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BMD
SI1
BIS2
BIS3
NON-MUSCLE DYSTROPHIN ISOFORMS: PROTEINS IN S E A R C H O F A F U N C T I O N
DIAGNOSTICS AND CARRIER TESTING IN DMD/BMD E. Bakker, A.J.L. Kneppers, P.A.M. Roest, M. Bout, F.B.L. Hogervorst, J.T. den Dunnen) G.J.B. van Ommen. Department of Human Genetics, Leiden University, The Netherlands.
Comi G.P., Robotti M., Salani S., Ricci C., Rigoletto C., Ausenda CD., Scarlato G. Institute of Clistical Neurology, University of Milan, Italy. The 2.3 Mbp dystrophin gene is transcribed in striated and smooth muscle as a 14 Kb mRNA coding for a 427 KDa protein, whose localization ath the sarcolemmal membrane and interaction with the components of the glycoprotein complex, extracellular matrix mad cellular cytoskeleton are well defined. The dystrophin role in preserving muscle integrity is relatively understood although fine meclmnismsleading to cell necrosis are still debated. Tiffs complex gene codes for at least six other proteins, beside that of muscle. Their expression may be tissue or developmentally regadated by splicing mechanisms. For some of these proteina candidate functions have been proposed such as a cognitive role for brain 427 and 140 Kda isoforms and retinal transmission for Dp260. Other proteins having a more diffuse tissue distribution such as DpT1 or one restricted to Schwmm cells and olfattory bulb (Dp116), definite insights on physiological role or pathological results of their absence have yet to be defined. Current knowledge in this field will be reviewed.
BIS5
BIS4 MOLKCULAR SCANNING MUTATIONS IN DMD/BMD
Since 1985, when we p e r f o r m e d the first p r e n a t a l diagnosis for DMD, molecular genetic diagnostic tests for D u c h e n n e and B e c k e r m u s c u l a r d y s t r o p h y have g r e a t l y evolved. The dystrophin gene was cloned in 1986. M u t a t i o n s are now d e t e c t e d in a p p r o x i m a t e l y 70% of the patients. In about 65% of the p a t i e n t s rearrangements, deletions or duplications, are detected in the d y s t r o p h i n gene. At present in about 5% of the cases a point mutation is b e i n g detected. In the r e m a i n i n g 30% of the cases baplotype analysis had to be used for carrier testing. For deletion/duplication analysis two Multiplex PCR's are used to s i m u l t a n e o u s l y test 18 exons for a deletion. S o u t h e r n blot analysis is routinely used to confirm the deletion or detect possible duplications and directly y i e l d the total size of the rearrangement. For haplotype analysis m a n y of the available (CA)n repeats in the d y s t r o p h i n gene are used. Point mutation detection is more complicated because the d y s t r o p h i n gene is e x t r e m e l y large, 2.5 Mb, w i t h a coding sequence of 14 kb, divided in 79 exons. All kinds cf m u t a t i o n d e t e c t i o n approaches have been a p p l i e d such as, SSCP on m u l t i p l e x PCR p r o d u c t s of exonic sequences, RT-PCR on lymphocytes followed by a protein (translation) t r u n c a t i o n test to detect frame shifting mutations. In one case a one kb insertion was detected in exon 47, which turned out to be a t r a n s p o s o n - l i k e sequence. R e c e n t l y a m o r e s o p h i s t i c a t e d technique has b e e n u s e d like a MyoD t r a n s f e c t i o n of fibroblasts of (deceased) patients, converting the cells into myoblasts. From these m y o b l a s t s R N A was isolated to p e r f o r m a PT-test. W i t h these later techniques we were able to detect frame shift mutations, amongst which two splice mutants. In total, over the past i0 years we have performed some 1400 carrier tests and 150 prenatal diagnoses. During this period some families were reinvestigated many times with newer markers or techniques until the DMD/BMD m u t a t i o n was d e t e c t e d or h a p l o t y p e analysis gave a reassuring result. The following types of c o m p l i c a t i o n s were e n c o u n t e r e d like, e x t r e m e l y late referrals, nonpaternity, germinal mosaicism, double recombinants, etc.
FOR
POINT
M. Ferrari, A.M. Bmbieri, N. Soriani, P. Carrera. I.R.C.C.S. H S. Raffaele, Lab. Genetiea Molecolam, MILANO About 60% of DMD/BMD patients cany deletions or duplications of entire exons of the gene. Up to now several tec~,h~-;quesfor nmtation detect/on have been employed We studied a total of 50 (33 DMD and 17 BMD) not reananged patients for the taesense of point mutations within the coding region of the dystrophin gene. Heteroduplex Analysis (HA) on genomic DNAs was performedon 37 exons of dystrophin gene with the conesponding exon-intmn boundaries. In addition HA and DGGE analysis was carried out on RT-PCR products of illegitimate tramcrilas; that was very useful allowing us to simultaneously analyse long fragnenls each comaining several exons. H e ~ e x bands c o n e s ~ either to novel variams or Ia'evioualy desorRmd polymosphisms were found in the following exons: 3, 13, 14, 17, 20, 23, 25, 44, 45, 48, 54, 55. Direct sequencing of hetemduplex bands revealed fwe new deleterious mutations: a) a G to A transition in the donor splice site of exon 44 in a DMD patient. Lack of exon 44 was confmned on transcripts, b) a non-sense mutation in exon 25 (Q1102Term) in a BMD patient; c) a frameshifl (C del) in exon 55 in a DMD patient, d) a ~ (GAGA del) in exon 20 in a DMD patient, e) a frameal~ (seven bp insertion for a CAA) in exon 23 in a DMD patient. Except for a) all these mutations result in l~emam~ termination, confirmed as a common featme within dystrophin point mutations.
MUTATION DETECTION BY DOUBLE STRAND CONFORMATION ANALYSIS PROVIDES RAPID MOLECULAR DIAGNOSIS FOR DUCHENNE AND BECKER MUSCULAR DYSTROPHIES Fawzy A. Saad Department of Genetics, Sakha Road ET-33516 Karl" E1 Sheikh, Egypt Approximately 65% of the DMD and BMD patients have intragenic deletions, about 6% of the patients have intragenic duplications, the remaining affected individuals are expected to have point mutations. The possibility of detecting the presence of single base substitutions in PCR amplicons by double strand conformation analysis (DSCA) has been recently reported (Saad et al. NAR 22:4352, 1994). The method is based on the principle that free double-stranded DNA in aqueous solution has an intrinsic curvature which is dependent on its nucleotide sequence. A single base substitution induces a modification of the curvature of the double helix and a consequent variation in the friction encountered by the DNA in a porous gel. This, in turn, should produce an electrophoretic mobility shift in nondenaturing polyacrylamide gels. DSCA has already proved to be able to detect new mutations and polymorphisms in the human dystrophin gene. Since the DSCA can be revealed in elec~'ophoretie separations of multiplex PCR products on polyacrylamide matrices, the same etectrophoretic run may allow the contemporaneous detection of intragenic-deletions, duplications and of point mutations.
BIS2
BIS3
NON-MUSCLE DYSTROPHIN ISOFORMS: PROTEINS IN S E A R C H O F A F U N C T I O N
DIAGNOSTICS AND CARRIER TESTING IN DMD/BMD E. Bakker, A.J.L. Kneppers, P.A.M. Roest, M. Bout, F.B.L. Hogervorst, J.T. den Dunnen) G.J.B. van Ommen. Department of Human Genetics, Leiden University, The Netherlands.
Comi G.P., Robotti M., Salani S., Ricci C., Rigoletto C., Ausenda CD., Scarlato G. Institute of Clistical Neurology, University of Milan, Italy. The 2.3 Mbp dystrophin gene is transcribed in striated and smooth muscle as a 14 Kb mRNA coding for a 427 KDa protein, whose localization ath the sarcolemmal membrane and interaction with the components of the glycoprotein complex, extracellular matrix mad cellular cytoskeleton are well defined. The dystrophin role in preserving muscle integrity is relatively understood although fine meclmnismsleading to cell necrosis are still debated. Tiffs complex gene codes for at least six other proteins, beside that of muscle. Their expression may be tissue or developmentally regadated by splicing mechanisms. For some of these proteina candidate functions have been proposed such as a cognitive role for brain 427 and 140 Kda isoforms and retinal transmission for Dp260. Other proteins having a more diffuse tissue distribution such as DpT1 or one restricted to Schwmm cells and olfattory bulb (Dp116), definite insights on physiological role or pathological results of their absence have yet to be defined. Current knowledge in this field will be reviewed.
BIS5
BIS4 MOLKCULAR SCANNING MUTATIONS IN DMD/BMD
Since 1985, when we p e r f o r m e d the first p r e n a t a l diagnosis for DMD, molecular genetic diagnostic tests for D u c h e n n e and B e c k e r m u s c u l a r d y s t r o p h y have g r e a t l y evolved. The dystrophin gene was cloned in 1986. M u t a t i o n s are now d e t e c t e d in a p p r o x i m a t e l y 70% of the patients. In about 65% of the p a t i e n t s rearrangements, deletions or duplications, are detected in the d y s t r o p h i n gene. At present in about 5% of the cases a point mutation is b e i n g detected. In the r e m a i n i n g 30% of the cases baplotype analysis had to be used for carrier testing. For deletion/duplication analysis two Multiplex PCR's are used to s i m u l t a n e o u s l y test 18 exons for a deletion. S o u t h e r n blot analysis is routinely used to confirm the deletion or detect possible duplications and directly y i e l d the total size of the rearrangement. For haplotype analysis m a n y of the available (CA)n repeats in the d y s t r o p h i n gene are used. Point mutation detection is more complicated because the d y s t r o p h i n gene is e x t r e m e l y large, 2.5 Mb, w i t h a coding sequence of 14 kb, divided in 79 exons. All kinds cf m u t a t i o n d e t e c t i o n approaches have been a p p l i e d such as, SSCP on m u l t i p l e x PCR p r o d u c t s of exonic sequences, RT-PCR on lymphocytes followed by a protein (translation) t r u n c a t i o n test to detect frame shifting mutations. In one case a one kb insertion was detected in exon 47, which turned out to be a t r a n s p o s o n - l i k e sequence. R e c e n t l y a m o r e s o p h i s t i c a t e d technique has b e e n u s e d like a MyoD t r a n s f e c t i o n of fibroblasts of (deceased) patients, converting the cells into myoblasts. From these m y o b l a s t s R N A was isolated to p e r f o r m a PT-test. W i t h these later techniques we were able to detect frame shift mutations, amongst which two splice mutants. In total, over the past i0 years we have performed some 1400 carrier tests and 150 prenatal diagnoses. During this period some families were reinvestigated many times with newer markers or techniques until the DMD/BMD m u t a t i o n was d e t e c t e d or h a p l o t y p e analysis gave a reassuring result. The following types of c o m p l i c a t i o n s were e n c o u n t e r e d like, e x t r e m e l y late referrals, nonpaternity, germinal mosaicism, double recombinants, etc.
FOR
POINT
M. Ferrari, A.M. Bmbieri, N. Soriani, P. Carrera. I.R.C.C.S. H S. Raffaele, Lab. Genetiea Molecolam, MILANO About 60% of DMD/BMD patients cany deletions or duplications of entire exons of the gene. Up to now several tec~,h~-;quesfor nmtation detect/on have been employed We studied a total of 50 (33 DMD and 17 BMD) not reananged patients for the taesense of point mutations within the coding region of the dystrophin gene. Heteroduplex Analysis (HA) on genomic DNAs was performedon 37 exons of dystrophin gene with the conesponding exon-intmn boundaries. In addition HA and DGGE analysis was carried out on RT-PCR products of illegitimate tramcrilas; that was very useful allowing us to simultaneously analyse long fragnenls each comaining several exons. H e ~ e x bands c o n e s ~ either to novel variams or Ia'evioualy desorRmd polymosphisms were found in the following exons: 3, 13, 14, 17, 20, 23, 25, 44, 45, 48, 54, 55. Direct sequencing of hetemduplex bands revealed fwe new deleterious mutations: a) a G to A transition in the donor splice site of exon 44 in a DMD patient. Lack of exon 44 was confmned on transcripts, b) a non-sense mutation in exon 25 (Q1102Term) in a BMD patient; c) a frameshifl (C del) in exon 55 in a DMD patient, d) a ~ (GAGA del) in exon 20 in a DMD patient, e) a frameal~ (seven bp insertion for a CAA) in exon 23 in a DMD patient. Except for a) all these mutations result in l~emam~ termination, confirmed as a common featme within dystrophin point mutations.
MUTATION DETECTION BY DOUBLE STRAND CONFORMATION ANALYSIS PROVIDES RAPID MOLECULAR DIAGNOSIS FOR DUCHENNE AND BECKER MUSCULAR DYSTROPHIES Fawzy A. Saad Department of Genetics, Sakha Road ET-33516 Karl" E1 Sheikh, Egypt Approximately 65% of the DMD and BMD patients have intragenic deletions, about 6% of the patients have intragenic duplications, the remaining affected individuals are expected to have point mutations. The possibility of detecting the presence of single base substitutions in PCR amplicons by double strand conformation analysis (DSCA) has been recently reported (Saad et al. NAR 22:4352, 1994). The method is based on the principle that free double-stranded DNA in aqueous solution has an intrinsic curvature which is dependent on its nucleotide sequence. A single base substitution induces a modification of the curvature of the double helix and a consequent variation in the friction encountered by the DNA in a porous gel. This, in turn, should produce an electrophoretic mobility shift in nondenaturing polyacrylamide gels. DSCA has already proved to be able to detect new mutations and polymorphisms in the human dystrophin gene. Since the DSCA can be revealed in elec~'ophoretie separations of multiplex PCR products on polyacrylamide matrices, the same etectrophoretic run may allow the contemporaneous detection of intragenic-deletions, duplications and of point mutations.