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The age when boys lose the ability to walk independently is one of the milestones in the progression of Duchenne muscular dystrophy (DMD). We have used this as a measure of disease severity in a group of 30 patients with DMD and six patients with intermediate Duchenne/Becker dystrophy (D/BMD). Dystrophin analysis was performed on tissue sections and western blots of muscle biopsy specimens from these patients and the relationships that were found between clinical severity and abundance of dystrophin labelling are reported. All patients with intermediate D/BMD had dystrophin labelling that was detected on sections and blots. Weak dystrophin labelling was found in sections from 21/30 DMD cases and on blots in 18/30 cases. Two non-exclusive patterns of dystrophin labelling were observed on sections: very clear labelling on a small percentage of fibres (usually < 1%) or very weak labelling on a much higher proportion (about 25%). The mean age at loss of mobility among the DMD patients with no dystrophin labelling on tissue sections was 7.9 years (range 6.3-9.5) while the mean age among those with some labelling was 9.9 years (range 8.0-11.9); this is a significant difference. Quantitative estimates of dystrophin abundance were obtained from densitometric analysis of dystrophin bands on blots. In the whole group of 36 patients, a significant positive relationship was found between the abundance of dystrophin and the age at loss of independent mobility. It is concluded that even the very low concentrations of dystrophin found in DMD patients may have some functional significance. [ABSTRACT FROM AUTHOR]

Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), two conditions which were previously believed to be completely separate entities. BM is a relatively mild dominantly inherited disorder characterised by proximal weakness and distal joint contractures. UCMD was originally described as an autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. Here we review the clinical phenotypes of BM and UCMD and their diagnosis and management, and provide an overview of the current knowledge of the pathogenesis of collagen VI related disorders. [ABSTRACT FROM AUTHOR]

Introduction: Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD). BM is a relatively mild dominantly inherited disorder with proximal weakness and distal joint contractures. UCMD is an autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. Methods: We developed a method for rapid direct sequence analysis of all 107 coding exons of the COL6 genes using single condition amplification/internal primer (SCAIP) sequencing. We have sequenced all three COL6 genes from genomic DNA in 79 patients with UCMD or BM. Results: We found putative mutations in one of the COL6 genes in 62% of patients. This more than doubles the number of identified COL6 mutations. Most of these changes are consistent with straightforward autosomal dominant or recessive inheritance. However, some patients showed changes in more than one of the COL6 genes, and our results suggest that some UCMD patients may have dominantly acting mutations rather than recessive disease. Discussion: Our findings may explain some or all of the cases of UCMD that are unlinked to the COL6 loci under a recessive model. The large number of single nucleotide polymorphisms which we generated in the course of this work may be of importance in determining the major phenotypic variability seen in this group of disorders. [ABSTRACT FROM AUTHOR]

About half of the children with classical congenital muscular dystrophy (CMD) show an absence in their skeletal muscle of laminin alpha2 chain, one of the components of the extracellular matrix protein, merosin. Linkage analysis implicated the laminin alpha2 chain gene (LAMA2) on chromosome 6q2, now confirmed by the discovery of mutations in the laminin alpha2 chain gene. We have further investigated the location of the LAMA2 locus on chromosome 6q2, using both linkage analysis in nine informative families and homozygosity mapping in 13 consanguineous families. Four of these families only had mild or moderate down regulation of laminin alpha2 chain expression and a milder phenotype; the rest had no protein or only a trace. Haplotype analysis in all the informative families, including those with partial laminin alpha2 expression, was compatible with linkage to chromosome 6q2. This observation expands the spectrum of the phenotype secondary to laminin alpha2 chain deficiency. Our results suggest that the LAMA2 locus is more centromeric than previously proposed. Recombinant events place the locus between markers D6S470 and D6S1620 in an interval of less than 3 cM. [ABSTRACT FROM PUBLISHER]

Autosomal recessive limb-girdle muscular dystrophies (AR LGMD) represent a group of muscle diseases with a wide spectrum of clinical signs, varying from very severe to mild. Four different loci that when mutated cause the AR LGMD phenotype have been mapped or cloned or both: in two of them the linked families seem to have a relatively mild phenotype (LGMD2a and LGMD2b), in the third one the reported linked families show a more severe clinical course (LGMD2c), while mutations in the fourth locus may cause severe or mild phenotypes (LGMD2d). The relative proportion of each of these genetic forms among the LGMD families and whether there are other genes that when mutated cause this phenotype is unknown. The closest available informative markers for each of the mapped AR LGMD genes have been tested in 13 Brazilian families with at least three affected patients. The findings from the present report confirm non-allelic heterogeneity for LGMD and suggest that in our population about 33% of the LGMD families are caused by mutations in the 15q gene, 33% in the 2p gene, 17% by mutations in the adhalin gene, and less than 10% may be by mutations at the 13q locus. They also suggest that there is at least one other gene responsible for this phenotype. In addition, the main clinical features of the different forms are discussed. [ABSTRACT FROM PUBLISHER]

This report is the second part of a trilogy from a multidisciplinary study which was undertaken to record the relationships between clinical severity and dystrophin gene and protein expression. The aim in part 2 was to correlate the effect of gene deletions on protein expression in individual patients with well defined clinical phenotypes. Among the DMD patients, most of the deletions/duplications disrupted the open reading frame, but three patients had in frame deletions. Some of the intermediate D/BMD patients had mutations which were frameshifting while others were in frame. All of the deletions/duplications in the BMD patients maintained the open reading frame and 25/26 deletions in typical BMD group 5 started with exon 45. The deletion of single exon 44 was the most common mutation in patients from groups 1 to 3. Dystrophin was detected in sections and blots from 58% of the DMD patients with a size that was compatible with synthesis from mRNA in which the reading frame had been restored. Certain deletions were particularly associated with the occurrence of limited dystrophin synthesis in DMD patients. For example, 9/11 DMD patients missing single exons had some detectable dystrophin labelling compared with 10/24 who had deletions affecting more than one exon. All patients missing single exon 44 or 45 had some dystrophin. Deletions starting or finishing with exons 3 or 51 (8/9) cases were usually associated with dystrophin synthesis whereas those starting or finishing with exons 46 or 52 (11/11) were not. Formal IQ assessments (verbal, performance, and full scores) were available for 47 patients. Mean IQ score among the DMD patients was 83 and no clear relationship was found between gene mutations and IQ. The mutations in patients with a particularly severe deficit of verbal IQ were spread throughout the gene. [ABSTRACT FROM PUBLISHER]

This multidisciplinary study was undertaken to record the variation in gene and protein expression in a large cohort of patients with well defined clinical phenotypes. The patients, whose ages ranged from 4 years to 66 years, spanned a wide range of disease severity. They represented the first 100 patients who had been examined in Newcastle, had undergone a muscle biopsy, and provided a blood sample for DNA analysis. The study had three aims: to observe any trends in the analyses across the clinical groups, to correlate gene and protein expression in individual patients, and to use the data collected to assess the relative usefulness of different techniques in the diagnosis and prognosis of patients with Duchenne and Becker dystrophy (DMD/BMD). In part 1, we describe the clinical assessment of the patients and the trends that were observed across the cohort. The patients were divided into seven groups. Group 1 had severe DMD (n = 21), group 2 had milder DMD (n = 20), group 3 were intermediate D/BMD patients (n = 9), group 4 had severe BMD (n = 5), and group 5 were more typical BMD patients (n = 31). Some patients were too young to be classified (n = 7) and a group of all the female patients were also classified separately (n = 7). The number of DMD and BMD patients was about equal, in accord with disease prevalence in the north of England, but an unusually high proportion were sporadic cases. Dystrophin labelling (performed with up to three antibodies) on both blots and sections increased gradually across the clinical groups. All histopathological indices, except the proportion of fat in biopsy sections, showed clear trends across the groups. [ABSTRACT FROM PUBLISHER]

This report is the third part of a trilogy from a multidisciplinary study which was undertaken to investigate gene and protein expression in a large cohort of patients with well defined and diverse clinical phenotypes. The aim of part 3 was to review which of the analytical techniques that we had used would be the most useful for differential diagnosis, and which would provide the most accurate indication of disease severity. Careful clinical appraisal is very important and every DMD patient was correctly diagnosed on this basis. In contrast, half of the sporadic BMD patients and all of the sporadic female patients had received different tentative diagnoses based on clinical assessments alone. Sequential observations of quantitative parameters (such as the time taken to run a fixed distance) were found to be useful clinical indicators for prognosis. Intellectual problems might modify the impression of physical ability in patients presenting at a young age. Histopathological assessment was accurate for DMD but differentiation between BMD and other disorders was more difficult, as was the identification of manifesting carriers. Our data on a small number of women with symptoms of muscle disease indicate that abnormal patterns of dystrophin labelling on sections may be an effective way of differentiating between female patients with a form of limb girdle dystrophy and those carrying a defective Xp21 gene. Dystrophin gene analysis detects deletions/duplications in 50 to 90% of male patients and is the most effective non-invasive technique for diagnosis. Quantitative Western blotting, however, would differentiate between all Xp21 and non-Xp21 male patients. In this study we found a clear relationship between increased dystrophin abundance (determined by densitometric analysis of blots) and clinical condition, with a correlation between dystrophin abundance and the age at loss of independent mobility among boys with DMD and intermediate D/BMD. This indicates that blotting is the most sensitive and accurate technique for diagnosis and prognosis. [ABSTRACT FROM PUBLISHER]

Among 85 patients with Duchenne and Becker muscular dystrophy, 29 were found to have mutations which disrupted the open reading frame for dystrophin. Thus any dystrophin detected in this group of patients should consist of the severely truncated polypeptides that represent prematurely terminated translation products. Dystrophin was detected in blots from 17/29 biopsies and the observed sizes of the polypeptides were compared with predicted sizes calculated in two ways: if translation was terminated at the stop codon generated by each frameshifting deletion, and if the reading frame was restored and translation proceeded. In every case the observed size matched the size predicted on the basis of a restored reading frame. This was in accord with immunocytochemical labelling of scattered dystrophin positive fibres which were found on serial sections labelled with antibodies to both the rod and C-terminal domains. Thus analysis at the protein level supports genetic evidence of exon skipping as a mechanism which restores frameshifting mutations in some fibres. [ABSTRACT FROM PUBLISHER]

The article presents phenotypic and genetic findings of 42 individuals from 21 families with mutations of valosin-containing protein (VCP) genes. Eighteen patients manifested a previously described mutation. The study measured serum creatine kinase (CK) levels in 19 patients. Some patients showed mild, unspecific myopathic changes.

Reference range for head circumference on the Tanner charts do not go beyond age 16. In this study the head circumference and heights of 354 adults in two British centres were measured. The centile charts constructed from these measurements show that adult head circumference is related to height. The mean head circumference of a male of average height is above the 97th centile for a 16 year old on the Tanner charts. The paediatric charts are therefore inappropriate for use in adult males. [ABSTRACT FROM AUTHOR]

A 10 month old girl presented with a history of constipation from early life. She was found to be hypercalcaemic with hypercalciuria and nephrocalcinosis. Her mild motor delay and hypotonia were thought to be linked to chronic hypercalcaemia, but when these features failed to improve despite normocalcaemia on a low calcium diet the possibility of neurocalcaemia disease was explored in more details. She was subsequently found to have spinal muscular atrophy type 2. We suspect that the hypercalcaemia with hypercalciuria observed in this case reflects altered bone turnover secondary to reduced muscular activity. [ABSTRACT FROM AUTHOR]

An abstract of the article "Clinical, Pathological and Genetic Findings in Two Patients with Myofibrillar Myopathy," by U. M. Sheerin, S. Omer, and K. Bushby.

Mutations in any of the three collagen 6A genes (COL6A1, COL6A2, and COL6A3) are responsible for Bethlem myopathy (BM) and Ullrich's congenital muscular dystrophy (UCMD). Diagnostic DNA testing in these conditions is difficult because of the large size of the genes involved and the frequent presence of polymorphisms. We have developed a method for rapid, robust, and economical direct sequence analysis of all three genes encoding collagen VI subunits, including 107 exons in total. SCALP (Single Condition Amplification/Internal Primer) sequencing avoids intermediate screening steps such as SSCP or DHPLC. Analysis is facilitated by semi-automated assembly software, with automated discrepancy base-calling followed by human review. SCALP analysis of the COL6A1-3 genes readily identifies premature stop codon mutations and small insertions/deletions as well as putative splice site and missense mutations. In addition we are documenting the location and frequency of a large number of single nucleotide polymorphisms, which may influence the phenotype. We will present the results obtained with this method of analysis in more than 75 patients with a clinical diagnosis of BM or UCMD. As SCALP was initially developed for dystrophin mutation analysis and this study constitutes its first application to an autosomal disease, we will discuss the use of this method in detecting mutations in heterozygote versus hemizygote probands. Several methods are routinely used to assay for gene copy number variation in genomic DNA, including quantitative fluorescent PCR, fluorescent microsatellite analysis and Southern blot analysis. We have investigated the potential of real time PCR to identify genomic DNA deletions and duplications in patients affected with hereditary neuropathy with liability to pressure palsies (HNPP) and hereditary sensory and motor neuropathy type 1A (HMSN1A). We have utilised the 5' nuclease extension assay (Taqman) to perform a diplex, dual-labelled assay for the PMP22 gene (labelled in TAMRA) and a diploid control fragment amplified from the MPZ gene (labelled in VIC). During a blind study of 100 individuals with either a normal, deleted or duplicated PMP22 genotype, major discrepancies were encountered when primer and probe batches were changed. Deleted, normal and duplicated thresholds were set, and the chance of miscalling a patient was calculated to be 1 in 740,000, using 8 sample replicates. When a new batch of primers and probes were used to complete the study, a large rate of miscalling was seen (4 out of 10 patients in one run). We believe that although the technique works well on a validated batch of reagents, changing the reagents could cause large error rates, necessitating the need to re-optimise the technique for each new set of primers and probes. [ABSTRACT FROM AUTHOR]

Background and Objective: Dysferlinopathies are a group of muscle disorders caused by mutations in the DYSF gene. Previous muscle imaging studies describe a selective pattern of muscle involvement in smaller patient cohorts, but a large imaging study across the entire spectrum of the dysferlinopathies had not been performed and previous imaging findings were not correlated with functional tests., Methods: We present cross-sectional T1-weighted muscle MRI data from 182 patients with genetically confirmed dysferlinopathies. We have analysed the pattern of muscles involved in the disease using hierarchical analysis and presented it as heatmaps. Results of the MRI scans have been correlated with relevant functional tests for each region of the body analysed., Results: In 181 of the 182 patients scanned, we observed muscle pathology on T1-weighted images, with the gastrocnemius medialis and the soleus being the most commonly affected muscles. A similar pattern of involvement was identified in most patients regardless of their clinical presentation. Increased muscle pathology on MRI correlated positively with disease duration and functional impairment., Conclusions: The information generated by this study is of high diagnostic value and important for clinical trial development. We have been able to describe a pattern that can be considered as characteristic of dysferlinopathy. We have defined the natural history of the disease from a radiological point of view. These results enabled the identification of the most relevant regions of interest for quantitative MRI in longitudinal studies, such as clinical trials., Clinical Trial Registration: NCT01676077., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background: Defects in glycosylation of alpha-dystroglycan (α-DG) cause autosomal-recessive disorders with wide clinical and genetic heterogeneity, with phenotypes ranging from congenital muscular dystrophies to milder limb girdle muscular dystrophies. Patients show variable reduction of immunoreactivity to antibodies specific for glycoepitopes of α-DG on a muscle biopsy. Recessive mutations in 18 genes, including guanosine diphosphate mannose pyrophosphorylase B ( GMPPB ), have been reported to date. With no specific clinical and pathological handles, diagnosis requires parallel or sequential analysis of all known genes., Methods: We describe clinical, genetic and biochemical findings of 21 patients with GMPPB -associated dystroglycanopathy., Results: We report eight novel mutations and further expand current knowledge on clinical and muscle MRI features of this condition. In addition, we report a consistent shift in the mobility of beta-dystroglycan (β-DG) on Western blot analysis of all patients analysed by this mean. This was only observed in patients with GMPPB in our large dystroglycanopathy cohort. We further demonstrate that this mobility shift in patients with GMPPB was due to abnormal N -linked glycosylation of β-DG., Conclusions: Our data demonstrate that a change in β-DG electrophoretic mobility in patients with dystroglycanopathy is a distinctive marker of the molecular defect in GMPPB., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Objective: Duchenne muscular dystrophy (DMD) is characterised by progressive muscle weakness. It has recently been reported that single nucleotide polymorphisms (SNPs) located in the SPP1 and LTBP4 loci can account for some of the inter-individual variability observed in the clinical disease course. The validation of genetic association in large independent cohorts is a key process for rare diseases in order to qualify prognostic biomarkers and stratify patients in clinical trials., Methods: Duchenne patients from five European neuromuscular centres were included. Information about age at wheelchair dependence and steroid use was gathered. Melting curve analysis of PCR fragments or Sanger sequencing were used to genotype SNP rs28357094 in the SPP1 gene in 336 patients. The genotype of SNPs rs2303729, rs1131620, rs1051303 and rs10880 in the LTBP4 locus was determined in 265 patients by mass spectrometry. For both loci, a multivariate analysis was performed, using genotype/haplotype, steroid use and cohort as covariates., Results: We show that corticosteroid treatment and the IAAM haplotype of the LTBP4 gene are significantly associated with prolonged ambulation in patients with DMD. There was no significant association between the SNP rs28357094 in the SPP1 gene and the age of ambulation loss., Conclusions: This study underlines the importance of replicating genetic association studies for rare diseases in large independent cohorts to identify the most robust associations. We anticipate that genotyping of validated genetic associations will become important for the design and interpretation of clinical trials., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Objective: GNE myopathy is a rare recessive myopathy associated with inclusion bodies on muscle biopsy. The clinical phenotype is associated with distal muscle weakness with quadriceps sparing. Most of the current information on GNE myopathy has been obtained through studies of Jewish and Japanese patient cohorts carrying founder mutations in the GNE gene. However, little is known about GNE myopathy in Europe where the prevalence is thought to be very low., Methods: Patients were referred through the National Specialist Commissioning Team service for limb-girdle muscular dystrophies at Newcastle (UK). All patients harbouring mutations in the GNE gene were recruited for our study. Detailed clinical and genetic data as well as muscle MRIs and muscle biopsies were reviewed., Results: We identified 26 patients harbouring mutations in the GNE gene. Two previously reported mutations (c.1985C>T, p.Ala662Val and c.1225G>T, p.Asp409Tyr) were prevalent in the Scottish, Northern Irish and Northern English populations; with 90% of these patients carrying at least one of the two mutations. Clinically, we confirmed the homogenous pattern of selective quadriceps sparing but noted additional features like asymmetry of weakness at disease onset., Conclusions: GNE myopathy is an important diagnosis to consider in patients presenting with distal leg muscle weakness. We report, for the first time, two common mutations in the north of Britain and highlight the broader spectrum of clinical phenotypes. We also propose that the prevalence of GNE myopathy may be underestimated due to the frequent absence of rimmed vacuoles in the muscle biopsy., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Objective: Titin gene (TTN) mutations have been described in eight families with hereditary myopathy with early respiratory failure (HMERF). Some of the original patients had features resembling myofibrillar myopathy (MFM), arguing that TTN mutations could be a much more common cause of inherited muscle disease, especially in presence of early respiratory involvement., Methods: We studied 127 undiagnosed patients with clinical presentation compatible with MFM. Sanger sequencing for the two previously described TTN mutations in HMERF (p.C30071R in the 119th fibronectin-3 (FN3) domain, and p.R32450W in the kinase domain) was performed in all patients. Patients with mutations had detailed review of their clinical records, muscle MRI findings and muscle pathology., Results: We identified five new families with the p.C30071R mutation who were clinically similar to previously reported cases, and muscle pathology demonstrated diagnostic features of MFM. Two further families had novel variants in the 119th FN3 domain (p.P30091L and p.N30145K). No patients were identified with mutations at position p.32450., Conclusions: Mutations in TTN are a cause of MFM, and titinopathy is more common than previously thought. The finding of the p.C30071R mutation in 3.9% of our study population is likely due to a British founder effect. The occurrence of novel FN3 domain variants, although still of uncertain pathogenicity, suggests that other mutations in this domain may cause MFM, and that the disease is likely to be globally distributed. We suggest that HMERF due to mutations in the TTN gene be nosologically classified as MFM-titinopathy.

Mutations in the dysferlin gene lead to limb girdle muscular dystrophy 2B, Miyoshi myopathy and distal anterior compartment myopathy. A cohort of 36 patients affected by dysferlinopathy is described, in the first UK study of clinical, genetic, pathological and biochemical data. The diagnosis was established by reduction of dysferlin in the muscle biopsy and subsequent mutational analysis of the dysferlin gene. Seventeen mutations were novel; the majority of mutations were small deletions/insertions, and no mutational hotspots were identified. Sixty-one per cent of patients (22 patients) initially presented with limb girdle muscular dystrophy 2B, 31% (11 patients) with a Miyoshi phenotype, one patient with proximodistal mode of onset, one patient with muscle stiffness after exercise and one patient as a symptomatic carrier. A wider range of age of onset was noted than previously reported, with 25% of patients having first symptoms before the age of 13 years. Independent of the initial mode of presentation, in our cohort of patients the gastrocnemius muscle was the most severely affected muscle leading to an inability to stand on tiptoes, and lower limbs were affected more severely than upper limbs. As previous anecdotal evidence on patients affected by dysferlinopathy suggests good muscle prowess before onset of symptoms, we also investigated pre-symptomatic fitness levels of the patients. Fifty-three per cent of the patients were very active and sporty before the onset of symptoms which makes the clinical course of dysferlinopathy unusual within the different forms of muscular dystrophy and provides a challenge to understanding the underlying pathomechanisms in this disease.