© 1992 Oxford University Press
Human Molecular Genetics, Vol. 1, No. 8
645-646
Identification of a 2 base pair nonsense mutation causing a cryptic splice site in a DMD patient A.V.Winnard2, Y.Jia-Hsu4 R.A.Gibbs4, J.R.Mendell1 and A.H.M.Burghes1-23* department of Neurology, department of Medical Biochemistry, College of Medicine, 3Department of Molecular Genetics, College of Biological Sciences, The Ohio State University, Columbus, OH and 4lnstitute for Molecular Genetics, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX, USA Received July 27, 1992; Revised and Accepted September 10, 1992
Sequence analysis of the lower molecular weight band indicated that the mutation creates a new acceptor site 2 bp 3' of the mutation causing the 3' end of exon 50 to be spliced into the middle of exon 51 (Figure 2C and D). The mutation alters the sequence from ATG TTCGAG GTA-ATG TTA TAG/GTA which conforms to the 3' acceptor sequence (Py)n N Py AG/G. This mutation creates an out-of-frame messenger RNA and therefore cannot account for the dystrophin of restored reading frame produced in the revertant fibers. It does, however, demonstrate how a change of only one base can alter splicing within exons. A mechanism such as this could account for the occurrence of some revertant fibers.
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Figure 1. Lane 1 = molecular weight marker, 2 = blank control, 3 = normal control, 4 = patient #71, 5 = DMD patient. RT-PCR was performed on muscle tissue mRNA/cDNA between bp 6970 and 7834 (exons 47-52) using primers 5' TTA CTG CTG GAA CAG TTG CCC CTG 3' and 5' GCT GGT CTT GTT TTT CAA ATT TTG GGC 3'. The PCR conditions were 2 - 5 /tl of cDNA in 50 mM KC1, 10 mM Tris (pH 8.3), 2.5 mM MgCl2, 0.01 % v/v Triton X-100, 0.01% v/v Tween-20, 0.01% w/v gelatin, 0.2 mM each dNTP, 10-30 pmol each primer and 1 unit Taq polymerase) Amplitaq, Perkin Elmer, Norwalk, CT): 94°C, 3 min: 45 step cycles of 94°C, 30 sec; 52°C, 45 sec; 72°C, 4 min, 30 sec: final 72°C, 7 min in Perkin Elmer Thermocycler 480. 25 iA were run on a 1% low melt TBE agarose gel.
*To whom correspondence should be addressed at: Department of Neurology, 452 Means Hall, 1654 Upham Drive, The Ohio State University, Columbus, OH 43210, USA
Downloaded from http://hmg.oxfordjournals.org/ at Russian Archive on December 17, 2013
Duchenne Muscular Dystrophy (DMD) patients are characteristically deficient for the DMD gene product dystrophin (1, 2). However, approximately 50% of patients show rare positive dystrophin staining fibers termed revertants (3, 4, 5, 6, 7). Immunohistochemical staining reveals the presence of these dystrophin positive fibers in deletion and non-deletion cases. The revertant fibers in deletion patients stain with amino and carboxyl antibodies but not with antibodies corresponding to the deleted epitope. In addition, the revertant fibers in some non-deletion cases stain with amino and carboxyl antibodies but do not stain with antibodies recognizing amino acids 2305—2554. This staining would indicate the presence of dystrophin of a restored reading frame. As DMD patients are typically out-of-frame, the reversion should by-pass the original mutation either by a deletion or an alteration of splicing. If the original mutation was a nonsense mutation or a small out-of-frame mutation, a secondary in-frame deletion occurring in the revertant fibers that deletes the original mutation could produce a truncated dystrophin protein. This truncated protein would not stain with antibodies corresponding to this deleted region. We examined one of these non-deletion patients (#71) with revertant fibers which stained positive for amino and carboxyl dystrophin antibodies but did not stain with the antibody corresponding to amino acids 2305 -2554 (7). This would indicate that the position of the mutation in this patient lies in or near the region of amino acids 2305-2554. Therefore, we analyzed this region using reverse transcription (RT)-PCR from muscle tissue RNA and direct sequencing. We found a 2bp G G - A T (TTG GAG-TTA TAG: Leu Glu-Leu STOP) nonsense mutation at base pair 7609 and 7610 in exon 51 (figure 2B). PCR amplification of the mRNA consistently demonstrated that 2 messages were present in this patient, one of the expected molecular weight and one of a slightly smaller molecular weight (Figure 1). Sequence analysis of the expected molecular weight band showed the 2 bp mutation and a normal splicing pattern (data not shown) (8). This 2 bp mutation was confirmed at the DNA level by PCR amplification of DNA isolated from muscle tissue (data not shown) and from lymphocytes (Figure 2B). Lymphocyte DNA from the patient's mother (#491) was analyzed using PCR, ASO analysis, and direct sequencing (Figure 2A). She is not a carrier of either a 1 bp polymorphism or a 1 or 2 bp mutation. Using PCR amplification of exon 51 and ASO analysis (data not shown), we screened 92 non- deleted DMD patients for the mutation. None of these patients were positive for the mutation, indicating that it is not a common mutation.
646 Human Molecular Genetics, Vol. 1, No. 8
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Figure 2. Panels A and B: Direct sequencing of PCR products amplified with an M13 tailed primer corresponding to bp 7534 in exon 51 and an intron primer between exons 51 and 52 (5' TGT AAA CGA ACG GCC AGT ACT CTG GTG AC A CAA CCT CTG 3' and 5' GGA GAG TAA AGT GAT TGG TGG AAA ATC 3') using the conditions listed in Figure 1. Direct sequencing was performed by first purifying the PCR products using Magic PCR preps (Promega, Madison, WI) according to manufacturer's instruction and then sequencing with 10 pmol of either the 32P end labeled sense primer or M13(-20) primer using the ,/mo/ DNA Seguencing System (Promega, Madison, WI) according to manufacturer's instructions. Asterisk indicate the 2 bp change. Panel A = #491, patient's mother. Panel B = #71, DMD patient. Panels C and D: Direct sequencing of PCR products in Figure 1. C = the sequence of the PCR product from lane 3, Figure 1. D = the sequence of the lower molecular weight band in lane 4, Figure 1. To separate the 2 PCR products in lane 4, Figure 1, the PCR products were purified 2 times over a 4.5% native acrylamide gel, band isolated and reamplified using the conditions in Figure 1. Sequencing was carried out as described for panels A and B, except that the sequencing primer was an internal primer corresponding to bp 7474 (5' AAG CAG CCT GAC CTA GCT CCT GGA 3')- Arrows indicate the exon 50/51 boundaries.
In conclusion, we used revertant fiber analysis to localize the mutation site and RT-PCR and sequencing to identify a novel 2 bp stop mutation in a DMD patient. ACKNOWLEDGEMENTS We wish to thank Thomas Prior for supplying a panel of non-deletion DMD patients for this study. This work was supported by the Muscular Dystrophy Association and by National Institutes of Health, grant R29-AR40015-01. A.V.W. is a recipient of a Proctor and Gamble Graduate Fellowship award.
REFERENCES 1. Hoffman,E.P., Brown,R.H.,Jr and Kunkel.L.M. (1987) Cell 51, 919-928. 2. Zubrzycka-Gaarn.E.E., Bulman.D.E., Karpati,G., Burghes.A.H.M., Belfall,M., Klamut.H.J., Talbot,J., et al. (1988) Nature 333, 466-469. 3. Shimizu,T.K., Matsumura.K., Hashimoto.K., Mannen,T., Ishigure,T., Eguchi,C, Nonaka,I., et al. (1988) Proc. Jpn. Acad. 64, 205-208. 4. Nicholson,L.V.B., Davidson.K., Johnson,M.A., Slater,M., Young.C, Battacharya.S. and Gardner-Medwin.D. (1989)7. Neurol. Sci. 94, 137-146. 5. Hoffman.E.P., Morgan.J.E., Watkins,S.C, and Partridge.T.A. (1990) J. Neurol. Sci. 99, 9—5. 6. Burrow,K., Coovert.D.D., Klein.C.J., Bulman,D.E., KisselJ.T., Rammohan.K.W., Burghes.A.H.M. etal. (1991) Neurology 4\, 662-666. 7. Klein.C.J., Coovert,D.D., Bulman,D.E., Ray.P.N., Mendell.J.R. and Burghes.A.H.M. (1992) Am. J. Hum. Genet. 50, 950-959. 8. Gibbs.R.A., Nguyen.P.N., Edwards.A., Civitello,A.B. and Caskey,C.T. (1990) Genomics 7, 235-244.
Downloaded from http://hmg.oxfordjournals.org/ at Russian Archive on December 17, 2013
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Human Molecular Genetics, Vol. 1, No. 8
645-646
Identification of a 2 base pair nonsense mutation causing a cryptic splice site in a DMD patient A.V.Winnard2, Y.Jia-Hsu4 R.A.Gibbs4, J.R.Mendell1 and A.H.M.Burghes1-23* department of Neurology, department of Medical Biochemistry, College of Medicine, 3Department of Molecular Genetics, College of Biological Sciences, The Ohio State University, Columbus, OH and 4lnstitute for Molecular Genetics, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX, USA Received July 27, 1992; Revised and Accepted September 10, 1992
Sequence analysis of the lower molecular weight band indicated that the mutation creates a new acceptor site 2 bp 3' of the mutation causing the 3' end of exon 50 to be spliced into the middle of exon 51 (Figure 2C and D). The mutation alters the sequence from ATG TTCGAG GTA-ATG TTA TAG/GTA which conforms to the 3' acceptor sequence (Py)n N Py AG/G. This mutation creates an out-of-frame messenger RNA and therefore cannot account for the dystrophin of restored reading frame produced in the revertant fibers. It does, however, demonstrate how a change of only one base can alter splicing within exons. A mechanism such as this could account for the occurrence of some revertant fibers.
12
3
4 5
Figure 1. Lane 1 = molecular weight marker, 2 = blank control, 3 = normal control, 4 = patient #71, 5 = DMD patient. RT-PCR was performed on muscle tissue mRNA/cDNA between bp 6970 and 7834 (exons 47-52) using primers 5' TTA CTG CTG GAA CAG TTG CCC CTG 3' and 5' GCT GGT CTT GTT TTT CAA ATT TTG GGC 3'. The PCR conditions were 2 - 5 /tl of cDNA in 50 mM KC1, 10 mM Tris (pH 8.3), 2.5 mM MgCl2, 0.01 % v/v Triton X-100, 0.01% v/v Tween-20, 0.01% w/v gelatin, 0.2 mM each dNTP, 10-30 pmol each primer and 1 unit Taq polymerase) Amplitaq, Perkin Elmer, Norwalk, CT): 94°C, 3 min: 45 step cycles of 94°C, 30 sec; 52°C, 45 sec; 72°C, 4 min, 30 sec: final 72°C, 7 min in Perkin Elmer Thermocycler 480. 25 iA were run on a 1% low melt TBE agarose gel.
*To whom correspondence should be addressed at: Department of Neurology, 452 Means Hall, 1654 Upham Drive, The Ohio State University, Columbus, OH 43210, USA
Downloaded from http://hmg.oxfordjournals.org/ at Russian Archive on December 17, 2013
Duchenne Muscular Dystrophy (DMD) patients are characteristically deficient for the DMD gene product dystrophin (1, 2). However, approximately 50% of patients show rare positive dystrophin staining fibers termed revertants (3, 4, 5, 6, 7). Immunohistochemical staining reveals the presence of these dystrophin positive fibers in deletion and non-deletion cases. The revertant fibers in deletion patients stain with amino and carboxyl antibodies but not with antibodies corresponding to the deleted epitope. In addition, the revertant fibers in some non-deletion cases stain with amino and carboxyl antibodies but do not stain with antibodies recognizing amino acids 2305—2554. This staining would indicate the presence of dystrophin of a restored reading frame. As DMD patients are typically out-of-frame, the reversion should by-pass the original mutation either by a deletion or an alteration of splicing. If the original mutation was a nonsense mutation or a small out-of-frame mutation, a secondary in-frame deletion occurring in the revertant fibers that deletes the original mutation could produce a truncated dystrophin protein. This truncated protein would not stain with antibodies corresponding to this deleted region. We examined one of these non-deletion patients (#71) with revertant fibers which stained positive for amino and carboxyl dystrophin antibodies but did not stain with the antibody corresponding to amino acids 2305 -2554 (7). This would indicate that the position of the mutation in this patient lies in or near the region of amino acids 2305-2554. Therefore, we analyzed this region using reverse transcription (RT)-PCR from muscle tissue RNA and direct sequencing. We found a 2bp G G - A T (TTG GAG-TTA TAG: Leu Glu-Leu STOP) nonsense mutation at base pair 7609 and 7610 in exon 51 (figure 2B). PCR amplification of the mRNA consistently demonstrated that 2 messages were present in this patient, one of the expected molecular weight and one of a slightly smaller molecular weight (Figure 1). Sequence analysis of the expected molecular weight band showed the 2 bp mutation and a normal splicing pattern (data not shown) (8). This 2 bp mutation was confirmed at the DNA level by PCR amplification of DNA isolated from muscle tissue (data not shown) and from lymphocytes (Figure 2B). Lymphocyte DNA from the patient's mother (#491) was analyzed using PCR, ASO analysis, and direct sequencing (Figure 2A). She is not a carrier of either a 1 bp polymorphism or a 1 or 2 bp mutation. Using PCR amplification of exon 51 and ASO analysis (data not shown), we screened 92 non- deleted DMD patients for the mutation. None of these patients were positive for the mutation, indicating that it is not a common mutation.
646 Human Molecular Genetics, Vol. 1, No. 8
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Figure 2. Panels A and B: Direct sequencing of PCR products amplified with an M13 tailed primer corresponding to bp 7534 in exon 51 and an intron primer between exons 51 and 52 (5' TGT AAA CGA ACG GCC AGT ACT CTG GTG AC A CAA CCT CTG 3' and 5' GGA GAG TAA AGT GAT TGG TGG AAA ATC 3') using the conditions listed in Figure 1. Direct sequencing was performed by first purifying the PCR products using Magic PCR preps (Promega, Madison, WI) according to manufacturer's instruction and then sequencing with 10 pmol of either the 32P end labeled sense primer or M13(-20) primer using the ,/mo/ DNA Seguencing System (Promega, Madison, WI) according to manufacturer's instructions. Asterisk indicate the 2 bp change. Panel A = #491, patient's mother. Panel B = #71, DMD patient. Panels C and D: Direct sequencing of PCR products in Figure 1. C = the sequence of the PCR product from lane 3, Figure 1. D = the sequence of the lower molecular weight band in lane 4, Figure 1. To separate the 2 PCR products in lane 4, Figure 1, the PCR products were purified 2 times over a 4.5% native acrylamide gel, band isolated and reamplified using the conditions in Figure 1. Sequencing was carried out as described for panels A and B, except that the sequencing primer was an internal primer corresponding to bp 7474 (5' AAG CAG CCT GAC CTA GCT CCT GGA 3')- Arrows indicate the exon 50/51 boundaries.
In conclusion, we used revertant fiber analysis to localize the mutation site and RT-PCR and sequencing to identify a novel 2 bp stop mutation in a DMD patient. ACKNOWLEDGEMENTS We wish to thank Thomas Prior for supplying a panel of non-deletion DMD patients for this study. This work was supported by the Muscular Dystrophy Association and by National Institutes of Health, grant R29-AR40015-01. A.V.W. is a recipient of a Proctor and Gamble Graduate Fellowship award.
REFERENCES 1. Hoffman,E.P., Brown,R.H.,Jr and Kunkel.L.M. (1987) Cell 51, 919-928. 2. Zubrzycka-Gaarn.E.E., Bulman.D.E., Karpati,G., Burghes.A.H.M., Belfall,M., Klamut.H.J., Talbot,J., et al. (1988) Nature 333, 466-469. 3. Shimizu,T.K., Matsumura.K., Hashimoto.K., Mannen,T., Ishigure,T., Eguchi,C, Nonaka,I., et al. (1988) Proc. Jpn. Acad. 64, 205-208. 4. Nicholson,L.V.B., Davidson.K., Johnson,M.A., Slater,M., Young.C, Battacharya.S. and Gardner-Medwin.D. (1989)7. Neurol. Sci. 94, 137-146. 5. Hoffman.E.P., Morgan.J.E., Watkins,S.C, and Partridge.T.A. (1990) J. Neurol. Sci. 99, 9—5. 6. Burrow,K., Coovert.D.D., Klein.C.J., Bulman,D.E., KisselJ.T., Rammohan.K.W., Burghes.A.H.M. etal. (1991) Neurology 4\, 662-666. 7. Klein.C.J., Coovert,D.D., Bulman,D.E., Ray.P.N., Mendell.J.R. and Burghes.A.H.M. (1992) Am. J. Hum. Genet. 50, 950-959. 8. Gibbs.R.A., Nguyen.P.N., Edwards.A., Civitello,A.B. and Caskey,C.T. (1990) Genomics 7, 235-244.
Downloaded from http://hmg.oxfordjournals.org/ at Russian Archive on December 17, 2013
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