Proc. Natl. Acad. Sci. USAVol. 89, pp. 2331-2335, March 1992Genetics

Point mutations in the dystrophin geneROLAND G. ROBERTS, MARTIN BOBROW, AND DAVID R. BENTLEYPaediatric 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 thatcause human disease is essential to determine the mechanismsof genetic variation and the function of gene domains and toperform precise carrier and prenatal diagnosis. The mutationsin one-third of Duchenne muscular dystrophy patients remainunknown as they do not involve gross rearrangements of thedystrophin gene. The size and complexity of the gene haveprohibited the systematic definition of point mutations. Wehave developed a method for the identification of these muta-tions by nested amplification, chemical mismatch detection,and sequencing of reverse transcripts of trace amounts ofdystrophin mRNA from peripheral blood lymphocytes. Anal-ysis of the entire coding region (11 kilobases) in seven patientshas resulted in detection of a sequence change in each case thatis clearly sufficient to cause the disease. All mutations shouldcause premature translational termination, and the resultingphenotypes are thus equivalent to those caused by frameshift-ing deletions. The results support a particular functionalimportance for the C-terminal region of dystrophin. Applica-tion of this approach to mutation detection will extend directcarrier and prenatal diagnosis to virtually every affectedfamily.

The spectrum of mutations that cause genetic disease exhibitsunusual variation in some genes. Mutations in the dystrophin(1) and steroid sulfatase (2) loci, for example, are predomi-nantly heterogeneous deletions, whereas mutations in anti-coagulant factors VIII and IX are mostly point mutations (3).Analysis of the types of structural alteration that affect thestructure, expression, stability, or function of the gene prod-uct can offer insight into the mechanisms by which suchmutations arise and the mode of action ofthe normal gene andits encoded protein.

Mutations in the human dystrophin gene are associatedwith common (1 in 3500 boys) X chromosome-linked mus-cular dystrophies of wide-ranging phenotype, from the mildBecker (BMD) form to the severe Duchenne (DMD) form.Because the disease frequency is maintained by recurrentmutation, many different mutants exist. Two-thirds of thesemutations consist of large deletions (4), which have a non-random distribution, being clustered in two hotspots. Ingeneral (5), deletions resulting in a translational frameshiftare generally associated with DMD, whereas those that mayremove large portions of the protein but maintain the readingframe are associated with BMD (6). The remaining one-thirdof mutations have not been characterized. Their identifica-tion represents a formidable challenge because of the largesize and complexity of the dystrophin gene [11 kilobases (kb)ofcoding sequence (7) distributed between 79 exons (R.G.R.,A. J. Coffey, M.B., and D.R.B., unpublished data) across 2.3megabases of genome (4)]. To date only one point mutationhas been reported (8), where immunological analysis oftruncated dystrophin from muscle biopsy material allowedprior localization of the mutation.

The ability to identify mutations on a routine basis allowsprecise establishment of carrier status in affected families andpermits accurate prenatal diagnosis. In families with no grossdystrophin gene rearrangement, diagnosis of carriers cur-rently relies on linkage analysis. Due to the high mutationrate, the mutation has often originated within a living familymember; hence relatives carrying a high-risk haplotype maynot carry the mutation. Furthermore, recombination occurswithin the dystrophin gene in up to 12% of meioses (9),necessitating the analysis of flanking markers. Direct diag-nosis would not be subject to either of these problems.We have analyzed the entire coding sequence of the

dystrophin gene from seven patients with DMD or interme-diate muscular dystrophy (IMD). In each case one mutationis clearly sufficient to cause the disease. The approach wehave used permits direct diagnosis to be extended to virtuallyany case ofDMD and BMD and is applicable to many othercomplex tissue-specific genes.

MATERIALS AND METHODSPatients. Patients with DMD or IMD were selected solely

on the basis that no deletion was detected by a multiplex PCR(10), which is capable of identifying 98% of deletions. Phe-notypes are indicated at the right of Fig. 3 (MR, mentalretardation), in the format "diagnosis, age ofdiagnosis/age ofconfinement to wheelchair/current age (years)" (-, stillmobile). Patient 2 only became wheelchair-bound at the ageof 14 years, 3 months, whereas patient 7 is still able to walktwo miles at the age of 10 years. Results of muscle dystrophinanalysis were available in two cases. No dystrophin wasdetected in muscle biopsy from patient 5 with polyclonalantiserum P6 (T. G. Sherratt and P. N. Strong, personalcommunication) (raised against amino acids 2814-3028) orfrom patient 4 with monoclonal antibody Dy4/6D3 (G. Dick-son, personal communication) (raised against a murine poly-peptide corresponding to amino acids 1164-1397) (11).

Reverse Transcription (RT) and Nested PCR. Total RNAwas prepared from peripheral blood lymphocytes (12). Sam-ples (200-500 ng) of total lymphocyte RNA were reverse-transcribed using primer DMDXb (X = 1-11; primer nomen-clature below). A PCR mix containing primers DMDXa andDMDXb was added to the products and 30 cycles of PCR(13-16) were performed. Two microliters of the products wasadded to a second mix containing primerDMDXc orDMDXeand primer DMDXd or DMDXf, and PCR was repeated (17).Eight microliters of the final product was electrophoresed ina 4% polyacrylamide minigel containing ethidium bromide.Products were gel-purified from 4% polyacrylamide minigelsusing DEAE membrane. Primers [nomenclature: X, reaction1-11; DMDXa and DMDXb (outer set), 5' and 3', respec-tively; DMDXc and DMDXd (inner set), 5' and 3', respec-tively; DMDXe, 5' inner primer for reaction Xb; DMDXf, 3'inner primer for reaction Xa] were as in ref. 15 except for the

Abbreviations: DMD, Duchenne muscular dystrophy; BMD, Beckermuscular dystrophy; IMD, intermediate muscular dystrophy; RT,reverse transcription; nt, nucleotide(s).

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promoter reaction (18) and the following: DMD1d, DMD7aand DMD7d, DMD8d, DMD10d-DMD10f, DMDSe andDMD5f, DMD11a-DMD11d (sequences available on re-quest).

Characterization of Mutations. For each mismatch reac-tion, a probe was amplified from cloned cDNA (1, 19) usingthe same primers as were used for secondary PCR. The probewas labeled using T4 polynucleotide kinase and [y-32PIATPand was hybridized to gel-purified products from the nestedRT-PCR. The hybrid mixture was then divided into two andsubjected to chemical modification of mismatched pyrimi-dine residues (20, 21) with osmium tetroxide (1 mM osmiumtetroxide/370 mM pyridine, 370C for 2 hr) and with hydrox-ylamine (2.3 M hydroxylamine hydrochloride/1.67 M dieth-ylamine, 370C for 2 hr). The DNA was cleaved adjacent tomodified residues with 1 M piperidine at 90'C for 30 min.Products were electrophoresed in a denaturing 5% polyacryl-amide gel and autoradiographed. Direct sequencing (22, 23)of regions identified by chemical mismatch detection wasperformed using PCR primers or internal oligonucleotides(sequences available on request).

Determination of Intron Sequence. In instances where mu-tations lay near the ends of exons or in introns, intronsequences were required for amplification of genomic DNA.These were determined by sequence analysis of vectorettePCR products (24) derived from yeast artificial chromosomeclones containing parts of the dystrophin gene (25) usingexon-specific primers (R.G.R., A. J. Coffey, M.B., andD.R.B., unpublished data).

RESULTSAmplification products from reverse-transcribed RNA (Fig.1A) or genomic DNA were screened for sequence differencesfrom normal cDNA (7, 19) using chemical mismatch detec-tion (20, 21) (Fig. 1B). In each case, the size of the chemicalcleavage product indicated the approximate position of themismatch. Changes were characterized by sequencing theamplified RT-PCR products (7, 19) (Fig. 2 A and C). Candi-

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date deleterious mutations were confirmed by direct se-quencing or restriction analysis of amplified genomic DNA(Fig. 2B).Sequence Differences in the Dystrophin Gene. Fourteen

distinct sequence changes were identified (Fig. 3B). Pointmutations generating in-frame termination codons werefound in four patients (patient 1; patient 2, Fig. 2A; patient 3;patient 5, Fig. 1B). Two of these changes (in patients 3 and5) are C -- T transitions in CpG dinucleotides. For example,mismatch analysis of reaction 10a with either osmium tetrox-ide or hydroxylamine in patient 5 resulted in the appearanceof a 342-nt band (Fig. 1B). Direct sequencing revealed a C --

T change at nucleotide 10316 in the cDNA sequence, whichconverts codon 3370 from an arginine codon to a terminationcodon (Fig. 3B). Similarly, mismatch analysis of reaction 9 inpatient 3 using hydroxylamine resulted in the appearance ofa band of 374 nt. Direct sequencing revealed a C -- Ttransition at nucleotide 9152, which converts codon 2982from arginine to a termination codon. In patient 7 a band of238 nt was detected in reaction lOb (Fig. 1B). This corre-sponded to deletion of the single nucleotide T10662, resultingin a frameshift mutation that should cause premature termi-nation of translation 10 amino acids after Pro3484 (Fig. 2C).

In patients 4 (Fig. 1A) and 6 the mutations were betrayedby aberrantly sized PCR products. The respective missingsequences were found by PCR to be absent from the genomeof patient 6 (exons 73-76; nucleotides 10537-11129) butpresent in that of patient 4. Intron sequences adjacent to theexon missing from the transcript of patient 4 (exon 68;nucleotides 10016-10182) were determined by analysis ofvectorette PCR products derived from yeast artificial chro-mosome clone 10-1 [which contains exons encoding the 3' 5.5kb of the transcript (25)]. Using primers specific for the intronsequences, the boundaries of the exon were amplified frompatient 4 and the splice signals were sequenced. The acceptorsite and branch site were intact, but the donor site GT wasmutated to GA (Fig. 2B). This result supports the "exondefinition" model (26, 27), in which mutation of a donor sitein the absence of nearby "cryptic" donor sites is expected to

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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.

2332 Genetics: Roberts et al.

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FIG. 2. (A) Direct sequence analysis ofa region -240 bp from the end of reaction 5b from patient 2, which was indicated by mismatch analysisto 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 normalsequence are presented. (B) Sequence of the boundary between exon 68 and intron 68, amplified from the genomic DNA of patient 4. The donorsplice 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 reaction10b of patient 7 corresponding to the mismatch band in Fig. 1B. The deletion of a T residue in codon 3485 shifts the translational reading frameto give a novel sequence of 11 amino acids followed by translational termination.

cause exon skipping. The loss of sequence from the tran-scripts causes frameshifts in both patients. Thus prematuretermination of translation is expected to occur in all sevencases.

Several changes appeared repeatedly and thus representpolymorphisms (Arg1745 -* His, Lys2366-* Gln, Asn2576 -)

Asn)-the first two have been reported previously as se-quence differences between two cDNA clones (28); the thirdwas reported in ref. 15. Lys2366 -* Gln is found in 26% ofchromosomes (29). Gly882-- Asp, Ser,007-- Ser, Thr1245 -*Ile, and Glu2585 -* Glu appear once only. These are likely tobe rare neutral variants, as they occupy positions that arepoorly conserved between species (30) and between repeatsin the same species (31).

Alternative Splicing May Moderate the Severe Phenotype.Fig. 4 shows products of reaction Sb from patient 2 and froma normal sample. The predominant product in the normalsample (360 bp) includes exons 38 and 39. There are threeproducts from patient 2. The faint middle band (360 bp)corresponds to a product that includes exon 38 and a copy ofexon 39 containing the mutation Gln1851 -s Term. Sequenceanalysis of the predominant smaller band (222 bp) shows thatit lacks exon 39 (nucleotides 5657-5794, amino acids 1817-1862). Loss of this exon thus eliminates the nonsense muta-tion from this alternatively spliced transcript, while thereading frame is maintained. A very small proportion ofalternatively spliced transcript was observed in normal sam-ples. The highest band was found to be an anomalouslymigrating heteroduplex molecule, commonly observed in thepresence of mixtures of related products that differ in length.

Patient 2 has an unexpectedly mild phenotype in view ofthe position of the termination codon in this case. Thealternatively spliced transcript that is found in lymphocyteswould encode a polypeptide with a small interstitial deletion.If this transcript (analogous to that found in BMD patients

with in-frame deletions) is present in his muscle tissue itwould account for his milder phenotype.

Direct Diagnosis. Sequence from intron 21 was determinedusing a vectorette PCR product from yeast artificial chromo-some clone 21-1 (25). Genomic DNA samples from relativesof patient 1 were amplified using primers specific for exon 21and intron 21. The products were gel purified and sequencedon the antisense strand using A and C reactions only (Fig. 5).The mutant sequence (A) is present in the patient (track 6A)and his mother (track SA), in conjunction with the wild-typesequence (track 5C), whereas the grandmother (tracks 4A,4C) and other family members have the wild-type sequence(C) alone. This defines the origin of mutation. Individual 1,who has inherited the same maternal haplotype as individual5 and had consequently been assigned a high carrier risk,does not have this mutation on the basis of direct diagnosis.

DISCUSSIONDystrophin is a large protein with homology to the a-actininand a-spectrin families (7). Although its precise function isunknown, a number of results (31-33) have suggested thatrod-like dystrophin homodimers may form a flexible hexag-onal lattice that is attached to the inner surface of the muscleplasma membrane via associations with other proteins. Inthis study, one mutation was found in each patient that leadsto premature translation termination and is therefore pre-sumed to have caused the disease. Together with the evi-dence from deletions and the mutation reported by Bulman etal. (8) (Glu157 -- Term), the results indicate that seriousdisruption of dystrophin function is generally achieved bytruncation.The mutations reported here result in loss of progressively

smaller segments of the C terminus. The severe phenotype ofpatient 6 demonstrates that loss of the C-terminal 242 aminoacids ofdystrophin is critical to function and/or stability. The

Genetics: Roberts et al. 2333

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Proc. Natl. Acad. Sci. USA 89 (1992)

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FIG. 3. (a) Diagram of dystrophin transcript. Shaded boxes represent untranslated regions. Open boxes represent regions translated intoprotein 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 belowdesignate the extent of each RT-PCR reaction. (b) Summary of results of chemical mismatch analysis and direct sequencing (Pt, patient). Boldvertical bars indicate changes detected by chemical mismatch. Beneath each bar is the effect of the change on amino acid sequence (the wild-typeresidue in three-letter notation, its position, and the residue predicted in the patient. Term, termination codon; fs, frameshift), with the natureof the nucleotide change in brackets. The extent of the predicted protein product is represented by the shaded bars. The stippled bar in patient2 (Pt 2) indicates the full-length product translated from the fraction of transcripts that lack exon 39. Phenotypes are indicated to the right ofthe diagram (see Materials and Methods).

phenotype of patient 7 is somewhat milder. His proteinshould lack only the last 201 amino acids (less than half of theC-terminal domain). This suggests that the region defined bythe structural difference between the truncated dystrophinproteins of patients 6 and 7-i.e., amino acids 3444-3485-isof functional importance.

Diagnosis of the one-third ofDMD cases without deletionshas hitherto only been possible using linked restriction frag-ment length polymorphisms. This indirect approach suffersthe risk of misdiagnosis as a result of the high degree ofcrossover observed in the dystrophin gene (11) and is notapplicable to isolated cases. Detection of mutations by theapproach described here enables efficient (one person-week

per patient) establishment of the appropriate assay in eachfamily for direct carrier and prenatal diagnosis. An example

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FIG. 4. Amplification products of reaction 5b from patient 2 anda normal sample. Structures of mRNAs are shown schematicallybeside the gel (exons numbered 37-40; solid boxes, regions of openreading frame amplified; stippled boxes, frameshifted region; filledcircle, termination codon).

FIG. 5. Analysis of the inheritance of the mutation Glu931 -I Termin the family of patient 1. Haplotypes at the DMD locus arerepresented by vertical bars under the pedigree. The arrowheadsindicate the position of the mutation.

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......... ...........-.1 .!cl:..,",...:.:"..:.,...i....:.7.f"...,...:"".,.:.,..7.:.,.,.t.. I.,...- 1.1.....-..-.-...-.......-., ...:.

2334 Genetics: Roberts et al.

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Proc. Natl. Acad. Sci. USA 89 (1992) 2335

is shown in Fig. 4, in which the carrier status of the patient'smother (positive status) and aunt (negative status) of thepatient was established conclusively. This strategy permitsdirect diagnosis of virtually every case of DMD and BMD.

This approach permits scanning of the complete codingsequence of a complex tissue-specific gene for a comprehen-sive range of mutations, including coding sequence changes,splice site mutations, and gross gene rearrangements, usinga venous blood sample as its starting material. The use ofmismatch detection to detect point mutations in amplifiedproducts of ectopic transcripts can also be applied to otherdiseases, as has been shown for example in hemophilia A (34)and Hunter syndrome (R. H. Flomen, P. M. Green, D.R.B.,F. Giannelli, and E. P. Green, unpublished data). Althoughits application to autosomal disorders may be complicated bythe additional presence of a normal gene, there is evidencethat ectopic dystrophin transcripts in female lymphocytesoriginate from both copies of the gene (ref. 14; R.G.R.,unpublished results) and that chemical mismatch analysis caneasily detect mutations in a heterozygous state (e.g., ref. 35).

The full-length cDNA was a generous gift from Dr. G. Dickson,Department of Experimental Pathology, Guy's Hospital. We thankMs. E. Manners for her help in compilation of clinical data. We aregreatly indebted to Prof. V. Dubowitz and Dr. J. Heckmatt, Depart-ment of Paediatrics and Neonatal Medicine at the HammersmithHospital, and to Drs. A. C. Berry, S. Hodgson, and S. Robb and Ms.T. F. M. Barby, Guy's Hospital, for clinical samples. This work wassupported by the Medical Research Council, the Generation Trust,the Muscular Dystrophy Group of Great Britain and NorthernIreland, and the Spastics Society.

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