Point Mutations in the Dystrophin Gene ROLAND G

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Proc. Natl. Acad. Sci. USA Vol. 89, pp. 2331-2335, March 1992 Genetics Point mutations in the dystrophin gene ROLAND G. ROBERTS, MARTIN BOBROW, AND DAVID R. BENTLEY Paediatric Research Unit, Division of Medical and Molecular Genetics, United Medical and Dental Schools of Guy's and St. Thomas's, Guy's Campus, London SE1 9RT, United Kingdom Communicated by Renato Dulbecco, December 16, 1991

ABSTRACT Derming the range of mutations in genes that The ability to identify mutations on a routine basis allows cause human disease is essential to determine the mechanisms precise establishment ofcarrier status in affected families and of genetic variation and the function of gene domains and to permits accurate prenatal diagnosis. In families with no gross perform precise carrier and prenatal diagnosis. The mutations dystrophin gene rearrangement, diagnosis of carriers cur- in one-third of Duchenne muscular dystrophy patients remain rently relies on linkage analysis. Due to the high mutation unknown as they do not involve gross rearrangements of the rate, the mutation has often originated within a living family dystrophin gene. The size and complexity of the gene have member; hence relatives carrying a high-risk haplotype may prohibited the systematic definition of point mutations. We not carry the mutation. Furthermore, recombination occurs have developed a method for the identification of these muta- within the dystrophin gene in up to 12% of meioses (9), tions by nested amplification, chemical mismatch detection, necessitating the analysis of flanking markers. Direct diag- and sequencing of reverse transcripts of trace amounts of nosis would not be subject to either of these problems. dystrophin mRNA from peripheral blood lymphocytes. Anal- We have analyzed the entire coding sequence of the ysis of the entire coding region (11 kilobases) in seven patients dystrophin gene from seven patients with DMD or interme- has resulted in detection of a sequence change in each case that diate muscular dystrophy (IMD). In each case one mutation is clearly sufficient to cause the disease. All mutations should is clearly sufficient to cause the disease. The approach we cause premature translational termination, and the resulting have used permits direct diagnosis to be extended to virtually phenotypes are thus equivalent to those caused by frameshift- any case of DMD and BMD and is applicable to many other ing deletions. The results support a particular functional complex tissue-specific genes. importance for the C-terminal region of dystrophin. Applica- tion of this approach to mutation detection will extend direct carrier and prenatal diagnosis to virtually every affected MATERIALS AND METHODS family. Patients. Patients with DMD or IMD were selected solely on the basis that no deletion was detected by a multiplex PCR The spectrum ofmutations that cause genetic disease exhibits (10), which is capable of identifying 98% of deletions. Phe- unusual variation in some genes. Mutations in the dystrophin notypes are indicated at the right of Fig. 3 (MR, mental (1) and steroid sulfatase (2) loci, for example, are predomi- retardation), in the format "diagnosis, age ofdiagnosis/age of nantly heterogeneous deletions, whereas mutations in anti- confinement to wheelchair/current age (years)" (-, still coagulant factors VIII and IX are mostly point mutations (3). mobile). Patient 2 only became wheelchair-bound at the age Analysis of the types of structural alteration that affect the of 14 years, 3 months, whereas patient 7 is still able to walk structure, expression, stability, or function of the gene prod- two miles at the age of 10 years. Results of muscle dystrophin uct can offer insight into the mechanisms by which such analysis were available in two cases. No dystrophin was mutations arise and the mode ofaction ofthe normal gene and detected in muscle biopsy from patient 5 with polyclonal its encoded protein. antiserum P6 (T. G. Sherratt and P. N. Strong, personal Mutations in the human dystrophin gene are associated communication) (raised against amino acids 2814-3028) or with common (1 in 3500 boys) X chromosome-linked mus- from patient 4 with monoclonal antibody Dy4/6D3 (G. Dick- cular dystrophies of wide-ranging phenotype, from the mild son, personal communication) (raised against a murine poly- Becker (BMD) form to the severe Duchenne (DMD) form. peptide corresponding to amino acids 1164-1397) (11). Because the disease frequency is maintained by recurrent Reverse Transcription (RT) and Nested PCR. Total RNA mutation, many different mutants exist. Two-thirds of these was prepared from peripheral blood lymphocytes (12). Sam- mutations consist of large deletions (4), which have a non- ples (200-500 ng) of total lymphocyte RNA were reverse- random distribution, being clustered in two hotspots. In transcribed using primer DMDXb (X = 1-11; primer nomen- general (5), deletions resulting in a translational frameshift clature below). A PCR mix containing primers DMDXa and are generally associated with DMD, whereas those that may DMDXb was added to the products and 30 cycles of PCR remove large portions of the protein but maintain the reading (13-16) were performed. Two microliters ofthe products was frame are associated with BMD (6). The remaining one-third added to a second mix containing primer DMDXc or DMDXe of mutations have not been characterized. Their identifica- and primer DMDXd or DMDXf, and PCR was repeated (17). tion represents a formidable challenge because of the large Eight microliters of the final product was electrophoresed in size and complexity ofthe dystrophin gene [11 kilobases (kb) a 4% polyacrylamide minigel containing ethidium bromide. ofcoding sequence (7) distributed between 79 exons (R.G.R., Products were gel-purified from 4% polyacrylamide minigels A. J. Coffey, M.B., and D.R.B., unpublished data) across 2.3 using DEAE membrane. Primers [nomenclature: X, reaction megabases of genome (4)]. To date only one point mutation 1-11; DMDXa and DMDXb (outer set), 5' and 3', respec- has been reported (8), where immunological analysis of tively; DMDXc and DMDXd (inner set), 5' and 3', respec- truncated dystrophin from muscle biopsy material allowed tively; DMDXe, 5' inner primer for reaction Xb; DMDXf, 3' prior localization of the mutation. inner primer for reaction Xa] were as in ref. 15 except for the

The publication costs of this article were defrayed in part by page charge Abbreviations: DMD, Duchenne muscular dystrophy; BMD, Becker payment. This article must therefore be hereby marked "advertisement" muscular dystrophy; IMD, intermediate muscular dystrophy; RT, in accordance with 18 U.S.C. §1734 solely to indicate this fact. reverse transcription; nt, nucleotide(s).

2331 Downloaded by guest on September 28, 2021 2332 Genetics: Roberts et al. Proc. Natl. Acad. Sci. USA 89 (1992) promoter reaction (18) and the following: DMD1d, DMD7a date deleterious mutations were confirmed by direct se- and DMD7d, DMD8d, DMD10d-DMD10f, DMDSe and quencing or restriction analysis of amplified genomic DNA DMD5f, DMD11a-DMD11d (sequences available on re- (Fig. 2B). quest). Sequence Differences in the Dystrophin Gene. Fourteen Characterization of Mutations. For each mismatch reac- distinct sequence changes were identified (Fig. 3B). Point tion, a probe was amplified from cloned cDNA (1, 19) using mutations generating in-frame termination codons were the same primers as were used for secondary PCR. The probe found in four patients (patient 1; patient 2, Fig. 2A; patient 3; was labeled using T4 polynucleotide kinase and [y-32PIATP patient 5, Fig. 1B). Two of these changes (in patients 3 and and was hybridized to gel-purified products from the nested 5) are C -- T transitions in CpG dinucleotides. For example, RT-PCR. The hybrid mixture was then divided into two and mismatch analysis of reaction 10a with either osmium tetrox- subjected to chemical modification of mismatched pyrimi- ide or hydroxylamine in patient 5 resulted in the appearance dine residues (20, 21) with osmium tetroxide (1 mM osmium of a 342-nt band (Fig. 1B). Direct sequencing revealed a C -- tetroxide/370 mM pyridine, 370C for 2 hr) and with hydrox- T change at nucleotide 10316 in the cDNA sequence, which ylamine (2.3 M hydroxylamine hydrochloride/1.67 M dieth- converts codon 3370 from an arginine codon to a termination ylamine, 370C for 2 hr). The DNA was cleaved adjacent to codon (Fig. 3B). Similarly, mismatch analysis ofreaction 9 in modified residues with 1 M piperidine at 90'C for 30 min. patient 3 using hydroxylamine resulted in the appearance of Products were electrophoresed in a denaturing 5% polyacryl- a band of 374 nt. Direct sequencing revealed a C -- T amide gel and autoradiographed. Direct sequencing (22, 23) transition at nucleotide 9152, which converts codon 2982 of regions identified by chemical mismatch detection was from arginine to a termination codon. In patient 7 a band of performed using PCR primers or internal oligonucleotides 238 nt was detected in reaction lOb (Fig. 1B). This corre- (sequences available on request). sponded to deletion of the single nucleotide T10662, resulting Determination of Intron Sequence. In instances where mu- in a frameshift mutation that should cause premature termi- tations lay near the ends of exons or in introns, intron nation of translation 10 amino acids after Pro3484 (Fig. 2C). sequences were required for amplification of genomic DNA. In patients 4 (Fig. 1A) and 6 the mutations were betrayed These were determined by sequence analysis of vectorette by aberrantly sized PCR products. The respective missing PCR products (24) derived from yeast artificial chromosome sequences were found by PCR to be absent from the genome clones containing parts of the dystrophin gene (25) using of patient 6 (exons 73-76; nucleotides 10537-11129) but exon-specific primers (R.G.R., A. J. Coffey, M.B., and present in that of patient 4. Intron sequences adjacent to the D.R.B., unpublished data). exon missing from the transcript of patient 4 (exon 68; nucleotides 10016-10182) were determined by analysis of vectorette PCR products derived from yeast artificial chro- RESULTS mosome clone 10-1 [which contains exons encoding the 3' 5.5 Amplification products from reverse-transcribed RNA (Fig. kb ofthe transcript (25)]. Using primers specific for the intron 1A) or genomic DNA were screened for sequence differences sequences, the boundaries of the exon were amplified from from normal cDNA (7, 19) using chemical mismatch detec- patient 4 and the splice signals were sequenced. The acceptor tion (20, 21) (Fig. 1B). In each case, the size of the chemical site and branch site were intact, but the donor site GT was cleavage product indicated the approximate position of the mutated to GA (Fig. 2B). This result supports the "exon mismatch. Changes were characterized by sequencing the definition" model (26, 27), in which mutation of a donor site amplified RT-PCR products (7, 19) (Fig. 2 A and C). Candi- in the absence of nearby "cryptic" donor sites is expected to (] b

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T10662- > (238 nt) FIG. 1. (A) Products of nested RT-PCR spanning nucleotides 9517-10469 from patient 4 and an individual with a wild-type donor site. bp, Base pairs. (B) Mismatch analysis of 10a and 10b products from patients 5 and 7. The band sizes pinpoint areas for direct sequencing. nt, Nucleotides. Downloaded by guest on September 28, 2021 Genetics: Roberts et al. Proc. Natl. Acad. Sci. USA 89 (1992) 2333

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- ~ C;jG5 ly 5' FIG. 2. (A) Direct sequence analysis ofa region -240 bp from the end ofreaction 5b from patient 2, which was indicated by mismatch analysis to differ from the wild-type sequence. The sequence shows a C -- T change 233 bp from the 5' end of 5b. Codons 1848-1853 of the normal sequence are presented. (B) Sequence of the boundary between exon 68 and intron 68, amplified from the genomic DNA of patient 4. The donor splice site is mutated, resulting in omission of exon 68 from the mRNA (Fig. 1A). (C) Sequence of a region -240 bp from the end of reaction 10b of patient 7 corresponding to the mismatch band in Fig. 1B. The deletion of a T residue in codon 3485 shifts the translational reading frame to give a novel sequence of 11 amino acids followed by translational termination. cause exon skipping. The loss of sequence from the tran- with in-frame deletions) is present in his muscle tissue it scripts causes frameshifts in both patients. Thus premature would account for his milder phenotype. termination of translation is expected to occur in all seven Direct Diagnosis. Sequence from intron 21 was determined cases. using a vectorette PCR product from yeast artificial chromo- Several changes appeared repeatedly and thus represent some clone 21-1 (25). Genomic DNA samples from relatives polymorphisms (Arg1745 -* His, Lys2366-* Gln, Asn2576 -) of patient 1 were amplified using primers specific for exon 21 Asn)-the first two have been reported previously as se- and intron 21. The products were gel purified and sequenced quence differences between two cDNA clones (28); the third on the antisense strand using A and C reactions only (Fig. 5). was reported in ref. 15. Lys2366 -* Gln is found in 26% of The mutant sequence (A) is present in the patient (track 6A) chromosomes (29). Gly882-- Asp, Ser,007-- Ser, Thr1245 -* and his mother (track SA), in conjunction with the wild-type Ile, and Glu2585 -* Glu appear once only. These are likely to sequence (track 5C), whereas the grandmother (tracks 4A, be rare neutral variants, as they occupy positions that are 4C) and other family members have the wild-type sequence poorly conserved between species (30) and between repeats (C) alone. This defines the origin of mutation. Individual 1, in the same species (31). who has inherited the same maternal haplotype as individual Alternative Splicing May Moderate the Severe Phenotype. 5 and had consequently been assigned a high carrier risk, Fig. 4 shows products of reaction Sb from patient 2 and from does not have this mutation on the basis of direct diagnosis. a normal sample. The predominant product in the normal sample (360 bp) includes exons 38 and 39. There are three DISCUSSION products from patient 2. The faint middle band (360 bp) Dystrophin is a large protein with homology to the a-actinin corresponds to a product that includes exon 38 and a copy of and a-spectrin families (7). Although its precise function is exon 39 containing the mutation Gln1851 -s Term. Sequence unknown, a number of results (31-33) have suggested that analysis of the predominant smaller band (222 bp) shows that rod-like dystrophin homodimers may form a flexible hexag- it lacks exon 39 (nucleotides 5657-5794, amino acids 1817- onal lattice that is attached to the inner surface of the muscle 1862). Loss of this exon thus eliminates the nonsense muta- plasma membrane via associations with other proteins. In tion from this alternatively spliced transcript, while the this study, one mutation was found in each patient that leads reading frame is maintained. A very small proportion of to premature translation termination and is therefore pre- alternatively spliced transcript was observed in normal sam- sumed to have caused the disease. Together with the evi- ples. The highest band was found to be an anomalously dence from deletions and the mutation reported by Bulman et migrating heteroduplex molecule, commonly observed in the al. (8) (Glu157 -- Term), the results indicate that serious presence of mixtures of related products that differ in length. disruption of dystrophin function is generally achieved by Patient 2 has an unexpectedly mild phenotype in view of truncation. the position of the termination codon in this case. The The mutations reported here result in loss of progressively alternatively spliced transcript that is found in lymphocytes smaller segments of the C terminus. The severe phenotype of would encode a polypeptide with a small interstitial deletion. patient 6 demonstrates that loss of the C-terminal 242 amino If this transcript (analogous to that found in BMD patients acids ofdystrophin is critical to function and/or stability. The Downloaded by guest on September 28, 2021 2334 Genetics: Roberts et al. Proc. Natl. Acad. Sci. USA 89 (1992) a

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FIG. 3. (a) Diagram of dystrophin transcript. Shaded boxes represent untranslated regions. Open boxes represent regions translated into protein domains (N-TER, a-actinin-like N-terminal domain; H, proline-rich hinge regions; 1-24, spectrin-related repeat domains; CYS, cysteine-rich domain homologous to C-terminal domain of a-actinin; C-TER, C-terminal domain, unique to dystrophin). The lines below designate the extent!~~~~~~~~~~~~~~~~~~~~~~~~~~~.of each RT-PCR reaction. (b) Summary of results of chemical mismatch analysis and direct sequencing (Pt, patient). Bold vertical bars indicate changes detected by chemical mismatch. Beneath each bar is the effect ofthe change on amino acid sequence (the wild-type residue in three-letter notation, its position, and the residue predicted in the patient. Term, termination codon; fs, frameshift), with the nature of the nucleotide change in brackets. The extent of the predicted protein product is represented by the shaded bars. The stippled bar in patient 2 (Pt 2) indicates the full-length product translated from the fraction of transcripts that lack exon 39. Phenotypes are indicated to the right of the diagram (see Materials and Methods). phenotype of patient 7 is somewhat milder. His protein per patient) establishment of the appropriate assay in each should lack only the last 201 amino acids (less than halfofthe family for direct carrier and prenatal diagnosis. An example C-terminal domain). This suggests that the region defined by the structural difference between the truncated dystrophin proteins of patients 6 and 7-i.e., amino acids 3444-3485-is of functional importance. Diagnosis ofthe one-third ofDMD cases without deletions I has hitherto only been possible using linked restriction frag- ment length polymorphisms. This indirect approach suffers the risk of misdiagnosis as a result of the high degree of crossover observed in the dystrophin gene (11) and is not 1 11 applicable to isolated cases. Detection of mutations by the approach described here enables efficient (one person-week

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FIG. 4. Amplification products of reaction 5b from patient 2 and a normal sample. Structures of mRNAs are shown schematically FIG. 5. Analysis ofthe inheritance ofthe mutation Glu931 -I Term beside the gel (exons numbered 37-40; solid boxes, regions of open in the family of patient 1. Haplotypes at the DMD locus are reading frame amplified; stippled boxes, frameshifted region; filled represented by vertical bars under the pedigree. The arrowheads circle, termination codon). indicate the position of the mutation. Downloaded by guest on September 28, 2021 Genetics: Roberts et al. Proc. Natl. Acad. Sci. USA 89 (1992) 2335

is shown in Fig. 4, in which the carrier status of the patient's 10. Abbs, S., Yau, S. C., Clark, S., Mathew, C. G. & Bobrow, M. mother (positive status) and aunt (negative status) of the (1991) J. Med. Genet. 28, 304-311. patient was established conclusively. This strategy permits 11. Nicholson, L. V. B., Davison, K., Falkous, G., Harwood, C., direct diagnosis of virtually every case of DMD and BMD. O'Donnell, E., Slater, C. R. & Harris, J. B. (1989) J. Neurol. This approach permits scanning of the complete coding Sci. 94, 125-136. sequence of a complex tissue-specific gene for a comprehen- 12. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, sive range of mutations, including coding sequence changes, 156-159. splice site 13. Saiki, R. K., Gelfand, D. H., Stoffel, S., Sharf, S. J., Higuchi, mutations, and gross gene rearrangements, using R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988) Science a venous blood sample as its starting material. The use of 239, 487-491. mismatch detection to detect point mutations in amplified 14. Roberts, R. G., Bentley, D. R., Barby, T. F. M., Manners, E. products of ectopic transcripts can also be applied to other & Bobrow, M. (1990) Lancet 336, 1523-1526. diseases, as has been shown for example in hemophilia A (34) 15. Roberts, R. G., Barby, T. F. M., Manners, E., Bobrow, M. & and Hunter syndrome (R. H. Flomen, P. M. Green, D.R.B., Bentley, D. R. (1991) Am. J. Hum. Genet. 49, 298-310. F. Giannelli, and E. P. Green, unpublished data). Although 16. Chelly, J., Kaplan, J. C., Maire, P., Gautron, S. & Kahn, A. its application to autosomal disorders may be complicated by (1988) Nature (London) 333, 858-860. the additional presence of a normal gene, there is evidence 17. Holding, C. & Monk, M. (1989) Lancet iH, 532-535. that ectopic dystrophin transcripts in female lymphocytes 18. Beggs, A. H., Koenig, M., Boyce, F. M. & Kunkel, L. M. originate from both copies of the gene (ref. 14; R.G.R., (1990) Hum. Genet. 86, 45-48. 19. Dickson, G., Love, D. R., Davies, K. E., Wells, K. E., Piper, unpublished results) and that chemical mismatch analysis can T. A. & Walsh, F. S. (1991) Hum. Genet. 88, 53-58. easily detect mutations in a heterozygous state (e.g., ref. 35). 20. Montandon, A. J., Green, P. M., Giannelli, F. & Bentley, D. R. (1989) Nucleic Acids Res. 17, 3347-3358. The full-length cDNA was a generous gift from Dr. G. Dickson, 21. Cotton, R. G. H., Rodrigues, N. R. & Campbell, R. D. (1988) Department of Experimental Pathology, Guy's Hospital. We thank Proc. Natl. Acad. Sci. USA 85, 4397-4401. Ms. E. Manners for her help in compilation of clinical data. We are 22. Green, P. M., Bentley, D. R., Mibashan, R. S., Nilsson, I. M. greatly indebted to Prof. V. Dubowitz and Dr. J. Heckmatt, Depart- & Giannelli, F. (1989) EMBO J. 8, 1067-1072. ment of Paediatrics and Neonatal Medicine at the Hammersmith 23. Winship, P. R. (1989) Nucleic Acids Res. 17, 1266. Hospital, and to Drs. A. C. Berry, S. Hodgson, and S. Robb and Ms. 24. Riley, J., Butler, R., Ogilvie, D., Finniear, R., Jenner, D., T. F. M. Barby, Guy's Hospital, for clinical samples. This work was Powell, S., Anand, R., Smith, J. C. & Markham, A. F. (1990) supported by the Medical Research Council, the Generation Trust, Nucleic Acids Res. 18, 2887-2890. the Muscular Dystrophy Group of Great Britain and Northern 25. Coffey, A. J., Roberts, R. G., Green, E. D., Cole, C. G., Ireland, and the Spastics Society. Butler, R., Anand, R., Giannelli, F. & Bentley, D. R. (1992) Genomics, in press. 1. Koenig, M., Hoffman, E. P., Bertelson, C. J., Monaco, A. P., 26. Robberson, B. L., Cote, G. J. & Berget, S. M. (1990) Mol. Feener, C. & Kunkel, L. M. (1987) Cell 50, 509-517. Cell. Biol. 10, 84-94. 2. Yen, P. H., Allen, E., Marsh, B., Mohandas, T., Wang, N., 27. Carstens, R. P., Fenton, W. A. & Rosenberg, L. R. (1991) Am. Taggart, R. T. & Shapiro, L. J. (1987) Cell 49, 443-454. J. Hum. Genet. 48, 1105-1114. 3. Green, P. M., Montandon, A. J., Bentley, D. R. & Giannelli, 28. Rosenthal, A., Speer, A., Billwitz, H., Cross, G. S., Forrest, F. (1991) Blood Coagulation Fibrinolysis 2, 539-565. S. M. & Davies, K. E. (1989) Nucleic Acids Res. 17, 5391. 4. den Dunnen, J. T., Grootscholten, P. M., Bakker, E., 29. Yau, S. C., Roberts, R. G., Bentley, D. R., Mathew, C. G. & Blonden, L. A. J., Ginjaar, H. B., Wapenaar, M. C., van Pas- Bobrow, M. (1991) Nucleic Acids Res. 19, 5803. sen, H. M. B., van Broeckhoven, C., Pearson, P. & van 30. Lemaire, C., Heilig, R. & Mandel, J. L. (1988) EMBO J. 7, Ommen, G. J. B. (1989) Am. J. Hum. Genet. 45, 835-847. 4157-4162. 5. Monaco, A. P., Bertelson, C. J., Liechti-Gallati, S., Moser, H. 31. Koenig, M. & Kunkel, L. M. (1990) J. Biol. Chem. 265f, & Kunkel, L. M. (1988) Genomics 2, 90-95. 4560-4566. 6. England, S. B., Nicholson, L. V. B., Johnson, M. A., Forrest, 32. Zubrzycka-Gaarn, E. E., Bulman, D. E., Karpati, G., S. M., Love, D. R., Zubrzycka-Gaarn, E. E., Bulman, D. E., Burghes, A. H. M., Belfall, B., Klamut, H. J., Talbot, J., Harris, J. B. & Davies, K. E. (1990) Nature (London) 343, Hodges, R. S., Ray, P. N. & Worton, R. G. (1988) Nature 180-182. (London) 333, 466-469. 7. Koenig, M., Monaco, A. P. & Kunkel, L. M. (1988) Cell 53, 33. Campbell, K. P. & Kahl, S. D. (1989) Nature (London) 338, 219-228. 259-262. 8. Bulman, D. E., Gangopadhyay, S. B., Bebchuck, K. G., Wor- 34. Naylor, J. A., Green, P. M., Montandon, A. J., Rizza, C. R. & ton, R. G. & Ray, P. N. (1991) Genomics 10, 457-460. Giannelli, F. (1991) Lancet 337, 635-639. 9. Abbs, S., Roberts, R. G., Mathew, C. G., Bentley, D. R. & 35. Montandon, A. J., Green, P. M., Bentley, D. R., Ljung, R., Bobrow, M. (1990) Genomics 7, 602-606. Nilsson, I. M. & Giannelli, F. (1990) Hum. Genet. 85, 200-204. Downloaded by guest on September 28, 2021