Genetic polymorphism in muscle biopsies of Duchenne and Becker muscular dystrophy patients.
Duchenne muscular dystrophy (DMD), with an incidence of one in 3500 male new borns, and its milder variant, Becker muscular dystrophy (BMD), are allelic X-linked recessive disorders, caused by mutations in the gene coding for dystrophin, a 427 kD cytoskeleton protein. There are no available molecular markers to differentiate these two. The purpose of this study was to study genetic polymorphism in muscular dystrophy and explore its potential in discriminating these two allelic forms of the disease. The results revealed unambiguously the presence of three transcripts : 598bp, 849bp and 1583bp long which are selectively expressed in the muscles afflicted with muscular dystrophy as compared to the normal muscle. 1583bp gene transcript was conspicuously present in the muscle tissues of both DMD and BMD patients whereas 598bp and 849bp long transcripts were exclusively present in DMD but not in BMD patients or normal human subjects. These gene transcripts had no sequence homology with dystrophin gene and these were also present in the families belonging to DMD and BMD patients. These results point to the fact that based upon the selective expression of these three gene transcripts, one could not only differentiate between DMD and BMD diseases at the molecular level, but also between normal and dystrophic muscle. Further, these findings also reveal that apart from dystrophin gene, these gene transcripts may also be responsible for the differential progression of DMD/BMD phenotype.
Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most commonly inherited muscular dystrophies. DMD, with an incidence of one in 3500 live male births, and its milder variant, BMD with an incidence of 7 in 100,000 live male births, are allelic X-linked recessive disorders, caused by mutations in the gene coding for dystrophin, a 427 kD cytoskeleton protein. The clinical spectrum of Xp.21 linked muscular dystrophy varies from severe fatal DMD, where the progressive loss of muscle function results in wheel chair dependence from about 7 years and death before 20 years, to mild BMD where a wheel chair may never be needed and a normal life span is frequently attained with 20%-30% of the affected boys having some degree of mental impairment.
These mutations are essentially heterogeneous in nature and bear no correlation between their position/size and severity in phenotype. These have been detected in the 2300 bp dystrophin gene1 located at Xp.21 of X chromosome with DMD exhibiting complete loss and BMD showing abnormal or reduced production of dystrophin protein. There have been a few reported cases of females having similar phenotype. The exact physiological role of this 3685 amino acid long protein is unknown. It is believed to act in association with other sarcolemmal glycoproteins (eg. dystroglycan) and acts as trans sarcolemmal linker between the sub sarcolemmal cytoskeleton and the extracellular matrix. It may also act as receptor or an ion channel. Dystrophin is found localized in membranes of cardiac cell, neurons and smooth muscle cells. Detection of mutations has been possible using cDNA probes, multiplex PCR, dystrophin staining, single strand conformation polymorphism (SSCP), heterduplex analysis, amplified fragment length polymorphism (AFLP) etc. All these studies have, however, been limited to the DMD locus while completely ignoring the part played by other genes in the progression of this disease.
Although it is well known that both DMD and BMD are caused by defects in the gene on the `X' chromosome located at Xp.21, the precise molecular mechanism by which the reported abnormalities of dystrophin lead to altered muscle fiber degeneration has not been clarified. There have been reports of detection of `inframe transcripts' responsible for frame restoring mechanism in frame shift mutations in BMD patients, resulting in milder phenotype. It is also known that DMD introns are unusually big (200kb) and remarkably conserved over the evolutionary scale. The additional transcritpion units are speculated to exist flanking these regions. The glaring gap in the inability to differentiate DMD from BMD at DNA level raised the issue about the generality of need to identify and characterize the differentially expressed gene transcripts in DMD/BMD patients and prompted us to undertake such a study to help relate genotype to its phenotype and assess its usefulness as a diagnostic tool, apart from exploring its role in altered muscle fiber degeneration.
There is a general agreement to the fact that the course of normal development as well as the pathological changes which accompany degenerative diseases, whether caused by a single gene mutation or a complex of multigene defects, are driven by selective changes in gene expression. Selective gene expression determines all life processes such as development and differentiation, homeostasis, response to insults, ageing and even apoptosis. Consequently, it became imperative to identify and isolate those genes that are differentially expressed in diseased states in order to understand the molecular processes responsible for genetic disease such as DMD and BMD.
For this, the available RAP-PCR technique (RNA arbitrarily primed PCR) was exploited to explore the existence of genes/gene transcripts (other than dystrophin) which are selectively expressed in DMD/BMD states. It followed the realization that a comprehensive, broader and objective approach towards the progression of the disease would be more befitting the understanding of the problem than restricting ourselves to DMD locus.
Work plan : The patients were selected as per the clinical characteristics, electrophysiological abnormalities and histological findings.
14 patients with DMD and 9 patients of BMD were studied in addition to 8 control cases (accident victims). One family of each DMD and BMD including at least two members of the each family were studied. Informed consent from each of the patients/normal human subjects studied, was taken. Muscle from normal healthy individuals (bereft of any muscle disease) involved in accidents and undergoing surgery was taken as control. Muscle biopsy was performed on quadriceps (vastus lateralis) at the junction of upper 2/3rd and lower 1/3rd from both control as well as DMD and BMD patients.
RNA-Dependent differential gene expression
a) RNA preparation : Muscle tissue was either stored in liquid Nitrogen or used fresh. About 1g of tissue was washed in normal saline and minced with sterile blade. It was then homogenized separately in solution D containing 4M Guanidium isothiocyanate, 25 mM sodium citrate (pH7) 0.51% sarkosyl and 0.1M 2 mercaptoethanol providing the necessary RNase inactivation. RNA was isolated using guanidium isothiocyanate method. mRNA was isolated using Qiagen's mRNA isolation kit. Isolated mRNA was quantitated spectrophotometrically in RNase free cuvettes separately for each sample. A260/A280 > 2.0denotes pure mRNA. Quantitation of mRNA was done by taking the absorbance at 260nm and O.D. of 1 was considered equal to 40 mg/ ml of RNA in solution.
b) cDNA Synthesis : About 100 ng of isolated mRNA from each sample was used as the starting template for complementary first strand synthesis. For this, two separate 25 æM arbitrary primers were used. The same procedure was simultaneously followed for the control reaction where instead of arbitrary primer stock, oligo d(T) primer stock (1æl of 85æM) was used for subsequent RT-PCR. The sequence of arbitrary primers used was :
A1 = 5'- AATCTA GAGCTCCT CCTC-3'
A2 = 5'- AATCTA GAGCTCCA GCAG-3'
c) PCR amplification of differentially expressed gene transcripts : Pre-PCR mix was prepared separately for each amplification reaction containing 10xTaq reaction buffer, 25mM MgCl2, 25mM each NTP, (æp), dATP (10 æCi/æl), Taq. polymerase (5U/ml), 25mM A1+A2primers. 1:10 diluted primed cDNA obtained from previous step. One cycle of low stringency amplification of cDNA and about 40 cycles for high stringency amplification were performed in each tube for best comparisons to be made. Low stringency cycle had 94oC for 1 min, 36oC for 5 min,and 72oC for 5 min followed by high stringency cycle constituting 94oC for 1 min, 52oC for 2 min and 72oC for 2 min followed by 10 min extension for 72oC.
A similar procedure was adopted for RT-PCR (control reaction) but instead of arbitrary primers, the control primer set for b actin gene was used. The PCR cycle was as follows : 94oC for 5 min, 60oC for 5 min, 72oC for 2 min (40 cycles) 94oC for 1 min, 60oC for 1 min followed by 10 min extension at 72oC. No radiolabelled oligos were used.
d) Electrophoresis of amplified PCR products : About 3æl of each sample was run in neighbouring lanes in-4% acrylamide -7 M urea sequencing gel prepared in 1 x TBE buffer. The gel was run at 400V and 65mA until xylene cyanol migrated at or near the bottom of the gel. For the control sample, about 12æl of amplified sample was removed from below the mineral oil and mixed with about 3æl of loading dye containing Bromophenol blue and Xylene cyanol. The sample was loaded in 3% agarose gel containing 1mg/ml of ethidium bromide and electrophoresed at 13OV. 'x 174/Hae III mol. wt. markers were run in parallel. Such control was performed for each set of PCR amplifications.
e) Gel Drying and autoradiography : The gel was removed carefully and vaccum dried on the gel drier at 85OC for 1" hours. Subsequently, it was exposed to Kodak X-OMAT AR film for three days at 20OC and kept at minus 20OC. The film was developed in the automatic developing machine and the gel patterns subjected to densitometric analysis.
f) Densitometric Analysis : All autoradiographs were subjected to computer aided densitometric analysis for each lane to identify differentially expressed gene transcripts in DMD/BMD patients and normal subjects. The densitometric scan was also exploited to identify the differentially expressed gene transcripts in the families of DMD/BMD patients. The corresponding intensity profile of the scanned electrophoretic bands was obtained.
g) Detection of dystrophin transcripts : Probe labelling and hybridization : Oligonucleotide hybridization is a procedure whereby a P- end - labelled probe hybridizes in solution to one strand of the amplified product.
The following DNA probes were used to perform dystrophin probing : -
The oligonucleotide probes were end labelled with [32P] ATP using polynucleotide kinase.8 The end labelled oligonucleotide were separated from incorporated ATP by spin column dialysis with G25 Sephadex. The probe was then annealed to target the sequence at 57oC for 15 minutes and electrophoresed in sequencing gel vaccum dried and subjected to autoradiograhy, as explained before. For this, each probe (25p mol.) was labelled using poly nucleotide kinase. The reaction was then stopped at 75oC for 10 minutes. The probe was then annealed to target the sequences of amplified DNA of DMD/BMD and normal human subjects at 57oC for 15 min. The dried gel was exposed to the X ray film and incubated it at - 20oC for 3 days. The hybridized sequence showed a band while the non hybridized sequence did not.
The study, addressed to understand the genetic polymorphism between muscle tissues derived from normal human subjects, Duchenne muscular dystrophy (DMD), and Becker's muscular dystrophy (BMD) patients, revealed following results :
1. A number of fingerprints corresponding to various RNA transcripts derived from normal human subjects, DMD, BMD and the families were detected [Figure 1] [Figure 2]
2. Examination of genetic polymorphism of RNA transcripts derived from normal human subjects, DMD and BMD revealed that gene transcripts about 598 bp, 849 bp and 1583 bp in size were conspicuously and selectively absent in gene transcripts from normal human subjects[Figure 3]. However, 1583 bp gene transcript was present in the muscle tissue derived from DMD and BMD patients [Figure 3].
These results unambiguously revealed that 1583 bp gene transcript is expressed only in DMD and BMD patients but not in normal human subjects whereas 849 bp and 598 bp gene transcripts are expressed only in DMD but not in either BMD or normal human subjects [Figure 3]. In order to ascertain whether or not these differentially expressed gene transcripts are reflected in the families of DMD and BMD patients, we studied the genetic polymorphism in these families as well. The results revealed that 1583 bp, 849 bp, 598 bp gene transcripts are also expressed in the families corresponding to DMD and BMD [Figure 4] while only 1583 bp gene transcript is expressed in BMD son and his mother [Figure 5]. However, in the families also, 1583 bp gene transcript was observed in both DMD and BMD patients and their respective mothers [Figure 4] [Figure 5] whereas 849 bp and 598 bp gene transcripts were morphism in these families as well. The results revealed that 1583 bp, 849 bp, 598 bp gene transcripts are also expressed in the families corresponding to DMD and BMD [Figure 4] while only 1583 bp gene transcript is expressed in BMD son and his mother [Figure 5]. However, in the families also, 1583 bp gene transcript was observed in both DMD and BMD patients and their respective mothers [Figure 4] [Figure 5] whereas 849 bp and 598 bp gene transcripts were only present in DMD but not in BMD son and his mother.
In order to explore whether or not the differentially expressed gene transcripts (such as 1583 bp, 849 bp and 598 bp) were identical to the dystrophin gene, studies were carried out by using various dystrophin DNA probes. The differentially expressed gene transcripts as identified in the present study had no sequence homology to the dystrophin gene indicating thereby that these differentially expressed gene transcripts belong to a separate set of genes specific to DMD and BMD states [Figure 6].
Although incontrovertible evidence suggests that Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by defects in dystrophin gene on the X chromosome located at Xp.21, the molecular mechanism responsible for this type of genetic aberration leading to DMD/BMD disease is far from clear.
The selective expression of genes is a key regulatory mechanism that controls biological processes. Depending upon the nature of the process, the spectrum of genes that a cell may express is subject to great variability. Comparison of gene expression in different types of cells can provide information about the signals that regulate normal cellular physiology. In diseased states, identification of selectively expressed genes may provide insight into how normal cellular responses are subverted. Therefore, RNA arbitrarily primed polymerase chain reaction (RAP-PCR) provides a technique allowing easy identification of differentially expressed genes having inherent capacity to yield information relating gene function with structure. Hence, in the present study, this technology was exploited to explore the existence of genes (other than dystrophin) which are selectively expressed in DMD/BMD states.
The results reported here unambiguously revealed the presence of three gene transcripts (598bp, 849bp and 1583bp) which are selectively expressed in the muscles afflicted with muscular dystrophy as compared to the normal muscle. 1583bp gene transcript was conspicuously present in the muscle tissues of both DMD and BMD patients whereas 849bp and 598bp was exclusively present in DMD but not in BMD or normal human subjects. These results point to the fact that based upon the selective expression of these three gene transcripts, one could not only differentiate between DMD and BMD diseases at the molecular level, but also between normal and muscular dystrophy [Table I]. These findings were further confirmed when the families belonging to DMD and BMD patients were examined for differential gene expression. 1583bp gene transcript was present only in the patients and their mothers but not in their fathers [Table II] [Table III]. Further, 598bp and 849bp gene transcripts were present again in patients and their mothers as well as exclusively present in DMD patient's family but not in BMD patient's family [Table II] [Table III]. They may have a crucial role in the pathogenesis of DMD and BMD. Further, studies were carried out in order to ascertain whether or not these three gene transcripts have any sequence resemblance with that of the dystrophin gene. Such studies did not reveal any relationship whatsoever [Figure 6]. This view is in conformity with the observation that all the three gene transcripts (598bp, 849bp and 1583bp) are expressed in the muscles of DMD patient which are known to lack the expression of dystrophin gene.
Based upon these results we propose that these three gene transcripts may have not only a crucial role in the initiation of DMD and BMD disease but also may have inherent capacity to act as a diagnostic marker for distinguishing DMD from BMD as well as muscular dystrophy from Normal human subjects. The findings reported here also open up the possibility that, other than dystrophin gene, there may be other genes responsible for DMD and BMD. However, one can not speculate at the moment as to how these three gene transcripts affect the dystrophin gene or initiate the disease process leading to DMD or BMD. More work is needed in order to explore this possibility.
We thank Department of Biotechnology, Govt of India for partial financial support, Dr. Sahid Jameel from ICGEB New Delhi for providing important reagants and Dr. Sher Ali from NII, New Delhi for his generous help in doing the densitometric scan for autoradiographs and Karunesh for computer aided calculations.