Summary


We have investigated the frequency of deletions in the dystrophin gene in 108 unrelated Duchenne and Becker muscular dystrophy (DMD/BMD) patients from southern Italy (DMD, n. 47; BMD, n. 61) and identified 89 deletions. The de novo mutation rate (about 30%), and the preferentially maternal origin of deletional mutations, analysed in families in which the maternal grandparents were available or their haplotypes could be unequivocally reconstructed, are in agreement with data reported for other populations. The correlation between BMD phenotype and type of deletion suggests that, in the distal rod domain region, the deletion size may not be as crucial as the particular combination of missing exons. In fact, we provide immunohistochemical and clinical evidence that in-frame deletion of the hinge III region in the distal rod domain results in a milder phenotype as compared with shorter deletions that do not include the hinge III region. Our data obtained in BMD patients, by confirming inferences arising from minigene transfection experiments in mdx mice, represent an important contribution to gene therapy approaches.

Analysis of Dystrophin Gene Deletions Indicates that the Hinge III Region of the Protein Correlates with Disease Severity

Background & objectives: Duchenne (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive disorders, caused by mutations in the dystrophin gene. Genetic diagnosis of the proband becomes crucial, and forms the base for carrier analysis, genetic counselling, prediction of natural history and prognosis, and eligibility for therapeutic strategies. Traditional multiplex PCR assay is the common method used in India to detect DMD gene deletions, mainly in the hot-spot region. Deletions of exons outside the usual 18 or 21 exons in the hot-spot, duplications and carrier analysis are often left without precise genetic diagnosis and require efficient dosage/quantitative analysis. In this study we evaluated the efficacy of using multiplex PCR (mPCR) of 30 exons followed by multiplex ligation-dependent probe amplification (MLPA), to study deletions and duplications in the DMD gene in patients clinically diagnosed as BMD/ DMD. Methods: Using an algorithm of mPCR and MLPA which was less invasive and cost-effective, we performed retrospective and prospective analysis on 150 male patients. Results: Multiplex PCR could pick up deletions in 103 of the 150 cases. MLPA was able to detect deletions and duplications including nine additional mutations. Further, the borders of the deletions and duplications were more accurately defined by this recent methodology, which enables one to determine the effect of the mutation on the reading frame. In all, including the single exon deletions, MLPA was efficient in accurately confirming mutations in 35 per cent of all cases. Ten novel mutations were identified in this study. Overall, this approach confirmed mutations in 75 per cent of the patients in our study. Interpretations & conclusions: The systematic approach/algorithm used in this study offers the best possible economical mutation analysis in the Indian scenario.

Use of multiplex ligation-dependent probe amplification (MLPA) for Duchenne muscular dystrophy (DMD) gene mutation analysis.

We have investigated the frequency of deletions in the dystrophin gene in 108 unrelated Duchenne and Becker muscular dystrophy (DMD/BMD) patients from southern Italy (DMD, n. 47; BMD, n. 61) and identified 89 deletions. The de novo mutation rate (about 30%), and the preferentially maternal origin of deletional mutations, analysed in families in which the maternal grandparents were available or their haplotypes could be unequivocally reconstructed, are in agreement with data reported for other populations. The correlation between BMD phenotype and type of deletion suggests that, in the distal rod domain region, the deletion size may not be as crucial as the particular combination of missing exons. In fact, we provide immunohistochemical and clinical evidence that in‐frame deletion of the hinge III region in the distal rod domain results in a milder phenotype as compared with shorter deletions that do not include the hinge III region. Our data obtained in BMD patients, by confirming inferences arising from minigene transfection experiments in mdx mice, represent an important contribution to gene therapy approaches.

Analysis of Dystrophin Gene Deletions Indicates that the Hinge III Region of the Protein Correlates with Disease Severity: Dystrophin Gene Deletions and Genotype-phenotype Correlation

The experiences in genetic counseling and prenatal diagnosis at a tertiary genetic center in India are described. Of 3500 subjects provided genetic counseling 28.7% were for prenatal diagnosis, 13.7% for mental retardation ±malformations, 11.5% for thalassemia, hemophilia and leukemia, 8.5% for neural tube defects and other malformations, and 8% for muscle dystrophy and spinal muscle atrophy. Chromosomal studies in blood (n = 5459) were for recurrent abortions (57.8%), delayed milestones (14.7%), malformations (11%), and infertility and amenorrhea (10.2%). Indications for amniotic fluid studies (n = 835) were advanced maternal age (35.7%), high risk result on triple test (21.3%), previous child with trisomy 21 (21.3%) and abnormalities seen on ultrasound (11.1%). Molecular studies were mostly for thalassemia (843, 24.3%), Duchenne muscular dystrophy (443, 12.5%), fragile X syndrome (367, 10.3%), spinal muscular atrophy (315, 8.9%), thrombophilia profile (233, 6.6%), triplet repeat disorders-spinocerebellar ataxias, Huntington disease and Friedreich ataxia-162 (4.6%), cystic fibrosis 140 (3.9 %) and mitochondrial disorders 101 (2.9%). Other disorders for which molecular diagnosis was done were intrauterine infections by PCR on the amniotic fluid, Prader Willi / Angelman syndromes, hemophilia, achondroplasia, congenital adrenal hyperplasia, and Apert syndrome etc. In biochemical studies triple marker tests were the most common (3239), followed by aminoacid chromatography (774). Among neurolipidosis metachromatic leukodystrophy was the commonest, followed by Krabbe’s disease, Tay Sach disease and Gaucher disease. Of the mucopolysacharidoses Hurler syndrome was the commonest, followed by Hunter syndrome. These data are compared with previous studies and a change towards increased prenatal diagnostic tests is observed. The commonest indication for amniocentesis has changed to advanced maternal age.Conclusion : Advanced molecular, cytogenetic and biochemical techniques have been a useful addition for genetic counseling and prenatal diagnosis in India.

Genetic counseling and prenatal diagnosis in India--experience at Sir Ganga Ram Hospital.

Background : Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy that affects young boys and the dystrophin gene on the X chromosome has been found to be associated with the disorder. Materials and Methods : In this prospective study, 112 clinically diagnosed DMD patients had muscle biopsy and were tested for exon deletions. Genotyping was also carried out at STR44, STR45, STR49 and STR 50 markers in 15 families. Results : Of the 112 clinically suspected DMD patients, the diagnosis of DMD was confirmed by histopathology and/or genetics in 101 patients. The mean age of onset was 3.1±1.44 years (1-6 years) and the mean age at presentation was 8.0±3.1 years (1.1-18.0 years). Delayed motor milestones were present in 63 (62.3%) patients. The mean creatine kinase value was 11822.64±8206.90 U/L (1240-57,700). Eighty-four patients had muscle biopsy and immunohistochemistry was done in 60 muscle samples, all of which demonstrated absence of dystrophin staining. Of the 60 dystrophin-negative cases, 73% showed deletion of at least one exon. Single exon deletion was found in 20.4%. Distal hotspot Exons 45, 47, 49 and 50 were the commonly deleted xenons and the deletion rates were 36%, 35%, 33.7% and 38.5% respectively. Conclusions : In this study population in south India the deletion rate was 73% and were more frequent in the distal end exon. With the availability of genetic analysis, the first investigation of choice in DMD should be genetic studies and muscle biopsy should be considered only if the genetic tests are negative or not available.

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Duchenne muscular dystrophy: A clinical, histopathological and genetic study at a neurology tertiary care center in southern India

We studied 160 cases of Duchenne muscular dystrophy (DMD) drawn from all parts of India, using multiplex PCR of 27 exons. Of these, 103 (64.4%) showed intragenic deletions. Most (69.7%) of the deletions involved exons 45-51. The phenotype of cases with deletion of single exons did not differ significantly from those with deletion of multiple exons. The distribution of deletions in studies from different countries was variable, but this was accounted for either by the small number of cases studied, or by fewer exons analyzed. It is concluded that there is likely to be no ethnic difference with respect to deletions in the DMD gene.

Minakshi Banerjee

Papers

Are there ethnic differences in deletions in the dystrophin gene