PubMed Health⌕ Search

Biomedical subjects

Caroline Sewry

Publications and source records attributed to Caroline Sewry.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of recessive RYR1 mutations in core myopathies.

We have characterized at the molecular level, three families with core myopathies carrying apparent recessive mutations in their RYR1 gene and studied the pharmacological properties of myotubes carrying endogenous mutations as well as the properties of mutant channels expressed in HEK293 cells. The proband of family 1 carried p.Ala1577Thr+p.Gly2060Cys in trans, having inherited a mutation from each parent. Immunoblot analysis of proteins from the patient's skeletal muscle revealed low levels of ryanodine receptor (RyR1) but neither substitution alone or in combination affected the functional properties of RyR1 channels in a discernable way. Two affected siblings in family 2 carried p.Arg109Trp+p.Met485Val substitutions in cis, inherited from the unaffected father. Interestingly, both affected siblings only transcribed the mutated paternal allele in skeletal muscle, whereas the maternal allele was silent. Single-channel measurements showed that recombinant, mutant RyR1 channels carrying both substitutions lost the ability to conduct Ca2+. In this case as well, low levels of RyR1 were present in skeletal muscle extracts. The proband of family 3 carried p.Ser71Tyr+p.Asn2283His substitutions in trans. Recombinant channels with Asn2283His substitution showed an increased activity, whereas recombinant channels with p.Ser71Tyr+p.Asn2283His substitution lost activity upon isolation. Taken together, our data suggest major differences in the ways RYR1 mutations may affect patients with core myopathies, by compromising RyR1 protein expression, stability and/or activity.

Amino Acid Substitution↗
PubMed2006

Flies deficient in Muscleblind protein model features of myotonic dystrophy with altered splice forms of Z-band associated transcripts.

Myotonic dystrophy (DM) is a dominantly inherited neuromuscular disorder characterised by muscle weakness and wasting. There are two forms of DM; both of which are caused by the expansion of repeated DNA sequences. DM1 is associated with a CTG repeat located in the 3' untranslated region of a gene, DMPK and DM2 with a tetranucleotide repeat expansion, CCTG, located in the first intron of a different gene, ZNF9. Recent data suggest a dominant RNA gain-of-function mechanism underlying DM, as transcripts containing either CUG or CCUG repeat expansions accumulate as foci in the nuclei of DM1 and DM2 cells respectively, where they exert a toxic effect, sequestering specific RNA binding proteins such as Muscleblind, which leads to splicing defects and the disruption of normal cellular functions. Z-band disruption is a well-known histological feature of DM1 muscle, which has also been reported in Muscleblind deficient flies. In order to determine whether there is a common molecular basis for this abnormality we have examined the alternative splicing pattern of transcripts that encode proteins associated with the Z-band in both organisms. Our results demonstrate that the missplicing of ZASP/LDB3 leads to the expression of an isoform in DM1 patient muscle, which is not present in normal controls, nor in other myopathies. Furthermore the Drosophila homologue, CG30084, is also misspliced, in Muscleblind deficient flies. Another Z-band transcript, alpha actinin, is misspliced in mbl mutant flies, but not in DM1 patient samples. These results point to similarities but subtle differences in the molecular breakdown of Z-band structures in flies and DM patients and emphasise the relevance of Muscleblind proteins in DM pathophysiology.

Actinin↗
PubMed2006

Identification of 45 novel mutations in the nebulin gene associated with autosomal recessive nemaline myopathy.

Nemaline myopathy (NM) is a clinically and genetically heterogeneous disorder of skeletal muscle caused by mutations in at least five different genes encoding thin filament proteins of the striated muscle sarcomere. We have previously described 18 different mutations in the last 42 exons of the nebulin gene (NEB) in 18 families with NM. Here we report 45 novel NEB mutations detected by denaturing high-performance liquid chromatography (dHPLC) and sequence analysis of all 183 NEB exons in NM patients from 44 families. Altogether we have identified, including the deletion of exon 55 identified in the Ashkenazi Jewish population, 64 different mutations in NEB segregating with autosomal recessive NM in 55 families. The majority (55%) of the mutations in NEB are frameshift or nonsense mutations predicted to cause premature truncation of nebulin. Point mutations (25%) or deletions (3%) affecting conserved splice signals are predicted in the majority of cases to cause in-frame exon skipping, possibly leading to impaired nebulin-tropomyosin interaction along the thin filament. Patients in 18 families had one of nine missense mutations (14%) affecting conserved amino acids at or in the vicinity of actin or tropomyosin binding sites. In addition, we found the exon 55 deletion in four families. The majority of the patients (in 49/55 families) were shown to be compound heterozygous for two different mutations. The mutations were found in both constitutively and alternatively expressed exons throughout the NEB gene, and there were no obvious mutational hotspots. Patients with more severe clinical pictures tended to have mutations predicted to be more disruptive than patients with milder forms.

Chromatography, High Pressure Liquid↗
PubMed2005

Defining alpha-skeletal and alpha-cardiac actin expression in human heart and skeletal muscle explains the absence of cardiac involvement in ACTA1 nemaline myopathy.

Mutations in alpha-skeletal actin (ACTA1) underlie several congenital muscle disorders including nemaline myopathy (NM). Almost all ACTA1-NM patients have normal cardiac function, and, even lethally affected congenital NM patients exhibit an unremarkable gestation with decreased foetal movement just prior to birth. Although alpha-skeletal actin is thought to be the predominant sarcomeric actin in human heart (Boheler KR, Carrier L, de la Bastie D, et al. Skeletal actin mRNA increases in the human heart during ontogenic development and is the major isoform of control and failing adult hearts. J Clin Invest 1991;88:323-30 ), ACTA1-NM patients almost never exhibit a cardiac phenotype. In this study, we define the relative expression of skeletal and cardiac actin proteins in human heart and skeletal muscle. We show that alpha-cardiac actin is the predominant sarcomeric isoform in human donor hearts and in early foetal skeletal muscle development. Skeletal actin is the predominant isoform from 25 to 27 weeks gestation and is the exclusive isoform expressed in muscle from infancy through to adulthood. These findings are consistent with clinical observations of NM patients and assist us to better understand the pathogenesis of inherited myopathies and cardiomyopathies with mutations in actin.

Actins↗
PubMed2005

Prenatal diagnosis in laminin alpha2 chain (merosin)-deficient congenital muscular dystrophy: a collective experience of five international centers.

The congenital muscular dystrophies (CMD) are clinically and genetically heterogeneous. The merosin (laminin alpha2 chain) deficient form (MDC1A), is characterized clinically by neonatal hypotonia, delayed motor milestones and associated contractures. It is caused by deficiency in the basal lamina of muscle fibers of the alpha2 chain of laminins 2 and 4 (LAMA2 gene at 6q22-23). Laminin alpha2 chain is also expressed in fetal trophoblast, which provides a suitable tissue for prenatal diagnosis in families where the index case has total deficiency of the protein. This article reports the collective experience of five centers over the past 10 years in 114 prenatal diagnostic studies using either protein analysis of the chorionic villus (CV) of the trophoblast plus DNA molecular studies with markers flanking the 6q22-23 region and intragenic polymorphisms (n=58), or using only DNA (n=44) or only protein (n=12) approaches. Of the 102 fetuses studied by molecular genetics, 27 (26%) were predicted to be affected while 75 (74%) were considered as unaffected, with 52 (51%) being heterozygous, thus conforming closely to an autosomal recessive inheritance. In 18 of the 27 affected fetuses, the trophoblast was studied by immunocytochemistry and there was a total or only traces deficiency of the protein in CV basement membrane in all. In 10 cases material from the presumably affected fetus was available for analysis after termination of the pregnancy and immunohistochemical study confirmed the diagnosis in all of them. Prenatal studies of 'at risk' pregnancies in the five centers produced neither false negative (merosin-deficiency in CVs in a normal fetus), nor false positive (normal merosin expression in CVs and affected child), indicating the reliability of the technique, when all the necessary controls are done. Our experience suggests that protein and DNA analysis can be used either independently or combined, according to the facilities of each center, to provide accurate prenatal diagnosis of the MDC1A, and have an essential role in genetic counseling.

Amniotic Fluid↗
PubMed2004

Effect of ryanodine receptor mutations on interleukin-6 release and intracellular calcium homeostasis in human myotubes from malignant hyperthermia-susceptible individuals and patients affected by central core disease.

In this study we report for the first time the functional properties of human myotubes isolated from patients harboring the native RYR1 I4898T and R4893W mutations linked to central core disease. We examined two aspects of myotube physiology, namely excitation-contraction and excitation-secretion coupling. Our results show that upon activation of the ryanodine receptor (RYR), myotubes release interleukin-6 (IL-6); this was dependent on de novo protein synthesis and could be blocked by dantrolene and cyclosporine. Myotubes from the two patients affected by central core disease showed a 4-fold increase in the release of the inflammatory cytokine IL-6, compared with cells derived from control or malignant hyperthermia susceptible individuals. All tested myotubes released calcium from intracellular stores upon stimulation via surface membrane depolarization or direct RYR activation by 4-chloro-m-cresol. The functional impact on calcium release of RYR1 mutations linked to central core disease or malignant hyperthermia is different: human myotubes carrying the malignant hyperthermia-linked RYR1 mutation V2168M had a shift in their sensitivity to the RYR agonist 4-chloro-m-cresol to lower concentrations, whereas human myotubes harboring C-terminal mutations linked to central core disease exhibited reduced [Ca2+]i increase in response to 4-chloro-m-cresol, caffeine, and KCl. Taken together, these results suggest that abnormal release of calcium via mutated RYR enhances the production of the inflammatory cytokine IL-6, which may in turn affect signaling pathways responsible for the trophic status of muscle fibers.

Caffeine↗
PubMed2004

Extreme variability of phenotype in patients with an identical missense mutation in the lamin A/C gene: from congenital onset with severe phenotype to milder classic Emery-Dreifuss variant.

BACKGROUND: Mutations of the LMNA gene, encoding the nuclear envelope proteins lamins A and C, have been associated with 7 distinct pathologic conditions. OBJECTIVE: To report 5 cases with the same missense mutation in exon 6 of the LMNA gene, resulting in an E358K substitution in the central rod domain. DESIGN: Case report. SETTING: Three muscle centers in England. PATIENTS: Five patients with missense mutations of the LMNA gene. RESULTS: All 5 individuals had muscle involvement, but the onset, severity, distribution of muscle weakness, and presence of associated features were highly variable. Three patients had humeroperoneal distribution of weakness and typical features of Emery-Dreifuss muscular dystrophy. Two other patients showed additional novel features. One had congenital onset and predominant axial weakness, with poor neck control and inability to sit independently at the age of 21 months. Another patient presented in childhood with an unusual pattern of muscle weakness, short stature, and midface hypoplasia with striking fat accumulation around the face and neck, in contrast to wasting of adipose tissue and muscle in the limbs. She developed both respiratory failure and cardiac arrhythmias in her late 20s. CONCLUSION: Our cases expand the clinical spectrum associated with mutations in the LMNA gene and further illustrate the overlapping phenotypes of the laminopathies.

Adult↗
PubMed2004

Functional characterisation of a mutant actin (Met132Val) from a patient with nemaline myopathy.

The mutation Met132Val in the ACTA1 gene was identified in a patient with mild nemaline myopathy (NM). We examined actin mRNA and protein from biopsy samples. Sixty-one percent of the mRNA from the biopsy was not cleaved with BstX1, indicating the presence of mutant messenger in vivo. Monomeric actin was extracted from 2.5 mg of mutant muscle and wild type muscle. A proportion of the NM actin did not polymerise in 50 mM KCl, 2.5 mM MgCl2 but all the wild-type actin did. NM actin was fully polymerised by 50 mM KCl, 2.5 mM MgCl2, 150 nM rhodamine-phalloidin. Thin filaments reconstituted with this co-polymer were different from wild-type. The NM actin produces faster sliding of thin filaments at pCa5 and higher relative isometric force. We conclude that the mutant mRNA and protein is expressed and that the mutation reduces polymerisability and alters thin filament function.

Actin Cytoskeleton↗
PubMed2004

Abnormalities in alpha-dystroglycan expression in MDC1C and LGMD2I muscular dystrophies.

We recently identified mutations in the fukutin related protein (FKRP) gene in patients with congenital muscular dystrophy type 1C (MDC1C) and limb girdle muscular dystrophy type 2I (LGMD2I). The sarcolemma of these patients typically displays an immunocytochemical reduction of alpha-dystroglycan. In this report we extend these observations and report a clear correlation between the residual expression of alpha-dystroglycan and the phenotype. Three broad categories were identified. Patients at the severe end of the clinical spectrum (MDC1C) were compound heterozygote between a null allele and a missense mutation or carried two missense mutations and displayed a profound depletion of alpha-dystroglycan. Patients with LGMD with a Duchenne-like severity typically had a moderate reduction in alpha-dystroglycan and were compound heterozygotes between a common C826A (Leu276Ileu) FKRP mutation and either a missense or a nonsense mutation. Individuals with the milder form of LGMD2I were almost invariably homozygous for the Leu276Ile FKRP mutation and showed a variable but subtle alteration in alpha-dystroglycan immunolabeling. Our data therefore suggest a correlation between a reduction in alpha-dystroglycan, the mutation and the clinical phenotype in MDC1C and LGMD2I which supports the hypothesis that dystroglycan plays a central role in the pathogenesis of these disorders.

Adult↗
PubMed2003

Clinical and functional effects of a deletion in a COOH-terminal lumenal loop of the skeletal muscle ryanodine receptor.

We have identified a patient affected by a relatively severe form of central core disease (CCD), carrying a heterozygous deletion (amino acids 4863-4869) in the pore-forming region of the sarcoplasmic reticulum calcium release channel. The functional effect of this deletion was investigated (i) in lymphoblastoid cells from the affected patient and her mother, who was also found to harbour the mutation and (ii) in HEK293 cells expressing recombinant mutant channels. Lymphoblastoid cells carrying the RYR1 deletion exhibit an 'unprompted' calcium release from intracellular stores, resulting in significantly smaller thapsigargin-sensitive intracellular Ca(2+) stores, compared with lymphoblastoid cells from control individuals. Blocking the RYR1 with dantrolene restored the intracellular calcium stores to levels similar to those found in control cells. Single channel and [(3)H]ryanodine binding measurements of heterologously expressed mutant channels revealed a reduced ion conductance and loss of ryanodine binding and regulation by Ca(2+). Heterologous expression of recombinant RYR1 peptides and analysis of their membrane topology demonstrate that the deleted amino acids are localized in the lumenal loop connecting membrane-spanning segments M8 and M10. We provide evidence that a deletion in the lumenal loop of RYR1 alters channel function and causes CCD.

Adolescent↗
PubMed2003

Large in-frame deletions of the rod-shaped domain of the dystrophin gene resulting in severe phenotype.

BACKGROUND: The prediction that Duchenne muscular dystrophy patients have out-of-frame deletions and Becker muscular dystrophy patients have in-frame deletions of the dystrophin gene holds well in the vast majority of cases. Large in-frame deletions involving the rod domain only have usually been associated with mild (BMD) phenotype. OBJECTIVES: To describe unusual cases with large in-frame deletions of the rod-shaped domain of the dystrophin gene associated with severe (Duchenne) clinical phenotype METHODS: Screening for dystrophin gene deletion was performed on genomic DNA by using multiplex polymerase chain reaction. Needle muscle biopsies from the quadriceps were obtained using a Bergström needle. The biopsies were stained with histologic and histochemical techniques as well as monoclonal antibodies to dystrophin 1, 2 and 3. RESULTS: In three children with large in-frame deletions of the rod domain (exons 10-44, 13-40 and 3-41), early-onset weakness and a disease course suggested the DMD phenotype. CONCLUSIONS: This observation emphasizes the uncertainty in predicting the Becker phenotype in a young patient based on laboratory evaluation, and that the clinical picture should always be considered.

Dystrophin↗
PubMed2002

Muscle development genes: their relevance in neuromuscular disorders.

Myogenesis is a complex cascade of events that involves the specification and differentiation of muscle precursor cells or myoblasts, their fusion to form primary and secondary myotubes and subsequent maturation into muscle fibres. In addition, the development of axial muscle requires the migration of muscle precursor cells. These events are under strict genetic control. The contribution of individual genes to this process has been highlighted both by the phenotype of mice with targeted inactivation of individual myogenic regulatory factors and by rare human disorders in which the involvement of these genes has been demonstrated. The inactivation of known myogenic regulatory genes is associated with abnormal regulation of skeletal muscle differentiation and has an effect on regeneration but does not cause progressive muscle weakness or wasting. This review summarises recent developments in this field and will be of particular relevance to those interested in neuromuscular disorders. We also examine the possibility that some rare human conditions associated with abnormal muscle formation may be due to genetic defects in one of the myogenic regulatory genes.

Animals↗
PubMed2002

Mutations in the nebulin gene can cause severe congenital nemaline myopathy.

Previously, we reported results indicating that nebulin was the gene causing the typical form of autosomal recessive nemaline (rod) myopathy. Here we describe the identification of mutations in the nebulin gene in seven offspring of five families affected by the severe congenital form of nemaline myopathy. One pregnancy was terminated on the grounds of foetal abnormality, while six affected infants died at ages ranging from the first day of life to 19 months. Only three of the six neonates were able to establish spontaneous respiration. Three had arthrogryposis. In three of the five families, the mutations were located in exon 184. These mutations are predicted to cause absence of the C-terminal part of nebulin.

Biopsy↗
PubMed2002

Muscle MRI findings in a three-generation family affected by Bethlem myopathy.

We report clinical and muscle magnetic resonance imaging (MRI) findings in three individuals (aged 6, 26 and 73 years) from a three-generation family with Bethlem myopathy, confirmed by molecular genetic analysis which showed an exon skipping mutation in the COL6A1 gene. The clinical severity ranged from mild proximal weakness and distal laxity in the younger patients, to inability to stand or walk and severe contractures in the 76-year-old grandmother. The pattern of muscle involvement showed variable severity in parallel with the severity of motor function impairment. Although there was a marked variability in the severity of the MRI findings, it was possible to recognize a specific pattern of muscle involvement in all three patients. This consisted of involvement of the peripheral region of the vastus lateralis and hamstrings muscles with relative sparing of their central part. This was best appreciated in the third decade of life, but could also be identified both in the younger patient with minimal MRI changes and in the oldest patient, despite her more severe and diffuse muscle involvement. This report suggests that muscle MRI could be used as an additional tool to establish the pattern and the degree of muscle involvement in patients with Bethlem myopathy. Further studies in a larger cohort are needed to evaluate the specificity of these findings.

Adult↗