Neurology India Medknow Publications on behalf of the Neurological Society of India ISSN: 0028-3886 EISSN: 1998-4022 Vol. 51, Num. 3, 2003, pp. 367-369

Neurology India, Vol. 51, No. 3, July, 2003, pp. 367-369

Re-evaluation of reading frame-shift hypothesis in Duchenne and Becker muscular dystrophy

Pandey GS , Kesari A, Mukherjee M, Mittal RD, Mittal B
Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow - 226014

Correspondence Address:
Department of Medical Genetics, Sanjay Gandhi PGI, Raebareli Road, Lucknow - 226014
balraj@sgpgi.ac.in

Code Number: ni03119

ABSTRACT

The reading frame hypothesis has been proposed to explain the molecular basis of two allelic forms of muscular dystrophies, Duchenne/Becker muscular dystrophy (D/BMD). To evaluate the hypothesis in Indian D/BMD patients, we analyzed deletion of dystrophin exons in 147 DMD and 19 BMD patients. Our studies showed deviation of more than 30% from the reading frame hypothesis in DMD patients (47/147). The present results implicate a need to reevaluate the reading frame hypothesis.

INTRODUCTION

Duchenne and Becker muscular dystrophies are allelic forms of X-linked recessive genetic disorders with a prevalence of 1 in 3500 live male births.[1] Approximately two-third patients show intragenic deletions ranging from one to several exons of the gene.[2],[3] The reading frame hypothesis of dystrophin gene mutations proposed by Monaco[4] explains two different phenotypes resulting from mutations in the same gene. The mutations that change the translational reading frame of the gene are responsible for the severe form DMD whereas mutations that maintain the translational reading frame of the gene result in a milder form BMD of the disease. This hypothesis has been used extensively to explain the clinical phenotype in about 92 % of the D/BMD patients.[5]

The patterns of gene deletions, distribution and proportion in the patients have been found to vary in different ethnic groups.[3],[6] In the present study, we have investigated the correlation between the reading frame of the gene to the clinical severity in D/BMD patients from North India. The present investigation, an extension of the earlier studies,[7] was carried out in 166 patients with D/BMD phenotype.

MATERIAL AND METHODS

Two hundred and eighty patients suspected to have D/BMD, enrolled in the Genetics and Neurology OPD of the Institute during the last 10 years, were selected for the study. The detailed clinical examination included information about the age of onset, family history, muscle strength, creatinine phosphokinase (CPK) levels8 and electromyography (EMG). The clinical phenotypes of these patients were classified as either DMD or BMD based on the clinical parameters such as severity of muscle weakness, age of onset of the disease, ambulation and the age at which patients became wheelchair-bound.9,10 Boys having an early age of onset and becoming wheelchair-bound at an early stage were grouped as DMD, while boys showing late onset of both phenotypes were grouped as BMD. DNA extraction, multiplex PCR and border-type analysis was done as described earlier. 7,11

RESULTS

Dystrophin gene deletions were observed in 196 of the 280 D/BMD. However, definite border-type analysis could be performed only in 166 patients, 147 had DMD and 19 had BMD [Table-1]. In the remaining 30 patients, border typing was not performed because the exact extent of gene deletions could not be determined. Deletions with similar borders at 5' and 3' ends, e.g., 1/1, 2/2, 3/3 were referred to as inframe deletions and those with dissimilar borders, e.g., 1/2, 2/1, 1/3, 3/1, 2/3, 3/2 were classified as frame-shift deletions.[11] The majority of BMD patients had an uniform pattern of gene deletions starting from exon 45 but the 3' extent varied from exon 46 to 52 [Figure-1]. All these BMD patients had inframe deletions except 2 who had out-of-frame deletions. Only 100 out of 147 DMD patients showed out-of-frame deletions but the remaining 47 maintained the reading frame of the dystrophin gene. The deletion of exons 45-46, 45-47, 45-48, 45-51, 45-53, 48-50 did not disrupt the frame but caused both BMD and DMD. Similarly, the deletion of exons 28, 44-45, 44-53, 47, 47-50, 47-51, 47-48 and 48-51 also gave DMD-like phenotype in spite of having inframe deletion.

DISCUSSION

Deviation from the reading frame hypothesis accounts for 10-30% of the D/BMD patients. Earlier it was believed that the majority of the deviations took place in patients harboring intragenic deletions in the proximal hotspot region of the dystrophin gene. This region codes for the actin-binding domain of dystrophin, which is critical for its functioning as an actin-binding cytoskeleton protein.[12],[13],[14],[15] However, in our case the deletion frequency in the proximal region accounts for around 15% of the patient population. Instead, the majority of deletions (80%) took place in the central hotspot region of the dystrophin gene, which codes for the rod domain consisting of spectrin-like repeat sequences. They have a structural role in the protein. Disease severity due to the deletion of the proximal rod portion may be very mild or even asymptomatic because very few deletions have been observed in the proximal region.[16] The large dystrophin gene also contains several downstream promoters, which along with alternate splicing can result in varied isoforms.[17] Splicing mutations in the dystrophin gene can result from small changes in the exon-intron boundary sequences. An intraexonal small mutation also induces exon skipping due to the disruption of exon recognition sequences that are necessary for the splicing of the upstream intron.[18] In our studies, we came across exceptional cases where patients showed DMD-like phenotype in spite of maintaining the reading frame. This may be due to exon skipping or deletion at the exon-intron boundaries. There may be a possibility of point mutation in addition to gross deletions that cannot be picked up by multiplex PCR.

In some cases we also observed frame shift mutations giving rise to a less severe form of the disease (BMD). This might be due to the over expression of the utrophin protein, an autosomal homologue of dystrophin.[19] It has been reported that the production of utrophin is significantly increased in the muscles of dystrophin-deficient mice and in boys with D/BMD.[20] This indicates that there is a natural attempt to produce utrophin by dystrophin-deficient muscles in order to compensate for dystrophin deficiency.[21] Therefore, some deviations from the reading frame hypothesis may be due to utrophin compensation in the muscle.

In conclusion, it appears that the reading frame hypothesis in its present form is not sufficient to explain the two allelic forms of D/BMD and there is a need to reevaluate the hypothesis.

A large number of D/BMD patients showing exceptions to the reading frame hypothesis highlight the difficulties in predicting the clinical progression of the disease based only on the DNA profile. For molecular diagnosis of D/BMD it is important to analyze not only genomic DNA, but also mRNA and its protein.

REFERENCES

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