Clinical and cardiovascular magnetic resonance profile of cardiomyopathy patients from South Africa: Pilot of the IMHOTEP study.

BACKGROUND: Cardiomyopathy is an important cause of heart failure, however, there is notable lack of data on causes and manifestations of cardiomyopathy in Africa. AIMS: The African Cardiomyopathy and Myocarditis Registry Program (IMHOTEP) aims to address the knowledge gap on etiology, treatment, and outcomes of cardiomyopathy in sub-Saharan Africa. METHODS AND RESULTS: We conducted a single-center pilot study to delineate the clinical and cardiovascular magnetic resonance (CMR) phenotypes of cardiomyopathy in South African patients. Assessment of the first 99 adult incident cases [mean age 36.8 ± 12.5 years; females 53.5%] enrolled in IMHOTEP showed that dilated cardiomyopathy (n = 77) was commonest, followed by hypertrophic (n = 13), restrictive (n = 5) and arrhythmogenic (n = 4) cardiomyopathies. A broad range of etiologies were encountered with secondary causes identified in 42% of patients. Onset of symptoms in the peripartum period was observed in 47% of women, and peripartum cardiomyopathy was diagnosed in 32.1% of women recruited. In addition to electrocardiography and echocardiography, CMR was performed in 67 cases and contributed diagnostically in a third of cases. Acute inflammation was rarely observed [2%] on CMR, however, late gadolinium enhancement (LGE) was noted in 92% of cases. CONCLUSION: We report a diverse spectrum of causes of cardiomyopathy in the South African population, with secondary, potentially treatable, etiologies in a significant proportion of cases. CMR was useful in delineating specific phenotypes and etiologies, influencing clinical care. A higher-than-expected burden of LGE was observed in this young patient cohort - the implications of which are yet to be determined.

Heterozygous CAPN3 missense variants causing autosomal-dominant calpainopathy in seven unrelated families.

AIMS: Recessive variants in CAPN3 gene are the cause of the commonest form of autosomal recessive limb girdle muscle dystrophy. However, two distinct in-frame deletions in CAPN3 (NM_000070.3:c.643_663del21 and c.598_621del15) and more recently, Gly445Arg and Arg572Pro substitutions have been linked to autosomal dominant (AD) forms of calpainopathy. We report 21 affected individuals from seven unrelated families presenting with an autosomal dominant form of muscular dystrophy associated with five different heterozygous missense variants in CAPN. METHODS: We have used massively parallel gene sequencing (MPS) to determine the genetic basis of a dominant form of limb girdle muscular dystrophy in affected individuals from seven unrelated families. RESULTS: The c.700G> A, [p.(Gly234Arg)], c.1327T> C [p.(Ser443Pro], c.1333G> A [p.(Gly445Arg)], c.1661A> C [p.(Tyr554Ser)] and c.1706T> C [p.(Phe569Ser)] CAPN3 variants were identified. Affected individuals presented in young adulthood with progressive proximal and axial weakness, waddling walking and scapular winging or with isolated hyperCKaemia. Muscle imaging showed fatty replacement of paraspinal muscles, variable degrees of involvement of the gluteal muscles, and the posterior compartment of the thigh and minor changes at the mid-leg level. Muscle biopsies revealed mild myopathic changes. Western blot analysis revealed a clear reduction in calpain 3 in skeletal muscle relative to controls. Protein modelling of these variants on the predicted structure of calpain 3 revealed that all variants are located in proximity to the calmodulin-binding site and are predicted to interfere with proteolytic activation. CONCLUSIONS: We expand the genotypic spectrum of CAPN3-associated muscular dystrophy due to autosomal dominant missense variants.

Altered myogenesis and premature senescence underlie human TRIM32-related myopathy.

TRIM32 is a E3 ubiquitin -ligase containing RING, B-box, coiled-coil and six C-terminal NHL domains. Mutations involving NHL and coiled-coil domains result in a pure myopathy (LGMD2H/STM) while the only described mutation in the B-box domain is associated with a multisystemic disorder without myopathy (Bardet-Biedl syndrome type11), suggesting that these domains are involved in distinct processes. Knock-out (T32KO) and knock-in mice carrying the c.1465G > A (p.D489N) involving the NHL domain (T32KI) show alterations in muscle regrowth after atrophy and satellite cells senescence. Here, we present phenotypical description and functional characterization of mutations in the RING, coiled-coil and NHL domains of TRIM32 causing a muscle dystrophy. Reduced levels of TRIM32 protein was observed in all patient muscle studied, regardless of the type of mutation (missense, single amino acid deletion, and frameshift) or the mutated domain. The affected patients presented with variable phenotypes but predominantly proximal weakness. Two patients had symptoms of both muscular dystrophy and Bardet-Biedl syndrome. The muscle magnetic resonance imaging (MRI) pattern is highly variable among patients and families. Primary myoblast culture from these patients demonstrated common findings consistent with reduced proliferation and differentiation, diminished satellite cell pool, accelerated senescence of muscle, and signs of autophagy activation.

A novel MYH7 founder mutation causing Laing distal myopathy in Southern Spain.

MYH7 gene mutations are associated with wide clinical and genetic heterogeneity. We report a novel founder mutation in MYH7 in Southern Spain (Andalucía). We studied two index patients and 24 family members from two apparently independent families by physical examination, serum creatine-kinase, muscle MRI, sequencing studies and genetic linkage analysis. Sixteen individuals were heterozygous for a (p.R1560P) variant in the MYH7 gene. Haplotype was consistent with a common ancestor for the two families. The patients displayed the classic Laing distal myopathy phenotype, with hanging first toe as the initial presentation, even in mildly affected patients who declared themselves asymptomatic, although neck flexor weakness was revealed as an early sign in some cases. MRI showed that the sartorius was the first muscle involved, even in two out of three asymptomatic carriers. Our findings support the novel variant p.R1560P in MYH7 as a founder mutation in Andalucía. The early involvement of the sartorius muscle in MRI may be useful as an indicator of affection status.

Variants in ACTG2 underlie a substantial number of Australasian patients with primary chronic intestinal pseudo-obstruction.

BACKGROUND: Primary chronic intestinal pseudo-obstruction (CIPO) is a rare, potentially life-threatening disorder characterized by severely impaired gastrointestinal motility. The objective of this study was to examine the contribution of ACTG2, LMOD1, MYH11, and MYLK mutations in an Australasian cohort of patients with a diagnosis of primary CIPO associated with visceral myopathy. METHODS: Pediatric and adult patients with primary CIPO and suspected visceral myopathy were recruited from across Australia and New Zealand. Sanger sequencing of the genes encoding enteric gamma-actin (ACTG2) and smooth muscle leiomodin (LMOD1) was performed on DNA from patients, and their relatives, where available. MYH11 and MYLK were screened by next-generation sequencing. KEY RESULTS: We identified heterozygous missense variants in ACTG2 in 7 of 17 families (~41%) diagnosed with CIPO and its associated conditions. We also identified a previously unpublished missense mutation (c.443C>T, p.Arg148Leu) in one family. One case presented with megacystis-microcolon-intestinal hypoperistalsis syndrome in utero with subsequent termination of pregnancy at 28 weeks' gestation. All of the substitutions identified occurred at arginine residues. No likely pathogenic variants in LMOD1, MYH11, or MYLK were identified within our cohort. CONCLUSIONS AND INFERENCES: ACTG2 mutations represent a significant underlying cause of primary CIPO with visceral myopathy and associated phenotypes in Australasian patients. Thus, ACTG2 sequencing should be considered in cases presenting with hypoperistalsis phenotypes with suspected visceral myopathy. It is likely that variants in other genes encoding enteric smooth muscle contractile proteins will contribute further to the genetic heterogeneity of hypoperistalsis phenotypes.

Ryanodine receptor type 3 (RYR3) as a novel gene associated with a myopathy with nemaline bodies.

BACKGROUND AND PURPOSE: Nemaline myopathy (NEM) has been associated with mutations in 12 genes to date. However, for some patients diagnosed with NEM, definitive mutations are not identified in the known genes, suggesting that there are other genes involved. This study describes compound heterozygosity for rare variants in ryanodine receptor type 3 (RYR3) gene in one such patient. METHODS AND RESULTS: Clinical examination of the patient at 22 years of age revealed a long narrow face, high arched palate and bilateral facial weakness. She had proximal weakness in all four limbs, mild scapular winging but no scoliosis. Muscle biopsy revealed wide variation in fibre size with type 1 fibre predominance and atrophy. Abundant nemaline bodies were located in perinuclear and subsarcolemmal areas, and within the cytoplasm. No likely pathogenic mutations in known NEM genes were identified. Copy number variation in known NEM genes was excluded by NEM-targeted comparative genomic hybridization array. Next-generation sequencing revealed compound heterozygous missense variants in the RYR3 gene. RYR3 transcripts are expressed in human fetal and adult skeletal muscle as well as in human brain and cauda equina samples. Immunofluorescence of human skeletal muscle revealed a 'single-row' appearance of RYR3, interspersed between the 'double rows' of ryanodine receptor type 1 (RYR1) at each A-I junction. CONCLUSION: The results suggest that variants in RYR3 may cause a recessive muscle disease with pathological features including nemaline bodies. We characterize the expression pattern of RYR3 in human skeletal muscle and brain, and the subcellular localization of RYR1 and RYR3 in human skeletal muscle.

Antimicrobial susceptibility of clinical isolates of Neisseria gonorrhoeae to alternative antimicrobials with therapeutic potential.

Background: The prevalence of MDR Neisseria gonorrhoeae is increasing globally and represents a public health emergency. Development and approval of new anti-gonococcal agents may take years. As a concurrent approach to developing new antimicrobials, the laboratory and clinical evaluation of currently licensed antimicrobials not widely used for the treatment of gonorrhoea may provide new options for the treatment of gonococcal infections. Objectives: To determine the in vitro activity of nine alternative, currently licensed and late-development antimicrobials with the potential to treat gonococcal infections against 112 clinical isolates of N. gonorrhoeae resistant to one or multiple antimicrobials. Methods: The MICs of conventional anti-gonococcal antimicrobials (penicillin, ceftriaxone, cefixime, azithromycin, ciprofloxacin, tetracycline and spectinomycin) and alternative antimicrobials (ertapenem, gentamicin, netilmicin, tigecycline, eravacycline, fosfomycin, linezolid, ceftazidime/avibactam and ceftaroline) were determined by agar dilution. Results: Ertapenem and the novel cephalosporins demonstrated similar MIC values to the third-generation cephalosporins, but increased MICs were observed for isolates with increased cefixime and ceftriaxone MICs. Tigecycline and eravacycline had MIC values below expected serum concentrations for all isolates tested. The aminoglycosides gentamicin and netilmicin were generally more potent than spectinomycin, with netilmicin demonstrating the greatest potency. Fosfomycin MICs were elevated compared with other agents, but remained within the MIC range for susceptible organisms, while linezolid MICs were generally higher than those for organisms considered resistant. Conclusions: Among potentially therapeutically useful alternative agents, the aminoglycosides, eravacycline, tigecycline and fosfomycin had good in vitro activity. The novel cephalosporins and ertapenem had comparable activity to cefixime and ceftriaxone.

Mitochondrial oligomers boost glycolysis in cancer stem cells to facilitate blebbishield-mediated transformation after apoptosis.

Apoptosis culminates in secondary necrosis due to lack of ATP. Cancer stem cells form spheres after apoptosis by evoking the blebbishield emergency program. Hence, determining how blebbishields avoid secondary necrosis is crucial. Here we demonstrate that N-Myc and VEGFR2 control transformation from blebbishields, during which oligomers of K-Ras, p27, BAD, Bax, and Bak boost glycolysis to avoid secondary necrosis. Non-apoptotic cancer cells also utilize oligomers to boost glycolysis, which differentiates the glycolytic function of oligomers from their apoptotic action. Smac mimetic in combination with TNF-α or TRAIL but not in combination with FasL abrogates transformation from blebbishields by inducing secondary necrosis. Thus blebbishield-mediated transformation is dependent on glycolysis, and Smac mimetics represent potential candidates to abrogate the blebbishield emergency program.

TPM3 deletions cause a hypercontractile congenital muscle stiffness phenotype.

OBJECTIVE: Mutations in TPM3, encoding Tpm3.12, cause a clinically and histopathologically diverse group of myopathies characterized by muscle weakness. We report two patients with novel de novo Tpm3.12 single glutamic acid deletions at positions ΔE218 and ΔE224, resulting in a significant hypercontractile phenotype with congenital muscle stiffness, rather than weakness, and respiratory failure in one patient. METHODS: The effect of the Tpm3.12 deletions on the contractile properties in dissected patient myofibers was measured. We used quantitative in vitro motility assay to measure Ca(2+) sensitivity of thin filaments reconstituted with recombinant Tpm3.12 ΔE218 and ΔE224. RESULTS: Contractility studies on permeabilized myofibers demonstrated reduced maximal active tension from both patients with increased Ca(2+) sensitivity and altered cross-bridge cycling kinetics in ΔE224 fibers. In vitro motility studies showed a two-fold increase in Ca(2+) sensitivity of the fraction of filaments motile and the filament sliding velocity concentrations for both mutations. INTERPRETATION: These data indicate that Tpm3.12 deletions ΔE218 and ΔE224 result in increased Ca(2+) sensitivity of the troponin-tropomyosin complex, resulting in abnormally active interaction of the actin and myosin complex. Both mutations are located in the charged motifs of the actin-binding residues of tropomyosin 3, thus disrupting the electrostatic interactions that facilitate accurate tropomyosin binding with actin necessary to prevent the on-state. The mutations destabilize the off-state and result in excessively sensitized excitation-contraction coupling of the contractile apparatus. This work expands the phenotypic spectrum of TPM3-related disease and provides insights into the pathophysiological mechanisms of the actin-tropomyosin complex.

Adult onset distal and proximal myopathy with complete ophthalmoplegia associated with a novel de novo p.(Leu1877Pro) mutation in MYH2.

An MYH2 mutation p.(Glu706Lys) was originally described in a family with autosomal dominant inheritance, where the affected family members presented with multiple congenital contractures and ophthalmoplegia, progressing to a proximal myopathy in adulthood. Another patient with a dominant mutation p.(Leu1870Pro) was described, presenting as a congenital myopathy with ophthalmoplegia. Here, we present a patient with symptoms beginning at age 16 years, of prominent distal but also proximal weakness, bulbar involvement and ophthalmoplegia. Initially, clinically classified as oculopharyngodistal myopathy, the patient was found to carry a novel, de novo MYH2 mutation c.5630T>C p.(Leu1877Pro). This expands the phenotype of dominant MYH2 myopathies with the clinical phenotype overlapping the oculopharyngodistal myopathy spectrum.

Production impact of a targeted selective treatment system based on liveweight gain in a commercial flock.

The sustainability of sheep production is hindered by anthelmintic resistance. Options to slow down or prevent resistance have been widely studied but their application in the field is still limited. In this study, the practical application and effect of a targeted selective treatment (TST) approach for the treatment of parasitic gastroenteritis was investigated in lambs (n = 385) over a 2 year period. At 14-day intervals during the grazing season, liveweight, breech soiling and anthelmintic treatments were individually recorded. Selection of lambs for anthelmintic treatment in the TST group was based on pre-calculated individual growth rates, with a matched cohort routinely treated (RT) with anthelmintic drug every 6 weeks. The adoption of a TST approach had no negative effect on the liveweight gains of the lambs, time to finishing or breech soiling measures compared to RT lambs; however a 50% decrease in anthelmintic treatment was observed in the TST group. The time to implement this system averaged 2 min per lamb. It is concluded that the TST could be suitable for commercial sheep farms, in association with automated weighing systems, potentially reducing selection for anthelmintic resistance, while having no negative effect on production.

Mutations in MYH7 cause Multi-minicore Disease (MmD) with variable cardiac involvement.

Central Core Disease (CCD) and Multi-minicore Disease (MmD) (the "core myopathies") have been mainly associated with mutations in the skeletal muscle ryanodine receptor (RYR1) and the selenoprotein N (SEPN1) gene. A proportion of cases remain unresolved. Mutations in MYH7 encoding the beta myosin heavy chain protein have been implicated in cardiac and, less frequently, skeletal muscle disorders. Here we report four patients from two families with a histopathological diagnosis of MmD, presenting in childhood with slowly progressive muscle weakness, more proximal in Family 1 and more distal in Family 2, and variable degrees of cardiorespiratory impairment evolving later in life. There was also a strong family history of sudden death in the first family. Muscle biopsies obtained in early childhood showed multiple minicores as the most prominent feature. Sequencing of the MYH7 gene revealed heterozygous missense mutations, c.4399C>G; p.Leu1467Val (exon 32) in Family 1 and c.4763G>C; p.Arg1588Pro (exon 34) in Family 2. These findings suggest MYH7 mutations as another cause of a myopathy with multiple cores, in particular if associated with dominant inheritance and cardiac involvement. However, clinical features previously associated with this genetic background, namely a more distal distribution of weakness and an associated cardiomyopathy, may only evolve over time.

Congenital fibre type disproportion associated with mutations in the tropomyosin 3 (TPM3) gene mimicking congenital myasthenia.

Congenital myopathy with fibre type disproportion (CFTD) has been associated with mutations in ACTA1, SEPN1, RYR1 and TPM3 genes. We report the clinico-pathological and electrophysiological features of 2 unrelated cases with heterozygous TPM3 mutation. Case 1 is a 19-year-old lady who presented with motor delay in infancy, respiratory failure in early teens requiring non-invasive ventilation despite being ambulant, ptosis, axial more than proximal weakness and scoliosis. Case 2 is a 7-year-old boy with hypotonia, feeding difficulties, motor delay and scoliosis, also requiring non-invasive ventilation while ambulant. Muscle biopsies in both cases showed fibre type disproportion. Muscle MRI (Case 1) showed mild uniformly increased interstitial tissue in and around the muscles. Sequencing of TPM3 in case 1 revealed a previously described heterozygous c.503G > A(pArg168His) missense variant in exon 5 and a novel heterozygous missense mutation c.521A > C(pGlu174Ala), also in exon 5, in case 2. A mild abnormality in the single fibre EMG was documented on electrophysiology in both cases. These cases highlight the neuromuscular transmission defect in CFTD secondary to TPM3 mutations.

[Myosin storage myopathy: a rare subtype of protein aggregate myopathies]. / Myosinspeichermyopathie: eine seltene Unterform der Proteinaggregationsmyopathien.

Myopathies with pathological protein aggregates comprise a numerically significant group of sporadic and hereditary muscle disorders. A rare disease entity within the group of protein aggregate myopathies is the myosin storage myopathy, which is caused by heterozygous mutations in the MYH7 gene which encodes the slow/beta-myosin heavy chain. We report the clinical, myopathological and MRI findings in the first German patient suffering from a myosin storage myopathy due to a heterozygous R 1845W missense mutation.

Adenovirus and adeno-associated virus-mediated delivery of human myophosphorylase cDNA and LacZ cDNA to muscle in the ovine model of McArdle's disease: expression and re-expression of glycogen phosphorylase.

At present there is no satisfactory treatment for McArdle's disease, deficiency of myophosphorylase. Injection of modified adenovirus 5 (AdV5) and adeno-associated virus 2 (AAV2) vectors containing myophosphorylase expression cassettes, into semitendinosus muscle of sheep with McArdle's disease, produced expression of functional myophosphorylase and some re-expression of the non-muscle glycogen phosphorylase isoforms (both liver and brain) in regenerating fibres. Expression of both non-muscle isoforms was also seen after control injections of AdV5LacZ vectors. There was up to an order of magnitude greater expression of phosphorylase after myophosphorylase vector injection than after LacZ controls (62% of sections with over 1000 positive muscle fibres, versus 7%). The results presented here suggest that the use of viral vector-mediated phosphorylase gene transfer may be applicable to the treatment of McArdle's disease and that sustained re-expression of the brain and liver isoforms should also be investigated as a possible treatment.

Macrolide resistance mechanisms among Streptococcus pneumoniae isolated over 6 years of Canadian Respiratory Organism Susceptibility Study (CROSS) (1998 2004).

BACKGROUND: Resistance to macrolides in Streptococcus pneumoniae arises primarily due to Erm(B) or Mef(A). Erm(B) typically confers high-level resistance to macrolides, lincosamides and streptogramin B (MLS(B) phenotype), whereas Mef(A) confers low-level resistance to macrolides only (M phenotype). The purpose of this study was to investigate the incidence of macrolide resistance mechanisms in Canadian isolates of S. pneumoniae obtained between 1998 and 2004. Furthermore, the genetic relatedness, serotype distribution and antibiotic susceptibility profile among S. pneumoniae isolates with dual erythromycin ribosomal methylase [Erm(B)] and efflux pump [Mef(A)] were analysed. METHODS: A total of 865 macrolide-resistant (erythromycin MIC > or = 1 mg/L) S. pneumoniae isolates were collected from the Canadian Respiratory Organism Susceptibility Study (CROSS) from 1998 to 2004. The presence of erm(B) and mef(A) was determined for each isolate by PCR; mutations in the genes coding for L4 and L22 ribosomal proteins and for 23S rRNA were identified by DNA sequencing. Each isolate containing both erm(B)- and mef(A)-mediated macrolide resistance was genotyped by PFGE and serotyped using the Quellung reaction with antisera. RESULTS: Of the 865 isolates studied, 404 (46.7%) were mef(A)-positive, 371 (42.9%) were erm(B)-positive, 50 (5.8%) were positive for both mef(A) and erm(B) and 40 (4.6%) were negative for both mef(A) and erm(B). Of the macrolide-resistant isolates negative for both mef(A) and erm(B), 22 (2.5%) contained 23S rRNA A2058G, A2059G or A2059C mutations, 7 (0.8%) contained 23S rRNA A2058G or A2059G mutations along with an S20N mutation in L4 ribosomal protein, and 1 isolate contained an E30K ribosomal protein mutation alone. Of the macrolide-resistant strains positive for both mef(A) and erm(B), 36 (72%) were multidrug-resistant (macrolide-, penicillin- and trimethoprim/sulfamethoxazole-resistant), 39 (78%) isolates belonged to serotype 19A or 19F and 36 (72%) belonged to one clonal complex (> or =80% genetic relatedness) genetically related to the Taiwan 19F-14 clone. CONCLUSIONS: The prevalence of efflux-based macrolide resistance in S. pneumoniae in Canada remained steady between 1998 and 2004. Macrolide resistance due to erm(B) decreased over the same time period, with a rapid increase in isolates with both erm(B) and mef(A) macrolide resistance.

Nemaline myopathy with exclusively intranuclear rods and a novel mutation in ACTA1 (Q139H).

Nemaline myopathies (NM) are a rare group of muscle disorders, but represent one of the most common forms of congenital myopathy. The clinical picture ranges from severe muscular hypotonia often leading to death during childhood to mild forms with long life expectancy. Diagnosis is made by muscle biopsy showing characteristic sarcoplasmic and sometimes intranuclear rod bodies. So far, disease-associated mutations have been detected in six genes without any simple correlation between genotype and phenotype or histological findings. We report a patient with a phenotype typical of congenital onset nemaline myopathy and exclusively intranuclear rods. Mutation analysis revealed a new heterozygous missense mutation in exon 3 of the ACTA1 gene (Q139H). Molecular modelling predicts that substitution of Q139 for H139 alters the amino acid side chains and hydrogen bonding which may alter the nucleotide binding cleft by adding 'bulk' to the mutated molecule. Two-dimensional gel electrophoresis demonstrates that mutant actin Q139H is expressed at approximately half the level of wild-type actin in the patient's muscle. We speculate that these alterations, although not directly affecting the nuclear export signal, negatively interfere with the nuclear export of the mutated protein and thereby cause retention of mutant actin and intranuclear rod formation.

'Cap myopathy': case report of a family.

We report the observation of an 18-year-old girl, whose clinical presentation was very suggestive of a congenital myopathy with neonatal onset. A congenital myopathy had been already diagnosed in her brother and in addition her half-cousin died diagnosed with a severe nemaline myopathy at age 4 years. A muscle biopsy performed on both siblings revealed histological and ultrastructural features of 'cap myopathy'. This case report suggests that 'cap myopathy' and some cases of nemaline myopathy with neonatal onset might be two phenotypic expressions of the same genetic disorder. These two entities could therefore, perhaps, be regarded as 'Z-line disorders' possibly caused by defective myofibrillogenesis.