Genotype-phenotype study in patients with valosin-containing protein mutations associated with multisystem proteinopathy.

Mutations in valosin-containing protein (VCP), an ATPase involved in protein degradation and autophagy, cause VCP disease, a progressive autosomal dominant adult onset multisystem proteinopathy. The goal of this study is to examine if phenotypic differences in this disorder could be explained by the specific gene mutations. We therefore studied 231 individuals (118 males and 113 females) from 36 families carrying 15 different VCP mutations. We analyzed the correlation between the different mutations and prevalence, age of onset and severity of myopathy, Paget's disease of bone (PDB), and frontotemporal dementia (FTD), and other comorbidities. Myopathy, PDB and FTD was present in 90%, 42% and 30% of the patients, respectively, beginning at an average age of 43, 41, and 56 years, respectively. Approximately 9% of patients with VCP mutations had an amyotrophic lateral sclerosis (ALS) phenotype, 4% had been diagnosed with Parkinson's disease (PD), and 2% had been diagnosed with Alzheimer's disease (AD). Large interfamilial and intrafamilial variation made establishing correlations difficult. We did not find a correlation between the mutation type and the incidence of any of the clinical features associated with VCP disease, except for the absence of PDB with the R159C mutation in our cohort and R159C having a later age of onset of myopathy compared with other molecular subtypes.

Seropositivity for NT5c1A antibody in sporadic inclusion body myositis predicts more severe motor, bulbar and respiratory involvement.

OBJECTIVES: To explore phenotypic differences between individuals with sporadic inclusion body myositis (sIBM) who are seropositive for the NT5c1A antibody compared with those who are seronegative. METHODS: Cross-sectional clinical, serological and functional analysis in 25 consecutive participants with sIBM. RESULTS: All participants met criteria for clinically defined or probable sIBM. 18 of 25 participants with sIBM (72%) were seropositive for the NT5c1A antibody. No differences between median age and duration of illness between the two groups were seen. Females have higher odds of being seropositive (OR=2.30). Participants with seropositive sIBM took significantly longer to get up and stand (p=0.012). There were no significant differences between the two groups in terms of distance covered on a 6 min walk. Seropositive participants were more likely to require assistive devices such as a walker or wheelchair for mobility (OR=23.00; p=0.007). A number of secondary (exploratory) outcomes were assessed. NT5c1A seropositive sIBM cases had lower total Medical Research Council (MRC) sum score and MRC sum score on the right (p=0.03 and 0.02, respectively). Participants with the NT5c1A antibody were significantly more likely to have symptoms of dysphagia (OR=10.67; p=0.03) and reduced forced vital capacity (p=0.005). Facial weakness occurred in 50% of seropositive participants while it was only seen in 14% of seronegative participants. CONCLUSIONS: Even though the small sample size limits definite conclusions, our cross-sectional study showed seropositivity to the NT5c1A antibody is associated with greater motor and functional disability in sIBM. The study also suggests more prominent bulbar, facial and respiratory involvement in individuals positive for NT5c1A antibodies.

Activation of the Wnt/ß-catenin signaling cascade after traumatic nerve injury.

Recent data have shown that preservation of the neuromuscular junction (NMJ) after traumatic nerve injury helps to improve functional recovery with surgical repair via matrix metalloproteinase-3 (MMP3) blockade. As such, we sought to explore additional pathways that may augment this response. Wnt3a has been shown to inhibit acetylcholine receptor (AChR) clustering via ß-catenin-dependent signaling in the development of the NMJ. Therefore, we hypothesized that Wnt3a and ß-catenin are associated with NMJ destabilization following traumatic denervation. A critical size nerve defect was created by excising a 10-mm segment of the sciatic nerve in mice. Denervated muscles were then harvested at multiple time points for immunofluorescence staining, quantitative real-time PCR, and western blot analysis for Wnt3a and ß-catenin levels. Moreover, a novel Wnt/ß-catenin transgenic reporter mouse line was utilized to support our hypothesis of Wnt activation after traumatic nerve injury. The expression of Wnt3a mRNA was significantly increased by 2 weeks post-injury and remained upregulated for 2 months. Additionally, ß-catenin was activated at 2 months post-injury relative to controls. Correspondingly, immunohistochemical analysis of denervated transgenic mouse line TCF/Lef:H2B-GFP muscles demonstrated that the number of GFP-positive cells was increased at the motor endplate band. These collective data support that post-synaptic AChRs destabilize after denervation by a process that involves the Wnt/ß-catenin pathway. As such, this pathway serves as a potential therapeutic target to prevent the motor endplate degeneration that occurs following traumatic nerve injury.

Genotype-phenotype studies of VCP-associated inclusion body myopathy with Paget disease of bone and/or frontotemporal dementia.

Valosin containing protein (VCP) disease associated with inclusion body myopathy, Paget disease of the bone and frontotemporal dementia is a progressive autosomal dominant disorder caused by mutations in Valosin containing protein gene. To establish genotype-phenotype correlations we analyzed clinical and biochemical markers from a database of 190 members in 27 families harboring 10 missense mutations. Individuals were grouped into three categories: symptomatic, presymptomatic carriers and noncarriers. The symptomatic families were further divided into ten groups based on their VCP mutations. There was marked intra and inter-familial variation; and significant genotype-phenotype correlations were difficult to establish because of small numbers. Nevertheless when comparing the two most common mutations, R155C mutation was found to be more severe, with an earlier onset of myopathy and Paget (p = 0.03). Survival analysis of all subjects revealed an average life span after diagnosis of myopathy and Paget of 18 and 19 years respectively, and after dementia only 6 years. R155C had a reduced survival compared to the R155H mutation (p = 0.03).We identified amyotrophic lateral sclerosis (ALS) was diagnosed in 13 individuals (8.9%) and Parkinson's disease in five individuals (3%); however, there was no genotypic correlation. This study represents the largest dataset of patients with VCP disease and expands our understanding of the natural history and provides genotype-phenotype correlations in this unique disease.

Slow development of ALS-like spinal cord pathology in mutant valosin-containing protein gene knock-in mice.

Pathological features of amyotrophic lateral sclerosis (ALS) include, in addition to selective motor neuron (MN) degeneration, the occurrence of protein aggregates, mitochondrial dysfunction and astrogliosis. SOD1 mutations cause rare familial forms of ALS and have provided the most widely studied animal models. Relatively recent studies implicating another protein, TDP-43, in familial and sporadic forms of ALS have led to the development of new animal models. More recently, mutations in the valosin-containing protein (VCP) gene linked to the human genetic disease, Inclusion Body Myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD), were found also to be associated with ALS in some patients. A heterozygous knock-in VCP mouse model of IBMPFD (VCP(R155H/+)) exhibited muscle, bone and brain pathology characteristic of the human disease. We have undertaken studies of spinal cord pathology in VCP(R155H/+) mice and find age-dependent degeneration of ventral horn MNs, TDP-43-positive cytosolic inclusions, mitochondrial aggregation and progressive astrogliosis. Aged animals (~24-27 months) show electromyography evidence of denervation consistent with the observed MN loss. Although these animals do not develop rapidly progressive fatal ALS-like disease during their lifespans, they recapitulate key pathological features of both human disease and other animal models of ALS, and may provide a valuable new model for studying events preceding onset of catastrophic disease.

Phase II screening trial of lithium carbonate in amyotrophic lateral sclerosis: examining a more efficient trial design.

OBJECTIVE: To use a historical placebo control design to determine whether lithium carbonate slows progression of amyotrophic lateral sclerosis (ALS). METHODS: A phase II trial was conducted at 10 sites in the Western ALS Study Group using similar dosages (300-450 mg/day), target blood levels (0.3-0.8 mEq/L), outcome measures, and trial duration (13 months) as the positive trial. However, taking riluzole was not a requirement for study entry. Placebo outcomes in patients matched for baseline features from a large database of recent clinical trials, showing stable rates of decline over the past 9 years, were used as historical controls. RESULTS: The mean rate of decline of the ALS Functional Rating Scale-Revised was greater in 107 patients taking lithium carbonate (-1.20/month, 95% confidence interval [CI] -1.41 to -0.98) than that in 249 control patients (-1.01/month, 95% CI -1.11 to -0.92, p = 0.04). There were no differences in secondary outcome measures (forced vital capacity, time to failure, and quality of life), but there were more adverse events in the treated group. CONCLUSIONS: The lack of therapeutic benefit and safety concerns, taken together with similar results from 2 other recent trials, weighs against the use of lithium carbonate in patients with ALS. The absence of drift over time and the availability of a large database of patients for selecting a matched historical control group suggest that use of historical controls may result in more efficient phase II trials for screening putative ALS therapeutic agents. CLASSIFICATION OF EVIDENCE: This study provided Class IV evidence that lithium carbonate does not slow the rate of decline of function in patients with ALS over 13 months.

Congenital myasthenic syndrome associated with epidermolysis bullosa caused by homozygous mutations in PLEC1 and CHRNE.

Mutations in the plectin gene (PLEC1) cause epidermolysis bullosa simplex (EBS), which may associate with muscular dystrophy (EBS-MD) or pyloric atresia (EBS-PA). The association of EBS with congenital myasthenic syndrome (CMS) is also suspected to result from PLEC1 mutations. We report here a consanguineous patient with EBS and CMS for whom mutational analysis of PLEC1 revealed a homozygous 36 nucleotide insertion (1506_1507ins36) that results in a reduced expression of PLEC1 mRNA and plectin in the patient muscle. In addition, mutational analysis of CHRNE revealed a homozygous 1293insG, which is a well-known low-expressor receptor mutation. A skin biopsy revealed signs of EBS, and an anconeus muscle biopsy showed signs of a mild myopathy. Endplate studies showed fragmentation of endplates, postsynaptic simplification, and large collections of thread-like mitochondria. Amplitudes of miniature endplate potentials were diminished, but the endplate quantal content was actually increased. The complex phenotype presented here results from mutations in two separate genes. While the skin manifestations are because of the PLEC1 mutation, footprints of mutations in PLEC1 and CHRNE are present at the neuromuscular junction of the patient indicating that abnormalities in both genes contribute to the CMS phenotype.

Rapid identification of mitochondrial DNA (mtDNA) mutations in neuromuscular disorders by using surveyor strategy.

Mutations of mitochondrial genome are responsible for respiratory chain defects in numerous patients. We have used a strategy, based on the use of a mismatch-specific DNA endonuclease named " Surveyor Nuclease", for screening the entire mtDNA in a group of 50 patients with neuromuscular features, suggesting a respiratory chain dysfunction. We identified mtDNA mutations in 20% of patients (10/50). Among the identified mutations, four are not found in any mitochondrial database and have not been reported previously. We also confirm that mtDNA polymorphisms are frequently found in a heteroplasmic state (15 different polymorphisms were identified among which five were novel).

PIP2 binding residues of Kir2.1 are common targets of mutations causing Andersen syndrome.

BACKGROUND: Mutations in KCNJ2, the gene encoding the inward-rectifying K+ channel Kir2.1, cause the cardiac, skeletal muscle, and developmental phenotypes of Andersen-Tawil syndrome (ATS; also known as Andersen syndrome). Although pathogenic mechanisms have been proposed for select mutations, a common mechanism has not been identified. METHODS: Seventeen probands presenting with symptoms characteristic of ATS were evaluated clinically and screened for mutations in KCNJ2. The results of mutation analysis were combined with those from previously studied subjects to assess the frequency with which KCNJ2 mutations cause ATS. RESULTS: Mutations in KCNJ2 were discovered in nine probands. These included six novel mutations (D71N, T75R, G146D, R189I, G300D, and R312C) as well as previously reported mutations R67W and R218W. Six probands possessed mutations of residues implicated in binding membrane-associated phosphatidylinositol 4,5-bisphosphate (PIP2). In total, mutations in PIP(2)-related residues accounted for disease in 18 of 29 (62%) reported KCNJ2 -based probands with ATS. Also reported is that mutation R67W causes the full clinical triad in two unrelated males. CONCLUSIONS: The novel mutations corresponding to residues involved in Kir2.1 channel-PIP2 interactions presented here as well as the overall frequency of mutations occurring in these residues indicate that defects in PIP2 binding constitute a major pathogenic mechanism of ATS. Furthermore, screening KCNJ2 in patients with the complex phenotypes of ATS was found to be invaluable in establishing or confirming a disease diagnosis as mutations in this gene can be identified in the majority of patients.

Ischemic stroke subtypes in Pakistan: the Aga Khan University Stroke Data Bank.

OBJECTIVE: Frequency of ischemic stroke subtypes is influenced by ethnic and geographic variables. Our objective was to identify various stroke subtypes and its determinants at a tertiary care hospital. METHODS: We prospectively collected data on ischemic stroke subtypes admitted to The Aga Khan University Hospital in Karachi. RESULTS: A total of 596 patients were enrolled in 22 months in the Aga Khan Universtiy Stroke Registry. These included 393 patients with Ischemic stroke, 126 patients with intracerebral hemorrhage, 50 patients with subarachnoid hemorrhage and others. The ischemic stroke group was classified according to the TOAST criteria and comprised of lacunar 168/393 (42.7%); large artery atherosclerosis 106/393 (26.9%); cardioembolic 24/393 (6.1%); undetermined 80/393 (20.3%); and other determined types 15/393 (3.8%). The high proportion of lacunar strokes in our population may be due to high burden of inadequately treated hypertension and diabetes. Clear cut cardioembolic stroke was relatively infrequent in our population. CONCLUSION: Lacunar stroke is the most common subtype of stroke in our patient population. This is most likely secondary to uncontrolled hypertension.

Predictors of mortality in brain abscess.

OBJECTIVE: Brain abscess carries significant morbidity and mortality. Our objective was to elucidate the clinical presentation of brain abscess and to assess predictors of mortality in these patients. METHODS: All patients with a brain abscess presenting to the Aga Khan University Hospital, a tertiary care referral center in Karachi, Pakistan, were studied retrospectively. Statistical analysis involved univariate analysis and a logistic regression model. RESULTS: Among the 66 patients analyzed, a distant metastatic focus of infection was the most commonly identified predisposing factor (29%). Otogenic infection was the commonest contiguous source and sinusitis was noticeably absent. Multiple abscesses were frequent (35%). Streptococci were the most common isolates (39%). Lumbar puncture was performed in 44% and steroids administered in 33%. Treatment was surgical in 58%. Most comatose patients were treated conservatively. Overall mortality was 29%. Univariate analysis identified comatose presentation and identification of a distant focus of infection as predictors of mortality. The logistic regression model, however, identified a distant focus of infection as the only independent predictor. CONCLUSION: Age greater than 30 years, corticosteroid use, multiple abscesses, performance of lumbar puncture and conservative management had no affect on outcome.

Myopathy with anti-Jo-1 antibodies: pathology in perimysium and neighbouring muscle fibres.

OBJECTIVE: To evaluate muscle pathology and clinical characteristics in patients with a myopathy and serum antibodies to the Jo-1 antigen (histidyl t-RNA synthetase). BACKGROUND: Anti-Jo-1 antibodies occur in syndromes that may include muscle weakness and pain, Raynaud's phenomenon, interstitial lung disease, arthritis, and a skin rash different from that seen in dermatomyositis. The muscle pathology is not well defined. METHODS: Case series. Review of charts, muscle biopsies, and laboratory records. Features of myopathology in 11 patients with anti-Jo-1 antibody associated myopathies were compared with other types of inflammatory myopathies. RESULTS: Myopathology in patients with anti-Jo-1 antibodies consistently included fragmentation of, and macrophage predominant inflammation in, perimysial connective tissue. Perifascicular myopathic changes, including atrophy, regenerating muscle fibres, and some muscle fibre necrosis, were most common in regions near the connective tissue pathology and were most prominent in patients with more severe weakness. Unlike many other inflammatory myopathies, inflammation in endomysial and perivascular regions was uncommon. By contrast with dermatomyositis, capillary density was normal. CONCLUSIONS: Myopathological changes in the anti-Jo-1 antibody syndrome include perimysial connective tissue fragmentation and inflammation, with muscle fibre pathology in neighbouring perifascicular regions. Myositis with anti-Jo-1 antibodies may result from an immune mediated disorder of connective tissue.

Factor XI deficiency-related spontaneous primary intraventricular hemorrhage.

Factor XI deficiency (plasma thromboplastin antecedent deficiency) is a rare autosomal-dominant disorder. Neurologic complications in factor XI deficiency are even rarer. We propose that the factor XI deficiency in our patient contributed to a primary intraventricular hemorrhage.