In this exciting era in the investigation of the muscular dystrophies, we are now in the process of identifying how specific gene mutations cause the clinical features observed in patients. Between the gene defect and the disease symptoms lies altered protein expression, which directly affects the pathophysiology of disease progression. A specific gene defect has been identified for Duchenne and myotonic dystrophy, and we are now in the interesting stage of working out exactly how gene and protein expression are related to clinical phenotype. Current Opinion in Rheumatology 1993, 5:706-711
We present a family in which the differential diagnosis between X linked Duchenne muscular dystrophy and autosomal recessive Duchenne-like muscular dystrophy was resolved in favour of the latter by analysis of dystrophin, which is the protein product of the Duchenne muscular dystrophy locus.
The synthesis of immunoglobulin and acetylcholine receptor antibody by lymphocytes extracted from the peripheral blood and thymus of patients with myasthenia gravis was investigated using the Marbrook culture system. Small amounts of immunoglobulin were synthesised by thymic lymphocytes cultured in medium without mitogen for 14 days and acetylcholine receptor antibody activity was detectable in cultures from 3 out of 5 patients with thymic hyperplasia. Addition of Pokeweed mitogen to the cultures increased both IgG and acetylcholine receptor antibody synthesis and under these conditions receptor antibody activity was detectable in all 5 thymic lymphocyte cultures studied. Peripheral blood lymphocytes also produced small amounts of IgG when cultured in the absence of mitogen but acetylcholine receptor antibody levels were undetectable. In the presence of Pokeweed mitogen, however, peripheral blood lymphocytes synthesised increased amounts of IgG and acetylcholine receptor antibody activity was detectable in cultures of lymphocytes from 3 out of 4 patients. The amount of acetylcholine receptor antibody per microgram IgG, the specific activity, was greater in cultures of thymic lymphocytes than the specific activity of IgG in serum, suggesting that the hyperplastic thymus may be a major site of acetylcholine receptor antibody synthesis. Therefore it should be possible to use the culture system described to investigate the processes involved in the breakdown of self-tolerance in myasthenia gravis.
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.
Polyclonal and monoclonal antibodies, which recognize different regions and epitopes of the dystrophin molecule, bind to a protein of Mr 400,000 which is present in extracts of mdx muscle from regions which contain neuromuscular junctions (NMJ) and is absent from those which do not. This NMJ-associated homologue of dystrophin has at least 2 epitopes which are different to usual Xp21 form of dystrophin expressed along the sarcolemma of muscle fibres in normal muscles. This protein is also expressed at the NMJ of a DMD patient who lacks the first 52 exons of the Xp21 dystrophin gene and it must therefore be translated from a different gene transcript.
Immunocytochemistry is a powerful tool for the elucidation of clinical problems, particularly when used in conjunction with complementary techniques such as immunoblotting and DNA analysis. We would like to present an experimental example which illustrates the way these three different disciplines were brought together to investigate the defective gene product in X-linked muscular dystrophy.
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.
N,2'-O-dimethyladenosine (m6Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m6Am mapping methods assume each gene uses a single start nucleotide. However, gene transcription usually involves multiple start sites, generating numerous 5' isoforms. Thus, gene levels annotations cannot capture the diversity of m6Am modification in the transcriptome. Here we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m6Am stoichiometry for each 5' isoform that initiates with adenosine. Using CROWN-seq, we map the m6Am landscape in nine human cell lines. Our findings reveal that m6Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m6Am stoichiometry. We find that m6Am is associated with increased transcript expression and provide evidence that m6Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m6Am in influencing transcription initiation.
This thesis is a report of immunological studies on the soluble protein inside human red blood cells. The differences shown by adult and fetal red cells in their physical structure, general metabolism and haemoglobin biochemistry were considered, together with the possibility of using red cells with their readily extractable intracellular soluble protein as an immunological model for tissue cells in general. Thus the two-fold aim of this research project was to investigate red cell protein as a tissue model and to look for novel fetal proteins in red cells by immunological methods. Antisera were prepared using red cell lysate protein as an immunogen. The antibody responses were determined principally by gel precipitation techniques, although some affinity chromatography (with analysis by polyacrylamide/SDS slab gel electrophoresis) and haemagglutination studies were also involved. Three sources of protein were used as immunogens in adult rabbits: unfractionated fetal lysate, unfractionated term cord lysate, and term cord lysate fractionated by ion-exchange chromatography so as to maximise recovery of non-haemoglobin red cell protein. Fetal lysate protein was also used to immunise two rabbits which had been neonatally tolerised to adult serum and red cells. In general, the results of the antibody analysis by gel precipitation could be divided into three categories of response: to contaminating serum proteins, to haemoglobin, and to other non-haemoglobin red cell proteins. Animals immunised with unfractionated lysates had a limited response to serum proteins whereas the ion-exchange fractionation concentrated certain serum proteins and this was reflected in an increased response to serum proteins. All the antisera showed a weak response to adult haemoglobin and a much stronger response to fetal haemoglobin possibly involving further antigenic determinants. A mobility antigen was detected in adult and fetal red cell lysates by two antisera; and an 2 mobility antigen, with erratic electrophoretic variants in fetal lysates, by a third. The existence of any novel non-haemoglobin protein in fetal red cells was not proved however. The results showed a rather limited antibody response to the many proteins known to be present in red blood cells. It was concluded that this could have been due to the inherent lack of immunogenicity associated with tissue cells, to the manner in which the immunogen was presented, or to the insensitivity of the methods used to detect the immune response.
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