I. B. Ginjaar

Integrated study of 100 patients with Xp21 linked muscular dystrophy using clinical, genetic, immunochemical, and histopathological data. Part 1. Trends across the clinical groups.

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.

The clinical and molecular genetic approach to Duchenne and Becker muscular dystrophy: an updated protocol.

Detection of large rearrangements in the dystrophin gene in Duchenne and Becker muscular dystrophy is possible in about 65-70% of patients by Southern blotting or multiplex PCR. Subsequently, carrier detection is possible by assessing the intensity of relevant bands, but preferably by a non-quantitative test method. Detection of microlesions in Duchenne and Becker muscular dystrophy is currently under way. Single strand conformational analysis, heteroduplex analysis, and the protein truncation test are mostly used for this purpose. In this paper we review the available methods for detection of large and small mutations in patients and in carriers and propose a systematic approach for genetic analysis and genetic counselling of DMD and BMD families, including prenatal and preimplantation diagnosis.

Integrated study of 100 patients with Xp21 linked muscular dystrophy using clinical, genetic, immunochemical, and histopathological data. Part 2. Correlations within individual patients.

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.

Integrated study of 100 patients with Xp21 linked muscular dystrophy using clinical, genetic, immunochemical, and histopathological data. Part 3. Differential diagnosis and prognosis.

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.

Predicted and observed sizes of dystrophin in some patients with gene deletions that disrupt the open reading frame.

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.

Monoclonal antibody evidence for structural similarities between the central rod regions of actinin and dystrophin

A monoclonal antibody, MANDYS141, binds to both dystrophin and actinin on Western blots (SDS‐denatured), but only to actinin in frozen sections of human muscle (native conformation). It differs from a polyclonal cross‐reacting antiserum in that it binds to several muscle isoforms of actinin (smooth, fast and slow) from man, mouse and chicken and recognises a quite different part of the proposed triple‐helical region of dystrophin (amino acids 1750–2248). The results suggest that structural homologies between actinin and dystrophin occur more than once in their central helical regions and provide experimental support for an actinin‐like central rod model for dystrophin.

Splicing mutations in DMD/BMD detected by RT-PCR/PTT: detection of a 19AA insertion in the cysteine rich domain of dystrophin compatible with BMD.

We have used an RNA based mutation detection method to screen the total coding region of the dystrophin gene of a Duchenne and a Becker muscular dystrophy patient in whom DNA based mutation detection methods have so far failed to detect mutations. By RT-PCR and the protein truncation test (PTT) we could identify point mutations in both cases. DMD patient DL184.3 has a T-->A mutation in intron 59 at position -9, creating a novel splice acceptor site for exon 60. As a result seven intronic bases are spliced into the mRNA, causing a frameshift and premature translation termination 20 codons downstream. Since this patient had died and only fibroblasts were available, we applied MyoD induced myodifferentiation of stored fibroblasts to enhance muscle specific gene expression. With the results of this mutation analysis, prenatal diagnosis could subsequently be performed in this family. BMD patient BL207.1 carries a G-->C mutation at position +5 of intron 64, disrupting the splice donor consensus sequence and activating a cryptic splice donor site 57bp downstream. The inclusion of these 57 intronic bases in the mRNA leaves the reading frame open and results in the insertion of 19 amino acids into the cysteine rich domain of dystrophin. Interestingly, this insertion in a part of the dystrophin considered to interact with the dystrophin binding complex of the sarcolemma is apparently compatible with mild BMD-like clinical features. Both mutations reported are missed by analysis of multiplex PCR products designed for deletion screening of the coding region. Extrapolation from existing point mutation detection efficiencies by DNA and RNA based methods emphasises that RNA based methods are more sensitive and that most of the remaining undetected mutations may affect splice or branch sites or create cryptic splice sites.