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