Cardiac arrhythmia and late-onset muscle weakness caused by a myofibrillar myopathy with unusual histopathological features due to a novel missense mutation in FLNC.

Myofibrillar myopathies (MFM) are mostly adult-onset diseases characterized by progressive morphological alterations of the muscle fibers beginning in the Z-disk and the presence of protein aggregates in the sarcoplasm. They are mostly caused by mutations in different genes that encode Z-disk proteins, including DES, CRYAB, LDB3, MYOT, FLNC and BAG3. A large family of French origin, presenting an autosomal dominant pattern, characterized by cardiac arrhythmia associated to late-onset muscle weakness, was evaluated to clarify clinical, morphological and genetic diagnosis. Muscle weakness began during adult life (over 30 years of age), and had a proximal distribution. Histology showed clear signs of a myofibrillar myopathy, but with unusual, large inclusions. Subsequently, genetic testing was performed in MFM genes available for screening at the time of clinical/histological diagnosis, and desmin (DES), αB-crystallin (CRYAB), myotilin (MYOT) and ZASP (LDB3), were excluded. LMNA gene screening found the p.R296C variant which did not co-segregate with the disease. Genome wide scan revealed linkage to 7q.32, containing the FLNC gene. FLNC direct sequencing revealed a heterozygous c.3646T>A p.Tyr1216Asn change, co-segregating with the disease, in a highly conserved amino acid of the protein. Normal filamin C levels were detected by Western-blot analysis in patient muscle biopsies and expression of the mutant protein in NIH3T3 showed filamin C aggregates. This is an original FLNC mutation in a MFM family with an atypical clinical and histopathological presentation, given the presence of significantly focal lesions and prominent sarcoplasmic masses in muscle biopsies and the constant heart involvement preceding significantly the onset of the myopathy. Though a rare etiology, FLNC gene should not be excluded in early-onset arrhythmia, even in the absence of myopathy, which occurs later in the disease course.

Four Caucasian patients with mutations in the fukutin gene and variable clinical phenotype.

Fukuyama congenital muscular dystrophy (FCMD) is frequent in Japan, due to a founder mutation of the fukutin gene (FKTN). Outside Japan, FKTN mutations have only been reported in a few patients with a wide spectrum of phenotypes from Walker-Warburg syndrome to limb-girdle muscular dystrophy (LGMD2M). We studied four new Caucasian patients from three unrelated families. All showed raised serum CK initially isolated in one case and muscular dystrophy. Immunohistochemical studies and haplotype analysis led us to search for mutations in FKTN. Two patients (two sisters) presented with congenital muscular dystrophy, mental retardation, and posterior fossa malformation including cysts, and brain atrophy at Brain MRI. The other two patients had normal intelligence and brain MRI. Sequencing of the FKTN gene identified three previously described mutations and two novel missense mutations. Outside Japan, fukutinopathies are associated with a large spectrum of phenotypes from isolated hyperCKaemia to severe CMD, showing a clear overlap with that of FKRP.

New morphologic and genetic findings in cap disease associated with beta-tropomyosin (TPM2) mutations.

OBJECTIVE: Mutations in the beta-tropomyosin gene (TPM2) are a rare cause of congenital myopathies with features of nemaline myopathy and cap disease and may also cause distal arthrogryposis syndromes without major muscle pathology. We describe the muscle biopsy findings in three patients with cap disease and novel heterozygous mutations in TPM2. METHODS: Three unrelated patients with congenital myopathy were investigated by muscle biopsy and genetic analysis. RESULTS: All three patients had early-onset muscle weakness of variable severity and distribution. Muscle biopsy demonstrated in all three patients near uniformity of type 1 fibers and an unusual irregular and coarse-meshed intermyofibrillar network. By electron microscopy, the myofibrils were broad and partly split, and the Z lines appeared jagged. In one of the patients caps structures were identified only by electron microscopy, and in one patient they were identified only in a second biopsy at adulthood. Three novel, de novo, heterozygous mutations in TPM2 were identified: a three-base pair deletion in-frame (p.Lys49del), a three-base pair duplication in-frame (p.Gly52dup), and a missense mutation (p.Asn202Lys). CONCLUSIONS: Mutations in TPM2 seem to be a frequent cause of cap disease. Because cap structures may be sparse, other prominent features, such as a coarse-meshed intermyofibrillar network and jagged Z lines, may be clues to correct diagnosis and also indicate that the pathogenesis involves defective assembly of myofilaments.

Prothrombin Scranton: substitution of an amino acid residue involved in the binding of Na+ (LYS-556 to THR) leads to dysprothrombinemia.

Several members of a family from Scranton, Pennsylvania were identified to have normal levels of prothrombin antigen while their prothrombin clotting activity was approximately 50% of normal. There has been no previous history of bleeding or other clinical manifestations in this family. The genomic DNA from the proband was amplified for all exons in the prothrombin gene and analyzed by single strand conformation polymorphism (SSCP)/heteroduplex analysis followed by DNA sequence analysis and restriction enzyme digestion. A mutation at nucleotide 20040 in exon 14 was identified and confirmed by restriction enzyme digestion. This mutation results in the substitution of Thr for Lys at amino acid 556. Amino acid 556 has been reported as one of the key residues for the binding of Na+ in the thrombin portion of the protein.

Lack of predictable site-dependent differences and time-dependent changes in postmortem concentrations of cocaine, benzoylecgonine, and cocaethylene in humans.

This study evaluated the stability of cocaine, benzoylecgonine, and cocaethylene in postmortem fluids in cases of cocaine-related death. Femoral and ventricular blood and cisternal cerebrospinal fluid were collected soon after death and again at the time of autopsy. In addition, iliac blood was collected at autopsy. There were no consistent patterns of site-specific differences for any of the analytes, and the central compartment showed both higher and lower concentrations than the peripheral. There was no consistent pattern of direction or magnitude of change in the concentrations with respect to time for any of the analytes. This is consistent with anecdotal reports from other workers and is believed to be a result of competing processes of tissue release and chemical and enzymatic degradation of the analytes. Postmortem cocaine and metabolite concentrations in blood are not necessarily reflective of the perimortem concentrations and should not be the primary consideration in determining the cause of death in suspected cocaine-related deaths.

Analysis of ecgonine and other cocaine biotransformation products in postmortem whole blood by protein precipitation-extractive alkylation and GC-MS.

Ecgonine is formed by the hydrolysis of both ester linkages in cocaine and is of interest as both a potential metabolite and a postmortem and postcollection artifact in biological specimens. Its extreme polarity and water solubility make its isolation very difficult, particularly from complex matrices such as postmortem whole blood. This procedure describes an initial protein precipitation followed by two derivatization steps, the first of which is to propylate organic acids and primary and secondary amines and the second of which is to form the p-nitrobenzoyl ester of organic alcohols. The resultant derivatized products are then subjected to cleanup using a standard extraction with n-butyl chloride as the solvent. Analysis of the extracts is performed by gas chromatography-mass spectrometry using deuterated internal standards, although the adducts would also be suitable for analysis by high-performance liquid chromatography. Significant quantities of ecgonine have been identified in postmortem whole blood samples using this method.

Postmortem distribution and redistribution of morphine in man.

This study evaluated both site dependent differences and time dependent changes in postmortem morphine concentrations in man. In 32 deaths involving morphine, left ventricular blood, femoral blood, and cisternal cerebrospinal fluid, were collected as soon after death as possible (T1), and collected again together with iliac blood at the time of autopsy (T2). Samples were analyzed for morphine by radioimmunoassay. No evidence was found for changes in morphine concentration with respect to time at either central or peripheral sites, or in the cerebrospinal fluid. Ventricular blood morphine concentrations were however consistently higher than those in the peripheral compartment, represented by either femoral or iliac blood. This was particularly true when the ventricular morphine concentration exceeded 0.300 mg/L. At peripheral sites, femoral and iliac blood morphine concentrations were well correlated with each other, making either an appropriate site for collection of peripheral blood for toxicological testing.