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.