Deletion analysis of the dystrophin gene in Duchenne and Becker muscular dystrophy patients: use in carrier diagnosis.

The dystrophin gene was analyzed in 8 Duchenne muscular dystrophy (DMD) and 10 Becker muscular dystrophy (BMD) unrelated families (22 subjects: 18 index cases and 4 sibs) for the presence of deletions by multiplex polymerase chain reaction (mPCR; 27 exons) and Southern hybridization using 8 cDMD probes. Deletions were identified in 5 DMD and 7 BMD patients (6 index cases and 1 sib). The concordance between the clinical phenotype and "reading frame hypothesis" was observed in 11/12 patients (92%). The female relatives of DMD/BMD patients with identifiable deletions were examined by quantitative mPCR. Carriers were identified in 7 families. We also describe a variation in the HindIII pattern with cDNA probe 8 and 11-14. Molecular characterization of the dystrophin gene in this study has been helpful in advising the patients concerning the inheritance of the condition, and carrier diagnosis of female relatives, and should also prove useful for prenatal diagnosis.

Relationship between myocardial counts and patient weight: adjusting the injected activity in myocardial perfusion scans.

It is generally accepted that the quality of myocardial images deteriorates with increasing patient weight. This is attributed to a reduction of counts detected from the myocardium. In this paper we have looked at the count reduction in obese patients and suggest a workable algorithm to increase the injected activity to compensate for this loss of count. In this prospective study, 64 consecutive patients with normal myocardial images were selected to include a weight range of 50-120 kg. The height, weight and gender of patients were noted. Each patient had two studies (total of 128 studies), one at rest and one following stress with adenosine and 20-40 W bicycle exercise. Total myocardial counts were calculated from the back-projected views. The total myocardial counts per MBq of the injected activity were calculated. There was no significant difference in the injected activity and the size of the heart (pixel length of heart) between stress and rest, or gender of the patient. The normalized myocardial counts were not different between men and women, but the counts were slightly, although not significantly, higher ( P=NS) with adenosine and exercise (mean of 243 x 10(3) counts) compared to rest images (229 x 10(3) counts). There was a significant progressive loss of counts in patients with increasing weight, body mass index or body surface area ( P<0.001). There was no significant difference in the changes in counts with weight between male and female, or rest and stress studies. The combined data from all the studies were used to calculate the correlation coefficient and the slope of the line for reduction of cardiac counts with a patient's weight, body mass index, and body surface area. The best correlation was with patient weight ( r=0.58, P<0.001). This was used to calculate the increase in injection activity with increasing weight to maintain the same average counts as achieved in a 70 kg patient with a 400 MBq injection. We suggest that the injection activity should increase from 100% for a 70 kg patient to 140% for 110 kg, 200% for 140 kg, and 250% for a 150 kg patient.

Deficiency of the 50 kDa dystrophin-associated-glycoprotein (adhalin) in an Indian autosomal recessive limb girdle muscular dystrophy patient : immunochemical analysis and clinical aspects.

Abnormalities of dystrophin are a common cause of muscular dystrophy and testing for dystrophin gene or protein has become a part of routine diagnostic evaluation of patients who present with progressive proximal muscle weakness, high serum creatine kinase concentrations, and histopathological evidence of a dystrophic process. Patients who have no dystrophin abnormalities are assumed to have autosomal recessive muscular dystrophy. In a family consisting of 5 sibs, 2 mentally normal brothers presented with abnormal gait and protrusion of chest and hips. Muscle biopsy from one of them showed dystrophic changes and reduced patchy binding of dystrophin. No detectable deletion was observed in the patient's DNA and his brother with cDMD probes. Dystrophin associated proteins, beta-dystroglycan showed discontinuous immunostaining in the sarcolemma and alpha-sarcoglycan (adhalin) was totally absent, while beta-, gamma-, and delta-sarcoglycans were highly reduced. Immunoblot analysis showed dystrophin of normal molecular weight but of decreased quantity, beta-dystroglycan was reduced by about 37% while alpha-sarcoglycan was completely absent. This study is a first attempt for a systematic clinical, genetic and molecular investigation of the autosomal recessive LGMD in India.

I. The modulatory effect in genotoxic responses due to age and duration of PHT-therapy in epileptic patients.

Sister chromatid exchange (SCE) frequency has been studied from the peripheral blood lymphocyte cultures of 42 epileptic patients on the anticonvulsant drug phenytoin (PHT) for 3 months and their follow-up (6 and 9 months), of 33 epileptics who had not started therapy (PHT-untreated), and of 40 normal healthy controls, all in the same age group, i.e., 10-30 years. PHT-treated epileptic patients at all three durations of therapy (3, 6, and 9 months) showed higher SCE frequency (P < 0.001) than healthy controls and PHT-untreated patients. There was no significant difference in SCE frequency between control and PHT-untreated patients, suggesting that disease is not associated with an increased frequency of SCEs. The frequency of SCEs seems to be influenced by an age factor, when older treated patients (21-30 years) showed higher SCE frequencies at 3 and 6 months (P < 0.001) and 9 months (P < 0.05) than the younger age group (10-20 years). SCE frequency increased linearly with the duration of therapy, i.e., from 3 months to 9 months. No correlation was found between SCE frequency and sex with respect to controls, PHT-untreated, and PHT-treated subjects. In conclusion, the modulating effect on SCE frequencies elicited by age and duration of therapy has been clearly demonstrated by SCE mean analysis. Teratogenesis Carcinog. Mutagen. 21:135-149, 2001.

II. An altered proliferation response due to the anticonvulsant phenytoin (PHT) in epileptic patients.

Lymphocyte proliferation kinetics (LPK) is an end point used in genetic toxicology that was proposed as an alternative for the screening of anticonvulsant drugs. The effect of phenytoin (PHT) was investigated on the mitotic and proliferation indices in cultured blood lymphocytes of 33 sporadically collected untreated and 42 PHT-treated epileptics, where the duration of treatment was 3, 6, and 9 months, and 40 control subjects (age range 10-30 years). PHT induced mitotic delays and decreased the mitotic index. A significant heterogeneity of the first, second and the third metaphases between treated and untreated groups was revealed. A reduction of the proliferation index (P < 0.001) and proliferation delay per cycle (P < 0.001) was also observed. There was little variation between the controls and untreated patients (P > 0.05). The results have confirmed that PHT can affect responses leading to genotoxicity. Teratogenesis Carcinog. Mutagen. 21:151-164, 2001.

Detection of deletion in the dystrophin gene of a patient with quadriceps myopathy.

A 43 year old male presented with slowly progressive weakness of limbs and hypertrophy of triceps, brachioradialis and calf muscles for four years. There was thinning of quadriceps muscles in both thighs. Histological study was compatible with Becker muscular dystrophy (BMD). Genomic DNA analysis showed a deletion of the Hind III fragments, spanning exons 45-47. A junction fragment of 11.0 kb was observed along with a deletion of a 3.4 kb PstI fragment containing exon 51 in the patient, and in one of his two sisters. The clinical and laboratory characteristics in this patient are in keeping with what has been described 'quadriceps myopathy' and fall within the phenotypic variants of BMD as has been shown by others.

Genotoxicity of the anticonvulsant drug phenytoin (PHT): a follow-up study of PHT-untreated epileptic patients. I. Sister chromatid exchange (SCE) analysis.

Phenytoin (PHT) is a widely prescribed antiepileptic drug. Its potential to interact with genetic material was investigated in a set of 30 epileptic patients (age 10-30 years) prior to and following the administration of PHT over a period of 9 months (grouped in a multiple of 3 months) and 40 control subjects in relation to age, sex, duration of drug therapy, and plasma concentration of PHT, using the sister chromatid exchange (SCE) frequency assay. Plasma levels of the phenytoin were measured by biochemical assay in epileptic patients before and after the PHT therapy. The peripheral blood lymphocytes were cultured and harvested at 72 h. The frequency of SCE was significantly higher (P < 0.001) in both age groups (10-20 and 21-30 years) for PHT-treated epileptics compared to PHT-untreated and control subjects. However, there were no considerable variations in SCE finding between the control and PHT-untreated patients. Between the two age groups, a significantly higher SCE frequency was observed in PHT-treated patients (P < 0.01) in the older age group (21-30 years). Mean SCE frequency did not differ between the male and female in the controls, PHT-untreated, or treated epileptics. Correlation between the plasma concentration of PHT and the incidence of SCE among 30 patients was insignificant. PHT monotherapy appears to have genotoxic effect as expressed by the induction of increased SCE rates in treated epileptics, while disease does not play any role in inducing genetic damage as shown by no difference in SCE frequencies between control subjects and PHT-untreated epileptic patients.

Genotoxicity of the anticonvulsant drug phenytoin (PHT): a follow-up study of PHT-untreated epileptic patients. II. Mitotic index (MI) and proliferation kinetics.

The mitotic index and proliferation rate index were investigated to determine the effect of phenytoin (PHT) in cultured blood lymphocytes of epileptics prior to and following administration of PHT over a period of 9 months (grouped in multiples of 3 months) and 40 control subjects (age range 10-30 years). Treatment with PHT brought inhibition of the mitotic index (MI) and proliferation rate index (PRI), which were significantly higher in treated subjects or which were more expressive in treated lymphocytes (P < 0.001) for all the three durations of treatment. In addition, statistically significant heterogeneity of first, second, and third metaphases between the treated, untreated, and control subjects was found. Mean PRI values were used to estimate cell cycle delays, showing the highest effect in treated lymphocytes (P < 0.001). There was no considerable variation between the control and untreated (P > 0.05). The study demonstrates that PHT may be potentially genotoxic and hence the usefulness of MI and PRI in monitoring epileptics on anticonvulsant treatment.

Molecular characterisation of Duchenne muscular dystrophy and phenotypic correlation.

Dystrophin gene was analysed in 32 unrelated DMD families (46 subjects: 32 index cases and 14 sibs) for the presence of deletions by mPCR for 27 exons and cDNA probes for the entire gene. Deletions were identified in 32 patients (25 index cases and seven sibs) from 25 families. The concordance between the clinical phenotype and 'reading frame' hypothesis was observed in 24 (75%) cases. Of these, nine patients were wheelchair bound between 8-12 years of age, nine (age range 5-10 years) showed progressive difficulty in walking and six (age range 1.6-4 years) had onset of muscle weakness. One patient (CH), who was wheelchair bound at 12 years, the effect of mutation on the ORF could not be ascertained due to the presence of a junction fragment. Seven patients had inframe deletions of which four were wheelchair bound by the age of 13 years, and three (age range 5-7 years) although, ambulatory had difficulty in walking. There were eight patients who showed no deletion, of which four became wheelchair bound by the age of 12 years, four, though still ambulatory, were unable to run and tired easily. Correlation between phenotype and genotype of these DMD patients demonstrates that genetic studies of lymphocyte DNA may not always reflect the situation in the tissue involved in dystrophin, i.e. muscle. We describe a common dystrophin gene polymorphism in the Indian population with cDNA 11-14 that alters the Hind III restriction sites. Novel RFLPs were observed in 26 patients and their family members. Whether this is a polymorphism or, related to the diseased phenotype needs confirmation.

Effects of methyl isocyanate on rat brain cells in culture.

Since the disaster in Bhopal, India, people exposed to methyl isocyanate (MIC) have complained of various disorders including neuromuscular dysfunction. In an attempt to get information about such dysfunction we have previously shown that MIC can affect muscle cells in culture. The present communication reports investigations into the effect of MIC on brain cells in culture. MIC was toxic to brain cells and the response was dose related. The observations were supported by light and electron microscopy.

Sister chromatid exchange in human chromosomes from normal individuals and epileptic patients on combinations of anticonvulsants.

Sister chromatid exchange (SCE) frequency, a sensitive indicator in mutagenicity testing, and mitotic index (MI) have been studied to observe genotoxic effects in epileptic patients on routine combinations of anticonvulsant therapy. All patients, both male and female and from various age groups, revealed an increased frequency of SCE per metaphase and a low MI (P less than 0.001) with respect to controls. A nonsignificant decrease in SCE frequency has been observed with an increase in the age of onset of epilepsy. Although the SCE frequency increased and the MI decreased in some groups with respect to the duration of epilepsy, there was no difference observed in SCE frequency with the duration of therapy.

Effects of methyl isocyanate on rat muscle cells in culture.

Since the Bhopal disaster, in which the causal agent was methyl isocyanate (MIC), exposed people have complained of various disorders including neuromuscular dysfunction. In an attempt to gain some information about the response of muscle tissue to MIC its effects were investigated in cells in culture isolated from muscle of 2 day old rats. After treatment with a range of MIC concentrations (0.025-0.5 microliter/5 ml culture) the total number of nuclei of the two main cell types (fibroblasts and myoblasts) and the number of nuclei in muscle fibres (myotubes) were recorded. At lower doses which had little effect on the total number of nuclei, the formation of muscle fibres--that is, fusion of muscle cells--was prevented as the proportion of nuclei in myotubes was decreased. At higher doses both cell types were killed. This would suggest either an effect on muscle differentiation or a selective toxicity towards myoblasts. The observations were supported by light and electron microscopy.

Mutagenic risk in epileptic patients before and after anticonvulsant therapy.

Therapy with anticonvulsant drugs has often been found to result in somatic chromosome aberrations in adult patients. There is also the possibility of epileptic fathers or mothers playing a role in the production of congenital malformations in their offspring. We have used the technique of sister chromatid exchange (SCE), a sensitive indicator of mutagenicity, to observe the mutagenic susceptibility in both male and female epileptic patients in different age groups prior to and after anticonvulsant therapy, and with respect to control. The frequency of SCE was significantly higher in all the age groups for treated and untreated cases compared with control. Between treated and untreated subjects in age group 26-50 years, a significantly higher SCE frequency was observed in the untreated patients (p less than 0.01). Similarly, untreated male patients showed a significantly higher SCE frequency (p less than 0.025) compared with treated male patients. Although the results of this study provide a general assessment of mutagenicity in epileptic patients that agrees with other studies and emphasizes the role of the disease in the higher occurrence of congenital malformation in their offspring, the importance of higher SCE frequency in untreated patients remains to be explained in further studies.

Mutagenic potential of anticonvulsant diphyenylhydantoin (DPH) on human lymphocytes in vitro.

Diphenylhydantoin (DPH)-treated human peripheral blood cultures were examined for sister chromatid exchange (SCE) frequency and mitotic index. A significantly enhanced SCE frequency in DPH-treated cultures was observed at a 15 micrograms/ml concentration (10-20 micrograms/ml DPH therapeutic range) and above. These results suggest an enhanced SCE response of lymphocytes in the therapeutic blood level of DPH. A significant linear fall in mitotic index was observed with increasing DPH concentrations. It is postulated that even in therapeutic doses DPH possibly affects nucleic acid metabolism.

Altered lipid composition of adipose tissue in human muscular dystrophy.

Lipids of subcutaneous adipose tissue from normal and dystrophic human samples were investigated with particular reference to the fatty acid composition of neutral lipids and phospholipids. The free fatty acid pattern did not show much change. Triglyceride, which together with cholesterol composed most of the neutral lipid fraction, showed significant changes in fatty acid composition by gas-liquid chromatography (GLC). There was a decrease in the total phospholipids. Analysis of the levels of individual phospholipids, however, showed an increase in sphingomyelin and ethanolamine phosphoglyceride contents. Fatty acid composition of the different phospholipid classes analyzed by GLC showed significant changes.

Effect of phenytoin on proliferation and differentiation of mouse muscle cells in vitro.

Effect of a commonly used anti-convulsant drug, phenytoin, was studied a four dose levels (5, 15, 50 and 100 micrograms/ml) on neonatal Bar Harbor mouse muscle in vitro. The cultures were exposed to the drug suspended in the growth medium at the end of 72 hrs, prior to the fusion of the cells. The effects of the drug were dose dependent. At high concentration (50 and 100 micrograms/ml), it hampered cell proliferation and prevented myoblasts from fusing to form multi-nucleated myotubes. Lower concentrations of phenytoin allowed cells fusion to occur, but the total cell count was decreased at 15 micrograms/ml.