders. We retrospectively re-assessed muscle biopsy findings in 23 patients with autosomal dominant lower motor neuron disease caused by p.G66V mutation in CHCHD10 (SMAJ), 10 X-linked spinal and bulbar muscular atrophy (SBMA) and 11 autosomal dominant c9orf72-mutated amyotrophic lateral sclerosis (c9ALS) patients. Distinct large fiber type grouping consisting of non-atrophic type IIA muscle fibers were 100% specific for the late-onset spinal muscular atrophies (SMAJ and SBMA) and were never observed in c9ALS. Common, but less specific findings included small groups of highly atrophic rounded type IIA fibers in SMAJ/SBMA, whereas in c9ALS, small group atrophies consisting of small-caliber angular fibers involving both fiber types were more characteristic. We also show that in the 2 slowly progressive motor neuron disorders (SMAJ and SBMA) the initial neurogenic features are often confused with considerable secondary "myopathic" changes at later disease stages, such as rimmed vacuoles, myofibrillar aggregates and numerous fibers reactive for fetal myosin heavy chain (dMyHC) antibodies. Based on our findings, muscle biopsy may be valuable in the diagnostic work-up of suspected motor neuron disorders in order to avoid a false ALS diagnosis in patients without clear findings of upper motor neuron lesions.

Diagnostically important muscle pathology in DNAJB6 mutated LGMD1D.

INTRODUCTION: Limb girdle muscular dystrophies are a large group of both dominantly and recessively inherited muscle diseases. LGMD1D is caused by mutated DNAJB6 and the molecular pathogenesis is mediated by defective chaperonal function leading to impaired handling of misfolded proteins which normally would be degraded. Here we aim to clarify muscle pathology of LGMD1D in order to facilitate diagnostic accuracy. After following six Finnish LGMD1D families, we analysed 21 muscle biopsies obtained from 15 patients at different time points after the onset of symptoms. All biopsies were obtained from the lower limb muscles and processed for routine histochemistry, extensive immunohistochemistry and electron microscopy. RESULTS: Histopathological findings were myopathic or dystrophic combined with rimmed vacuolar pathology, and small myofibrillar aggregates. These myofibrillar inclusions contained abnormal accumulation of a number of proteins such as myotilin, αB-crystallin and desmin on immunohistochemistry, and showed extensive myofibrillar disorganization with excess of Z-disk material on ultrastructure. Later in the disease process the rimmed vacuolar pathology dominated with rare cases of pronounced larger pleomorphic myofibrillar aggregates. The rimmed vacuoles were reactive for several markers of defect autophagy such as ubiquitin, TDP-43, p62 and SMI-31. CONCLUSIONS: Since DNAJB6 is known to interact with members of the chaperone assisted selective autophagy complex (CASA), including BAG3 - a known myofibrillar myopathy causing gene, the molecular muscle pathology is apparently mediated through impaired functions of CASA and possibly other complexes needed for the maintenance of the Z-disk and sarcomeric structures. The corresponding findings on histopathology offer clues for the diagnosis.

PP2A Inhibitor PME-1 Drives Kinase Inhibitor Resistance in Glioma Cells.

Glioblastoma multiforme lacks effective therapy options. Although deregulated kinase pathways are drivers of malignant progression in glioblastoma multiforme, glioma cells exhibit intrinsic resistance toward many kinase inhibitors, and the molecular basis of this resistance remains poorly understood. Here, we show that overexpression of the protein phosphatase 2A (PP2A) inhibitor protein PME-1 drives resistance of glioma cells to various multikinase inhibitors. The PME-1-elicited resistance was dependent on specific PP2A complexes and was mediated by a decrease in cytoplasmic HDAC4 activity. Importantly, both PME-1 and HDAC4 associated with human glioma progression, supporting clinical relevance of the identified mechanism. Synthetic lethality induced by both PME-1 and HDAC4 inhibition was dependent on the coexpression of proapoptotic protein BAD. Thus, PME-1-mediated PP2A inhibition is a novel mechanistic explanation for multikinase inhibitor resistance in glioma cells. Clinically, these results may inform patient stratification strategies for future clinical trials with selected kinase inhibitors in glioblastoma multiforme. Cancer Res; 76(23); 7001-11. ©2016 AACR.

Characterization of a novel cell line established from a patient with Herceptin-resistant breast cancer.

Clinical resistance to the HER-2 oncogene-targeting drug trastuzumab (Herceptin) exists, but studies of the resistance mechanisms are hampered by the lack of suitable experimental model systems. We established a carcinoma cell line (designated JIMT-1) from a pleural metastasis of a 62-year old patient with breast cancer who was clinically resistant to trastuzumab. JIMT-1 cells grow as an adherent monolayer and form xenograft tumors in nude mice. JIMT-1 cells have an amplified HER-2 oncogene, which showed no identifiable mutations in its coding sequence. JIMT-1 cells overexpress HER-2 mRNA and protein, and the levels of HER-1, HER-3, and HER-4 mRNA and protein were similar to the trastuzumab-sensitive cell line SKBR-3. The cell line lacks expression of hormone receptors (estrogen receptors and progesterone receptors) and is phenotypically of epithelial progenitor cell origin, as evidenced by immunohistochemical positivity for both cytokeratins 5/14 and 8/18. JIMT-1 cells were insensitive to trastuzumab and another HER-2-inhibiting drug, pertuzumab (2C4), in vitro and in xenograft tumors. Small molecule tyrosine kinase inhibitors Ci1033 and ZD1839 inhibited the JIMT-1 cell growth but to a lesser degree than in trastuzumab-sensitive BT-474 cells. The lack of growth inhibition was rationalized by the unaltered Akt phosphorylation in JIMT-1 cells. Erk1/2 phosphorylation was slightly reduced but still evident in JIMT-1 cells. We conclude that the JIMT-1 cell line provides a valuable experimental model for studies of new trastuzumab-resistance mechanisms.

Gene expression profiling in tibial muscular dystrophy reveals unfolded protein response and altered autophagy.

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.

Recessive myosin myopathy with external ophthalmoplegia associated with MYH2 mutations.

Myosin myopathies comprise a group of inherited diseases caused by mutations in myosin heavy chain (MyHC) genes. Homozygous or compound heterozygous truncating MYH2 mutations have been demonstrated to cause recessive myopathy with ophthalmoplegia, mild-to-moderate muscle weakness and complete lack of type 2A muscle fibers. In this study, we describe for the first time the clinical and morphological characteristics of recessive myosin IIa myopathy associated with MYH2 missense mutations. Seven patients of five different families with a myopathy characterized by ophthalmoplegia and mild-to-moderate muscle weakness were investigated. Muscle biopsy was performed to study morphological changes and MyHC isoform expression. Five of the patients were homozygous for MYH2 missense mutations, one patient was compound heterozygous for a missense and a nonsense mutation and one patient was homozygous for a frame-shift MYH2 mutation. Muscle biopsy demonstrated small or absent type 2A muscle fibers and reduced or absent expression of the corresponding MyHC IIa transcript and protein. We conclude that mild muscle weakness and ophthalmoplegia in combination with muscle biopsy demonstrating small or absent type 2A muscle fibers are the hallmark of recessive myopathy associated with MYH2 mutations.

Most expression and splicing changes in myotonic dystrophy type 1 and type 2 skeletal muscle are shared with other muscular dystrophies.

The prevailing pathomechanistic paradigm for myotonic dystrophy (DM) is that aberrant expression of embryonic/fetal mRNA/protein isoforms accounts for most aspects of the pleiotropic phenotype. To identify aberrant isoforms in skeletal muscle of DM1 and DM2 patients, we performed exon-array profiling and RT-PCR validation on the largest DM sample set to date, including Duchenne, Becker and tibial muscular dystrophy (NMD) patients as disease controls, and non-disease controls. Strikingly, most expression and splicing changes in DM patients were shared with NMD controls. Comparison between DM and NMD identified almost no significant differences. We conclude that DM1 and DM2 are essentially identical for dysregulation of gene expression, and DM expression changes represent a subset of broader spectrum dystrophic changes. We found no evidence for qualitative splicing differences between DM1 and DM2. While some DM-specific splicing differences exist, most of the DM splicing differences were also seen in NMD controls. SSBP3 exon 6 missplicing was observed in all diseased muscle and led to reduced protein. We conclude there is no widespread DM-specific spliceopathy in skeletal muscle and suggest that missplicing in DM (and NMD) may not be the driving mechanism for the muscle pathology, since the same pathways show expression changes unrelated to splicing.

Co-segregation of DM2 with a recessive CLCN1 mutation in juvenile onset of myotonic dystrophy type 2.

Myotonic dystrophy type 2 (DM2) is a common adult onset muscular dystrophy caused by a dominantly transmitted (CCTG)( n ) expansion in intron 1 of the CNBP gene. In DM2 there is no obvious evidence for an intergenerational increase of expansion size, and no congenital cases have been confirmed. We describe the clinical and histopathological features, and provide the genetic and molecular explanation for juvenile onset of myotonia in a 14-year-old female with DM2 and her affected mother presenting with a more severe phenotype despite a later onset of symptoms. Histological and immunohistochemical findings correlated with disease severity or age at onset in both patients. Southern blot on both muscle and blood samples revealed only a small increase in the CCTG repeat number through maternal transmission. Fluorescence in situ hybridization, in combination with MBNL1 immunofluorescence on muscle sections, showed the presence of mutant mRNA and MBNL1 in nuclear foci; the fluorescence intensity and its area appeared to be similar in the two patients. Splicing analysis of the INSR, CLCN1 and MBNL1 genes in muscle tissue demonstrates that the level of aberrant splicing isoforms was lower in the daughter than in the mother. However, in the CLCN1 gene, a heterozygous mutation c.501C>G p.F167L was present in the daughter's DNA and found to be maternally inherited. Biomolecular findings did not explain the unusual young onset in the daughter. The co-segregation of DM2 with a recessive CLCN1 mutation provided the explanation for the unusual clinical findings.

New immunohistochemical method for improved myotonia and chloride channel mutation diagnostics.

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.

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.