Anna Fidziańska

Functional and clinical characterization of KCNJ2 mutations associated with LQT7 (Andersen syndrome)

Andersen syndrome (AS) is a rare, inherited disorder characterized by periodic paralysis, long QT (LQT) with ventricular arrhythmias, and skeletal developmental abnormalities. We recently established that AS is caused by mutations in KCNJ2, which encodes the inward rectifier K(+) channel Kir2.1. In this report, we characterized the functional consequences of three novel and seven previously described KCNJ2 mutations using a two-microelectrode voltage-clamp technique and correlated the findings with the clinical phenotype. All mutations resulted in loss of function and dominant-negative suppression of Kir2.1 channel function. In mutation carriers, the frequency of periodic paralysis was 64% and dysmorphic features 78%. LQT was the primary cardiac manifestation, present in 71% of KCNJ2 mutation carriers, with ventricular arrhythmias present in 64%. While arrhythmias were common, none of our subjects suffered sudden cardiac death. To gain insight into the mechanism of arrhythmia susceptibility, we simulated the effect of reduced Kir2.1 using a ventricular myocyte model. A reduction in Kir2.1 prolonged the terminal phase of the cardiac action potential, and in the setting of reduced extracellular K(+), induced Na(+)/Ca(2+) exchanger-dependent delayed afterdepolarizations and spontaneous arrhythmias. These findings suggest that the substrate for arrhythmia susceptibility in AS is distinct from the other forms of inherited LQT syndrome.

Mutations in the sarcoglycan genes in patients with myopathy.

BACKGROUND Some patients with autosomal recessive limb-girdle muscular dystrophy have mutations in the genes coding for the sarcoglycan proteins (alpha-, beta-, gamma-, and delta-sarcoglycan). To determine the frequency of sarcoglycan-gene mutations and the relation between the clinical features and genotype, we studied several hundred patients with myopathy. METHODS Antibody against alpha-sarcoglycan was used to stain muscle-biopsy specimens from 556 patients with myopathy and normal dystrophin genes (the gene frequently deleted in X-linked muscular dystrophy). Patients whose biopsy specimens showed a deficiency of alpha-sarcoglycan on immunostaining were studied for mutations of the alpha-, beta-, and gamma-sarcoglycan genes with reverse transcription of muscle RNA, analysis involving single-strand conformation polymorphisms, and sequencing. RESULTS Levels of alpha-sarcoglycan were found to be decreased on immunostaining of muscle-biopsy specimens from 54 of the 556 patients (10 percent); in 25 of these patients no alpha-sarcoglycan was detected. Screening for sarcoglycan-gene mutations in 50 of the 54 patients revealed mutations in 29 patients (58 percent): 17 (34 percent) had mutations in the alpha-sarcoglycan gene, 8 (16 percent) in the beta-sarcoglycan gene, and 4 (8 percent) in the gamma-sarcoglycan gene. No mutations were found in 21 patients (42 percent). The prevalence of sarcoglycan-gene mutations was highest among patients with severe (Duchenne-like) muscular dystrophy that began in childhood (18 of 83 patients, or 22 percent); the prevalence among patients with proximal (limb-girdle) muscular dystrophy with a later onset was 6 percent (11 of 180 patients). CONCLUSIONS Defects in the genes coding for the sarcoglycan proteins are limited to patients with Duchenne-like and limb-girdle muscular dystrophy with normal dystrophin and occur in 11 percent of such patients.

Architectural abnormalities in muscle nuclei. Ultrastructural differences between X-linked and autosomal dominant forms of EDMD

OBJECTIVES The aim of our study was to compare the ultrastructure of myonuclei in both forms of Emery-Dreifuss dystrophy (EDMD)-X-linked and dominantly autosomally transmitted. The muscle biopsies were taken from rectus femoris in four X-linked EDMD cases and three ADEDMD cases. METHODS The biopsies were evaluated using immunocytochemical staining to establish emerin or A/C lamins deficiency. The muscle ultrastructure, especially that of nuclei, was analysed to find out whether there are differences between the two forms of EDMD. RESULTS In both forms of EDMD, there was an aberrant nuclear architecture. In the X-linked form, the breakdown of fragile nuclear membrane and presence of nucleoplasm extrusion were a distinct feature. In the AD from, there was chromatin reorganization and loss of nucleoplasm volume.

In vivo and in vitro examination of the functional significances of novel lamin gene mutations in heart failure patients

Context: Lamin A/C (LMNA) gene variations have been reported in more than one third of genotyped families with dilated cardiomyopathy (DCM). However, the relationship between LMNA mutation and the development of DCM is poorly understood. Methods and results: We found that end stage DCM patients carrying LMNA mutations displayed either dramatic ultrastructural changes of the cardiomyocyte nucleus (D192G) or nonspecific changes (R541S). Overexpression of the D192G lamin C dramatically increased the size of intranuclear speckles and reduced their number. This phenotype was only partially reversed by coexpression of the D192G and wild type lamin C. Moreover, the D192G mutation precludes insertion of lamin C into the nuclear envelope when co-transfected with the D192G lamin A. By contrast, the R541S phenotype was entirely reversed by coexpression of the R541S and wild type lamin C. As lamin speckle size is known to be correlated with regulation of transcription, we assessed the SUMO1 distribution pattern in the presence of mutated lamin C and showed that D192G lamin C expression totally disrupts the SUMO1 pattern. Conclusion: Our in vivo and in vitro results question the relationship of causality between LMNA mutations and the development of heart failure in some DCM patients and therefore, the reliability of genetic counselling. However, LMNA mutations producing speckles result not only in nuclear envelope structural damage, but may also lead to the dysregulation of cellular functions controlled by sumoylation, such as transcription, chromosome organisation, and nuclear trafficking.

“Cap disease” New congenital myopathy

In a 7-year-old boy with delayed motor development, a congenital non progressive myopathy was diagnosed. Histochemical and ultrastructural examination of a muscle specimen revealed unusual pathologic findings. In 70% of muscle fibers, peripherally located zones lacking in ATPase activity and consisting of abnormally arranged myofibrils were observed. The characteristic position of the peripherally located myofibrils and their abnormal sarcomere pattern seem to point to an error in the fusion as well as in the synthesis of muscle protein.

Genetic and ultrastructural studies in dilated cardiomyopathy patients: a large deletion in the lamin A/C gene is associated with cardiomyocyte nuclear envelope disruption

Major nuclear envelope abnormalities, such as disruption and/or presence of intranuclear organelles, have rarely been described in cardiomyocytes from dilated cardiomyopathy (DCM) patients. In this study, we screened a series of 25 unrelated DCM patient samples for (a) cardiomyocyte nuclear abnormalities and (b) mutations in LMNA and TMPO as they are two DCM-causing genes that encode proteins involved in maintaining nuclear envelope architecture. Among the 25 heart samples investigated, we identified major cardiomyocyte nuclear abnormalities in 8 patients. Direct sequencing allowed the detection of three heterozygous LMNA mutations (p.D192G, p.Q353K and p.R541S) in three patients. By multiplex ligation-dependant probe amplification (MLPA)/quantitative real-time PCR, we found a heterozygous deletion encompassing exons 3–12 of the LMNA gene in one patient. Immunostaining demonstrated that this deletion led to a decrease in lamin A/C expression in cardiomyocytes from this patient. This LMNA deletion as well as the p.D192G mutation was found in patients displaying major cardiomyocyte nuclear envelope abnormalities, while the p.Q353K and p.R541S mutations were found in patients without specific nuclear envelope abnormalities. None of the DCM patients included in the study carried a mutation in the TMPO gene. Taken together, we found no evidence of a genotype–phenotype relationship between the onset and the severity of DCM, the presence of nuclear abnormalities and the presence or absence of LMNA mutations. We demonstrated that a large deletion in LMNA associated with reduced levels of the protein in the nuclear envelope suggesting a haploinsufficiency mechanism can lead to cardiomyocyte nuclear envelope disruption and thus underlie the pathogenesis of DCM.

Calpain III mutation analysis of a heterogeneous limb–girdle muscular dystrophy population

Objective: To determine the frequency of calpain III mutations in a heterogeneous limb–girdle muscular dystrophy (LGMD) population. Background: Mutations of the calpain III gene have been shown to cause a subset of autosomal recessive LGMDs. Patient populations studied to date have been primarily of French and Spanish origin, in which calpain III may cause 30% of autosomal recessive MDs. The incidence of calpain III mutations in non–French/Spanish MD patients has not been studied thoroughly. No sensitive and specific biopsy screening methods for detecting patients with abnormal calpain III protein are available. Thus, detection of patients relies on direct detection of gene mutations. Methods: The authors studied the calpain III gene in 107 MD patient muscle biopsies exhibiting normal dystrophin. Muscle biopsy RNA was produced for each patient, and the entire calpain III complementary DNA was screened for mutations by reverse-transcriptase PCR/single-strand conformation polymorphism using three different conditions. Results: The authors identified nine patients (eight unrelated) with causative mutations. Six of the seven distinct mutations identified are novel mutations and have not been described previously. Conclusion: The results suggest that approximately 9.2% of patients in the heterogeneous population with an LGMD diagnosis will show mutations of the calpain III gene. Interestingly, two patients were heterozygous for a single mutation at the DNA level, whereas only the mutant allele was observed at the RNA level. This suggests that there are undetectable, nondeletion mutations that ablate expression of the calpain III gene.

TPM3 mutation in one of the original cases of cap disease.

Cap disease is a rare congenital skeletal muscle disorder characterized by the presence of cap-like structures which are well demarcated and peripherally located under the sarcolemma and show abnormal accumulation of sarcomeric proteins.1,2 Clinical features are early onset of hypotonia and nonprogressive or slowly progressive muscle weakness. Respiratory problems are common. Five dominant mutations have been reported in association with cap disease, all in the β-tropomyosin (βTm) gene ( TPM2 ).3-5 We report a mutation in the αTmslow (γTm) gene ( TPM3 ) in a patient with cap disease, supporting the concept that cap disease is genetically heterogeneous and closely related to nemaline myopathy.4 ### Case report. This 38-year-old woman was first described in 2002 (patient K.D.).2 There was no family history of neuromuscular disorder. She was born at term after an uncomplicated pregnancy, labor, and delivery. She had somewhat delayed motor milestones and at age 5 years mild motor difficulties were noted. She could not keep up with her peers and could not run. At age 18 years, a muscle biopsy was performed and the diagnosis of congenital myopathy (cap disease) was established (figure). Nemaline rods were not identified. EMG showed a myopathic pattern. She was admitted to hospital with pneumonia when she was 21 years old …

Coexistence of X-linked recessive Emery-Dreifuss muscular dystrophy with inclusion body myositis-like morphology

We reported three cases (two familial and one sporadic) of X-linked Emery-Dreifuss muscular dystrophy (EDMD), genetically documented. Two patients demonstrated a typical inclusion body myositis (IBM)-like morphology. The third patient had only minor changes. Patients had elbow and ankle contractures, progressive wasting of humeroperoneal muscles and cardiac failure (pacemaker implantation in all). There was a mutation within the Xq28 gene and complete absence of emerin in the nuclear membrane. Mononuclear cell infiltrations, rimmed vacuoles, amyloid deposits, as well as cytoplasmic and nuclear tubulofilamentous muscle inclusions were most unusual findings. Coexistence of IBM-like morphology and X-linked recessive EDMD might indicate that pathological features of IBM are nonspecific and may be present in other neuromuscular disorders.

Cap disease--a failure in the correct muscle fibre formation.

Four unrelated patients are reported with muscle hypotonia, weakness, skeletal dysmorphism and respiratory insufficiency since childhood. Muscle tissues were found to contain a number of muscle fibres with abnormal structure. Peripherally located structures such as a cap lacking in ATP-ase and fast myosin activity, rich in desmin, tropomyosin and alpha-actinin consisted of abnormally arranged myofibrils. The position of the peripherally situated myofibrils, as well as their abnormal sarcomere pattern, seems to point to an error in fusion and muscle protein synthesis. Whether our cases of congenital myopathy with cap structures are of hereditary origin or of a sporadic type remains unknown, so far. It seems that the result of our study, as well as data presented in the literature, allows us to divede cap disease into two forms: fatal and nonfatal. The morphological changes in the muscle fibres are identical in all the presented cases but the number of muscle fibres with cap structures is much higher in the fatal form.