Automated sequence screening of the entire dystrophin cdna in Duchenne dystrophy Point mutation detection.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 141B:44 – 50 (2006) Automated Sequence Screening of the Entire Dystrophin cDNA in Duchenne Dystrophy: Point Mutation Detection Sherifa Ahmed Hamed* and Eric P. Hoffman Research Center for Genetic Medicine, Children’s National Medical Center, George Washington University, Washington, DC This is the first report of direct sequencing of the complete 11 kb coding sequence of the dystrophin gene affording high sensitivity for all types of mutations of both coding sequence and splicing. Direct automated capillary gel sequence analysis of dystrophin reverse-transcribed polymerase chain reaction (RT-PCR) products was carried out in 15 Duchenne muscular dystrophy (DMD) patient muscle biopsies (170,000 bp sequenced). We identified mutations in 67% of patients tested (10/15); including premature stop codons (n ¼ 5) and small deletions/duplications (n ¼ 5). Mutation-negative patients (n ¼ 5) were also negative for promoter mutations. All were tested for the possibility of transcription abnormalities using quantitative multiplex fluorescence polymerase chain studies (QMF-PCR), however, equal ratios of mRNA transcripts were identified at the 50 and 30 regions, with mild reduction in overall quantity, suggesting that transcription abnormalities were less likely. We suggested that such patients might have a problem with the 3.5 kb 30 UTR, polyA site or undetected stop codons. It is also possible that splicing defects could result in addition of intron sequence which could lead to preferential amplification of low level residual normal transcript skipping. ß 2005 Wiley-Liss, Inc. KEY WORDS: Duchenne muscular dystrophy; point mutation; automated sequencing; quantitative multiplex polymerase chain reaction INTRODUCTION Duchenne muscular dystrophy (DMD) is the most common childhood lethal disorder worldwide [Worton and Thompson, 1988]. The responsible gene and protein were the first to be isolated by positional cloning in 1987 [Hoffman et al., 1987; Grant sponsor: Egyptian Government (fellowship to SAH); Grant sponsor: National Institute of Health (to EPH); Grant number: 5RO1 NS29525. *Correspondence to: Dr. Sherifa Ahmed Hamed, M.D., Neurology Consultant, Saudi German Hospital—Aseer, Khamis Mushayt, Saudia Arabia, P.O. Box 2553, Khamis Mushayt. E-mail: email@example.com Received 15 January 2005; Accepted 18 July 2005 DOI 10.1002/ajmg.b.30234 ß 2005 Wiley-Liss, Inc. Koenig et al., 1987; Monaco et al., 1988]. The gene is approximately 2.5 Mbp, and remains the only human megagene. There are multiple gene promoters and alternatively spliced isoforms [Ahn and Kunkel, 1993], however, the fulllength 427 kDa dystrophin protein driven by the muscle promoter appears the most critical for health. Approximately 55% of DMD patients show deletion mutation of one or more exons of the gene [Koenig et al., 1987; den Dunnen et al., 1989; Hoffman, 1993]. In families segregating deletion mutation, prenatal diagnosis and identification of female carriers is straightforward. Linkage analysis can be done in non-deletion families, however, the high spontaneous mutation rate, early death of patients, 10% intragenic recombination rate, and high rate of gonadal mosaicism in both sexes serve to complicate interpretation of results [Oudet et al., 1992; Saito et al., 1995; Hoffman, 1997]. To date many studies have addressed the nature of non-deletion mutations in Duchenne dystrophy. All have relied on a screening method that first localize a potential mutation by electrophoretic shift of PCR, reverse transcribed PCR product (RT-PCR), or protein fragment, then subsequent sequencing of the aberrant band. Deletion testing of muscular dystrophy patients is done routinely in hundreds of laboratories, however few, if any, molecular diagnostic laboratories have attempted to implement routine point mutation screening. There have been many issues hindering direct mutation detection in non-deletion patients: (1) no mutation hot spots or frequently recurring mutations have been identified to date, (2) use of genomic DNA would require over 79 exon-specific amplifications per patient, (3) use of RNA from either muscle biopsy or peripheral blood cells reduces the number of PCR reactions needed, however RNA is more problematic during work, and very low level of dystrophin mRNA in peripheral blood requires nested PCR which may induce artifacts, (4) methods of heteroduplex analysis (HAD), single stranded conformation polymorphism (SSCP) [Lenk et al., 1993; KeKou et al., 1999], or chemical mismatch cleavage (CMC) [Roberts et al., 1992] are known to be relatively insensitive, (5) the dystrophin gene has many polymorphisms (http://www.dmd.nl/), hence, it can be difficult to distinguish between a band shift from mutations versus polymorphisms, and (6) protein truncation test (PTT) [Roest et al., 1993], which seems the most sensitive method to date, is technically challenging, requiring RNA isolation, complementary DNA (cDNA) production, multiple nested RT-PCR reactions, transcription-coupled translation reactions for each RTPCR product, and denaturing gradient gel electrophoresis (DGGE), prior to any sequence analysis of potential mutationcontaining gene fragments [Tubiello et al., 1995]. We reasoned that direct sequencing of RT-PCR products might afford the most sensitive and specific method of detecting non-deletion mutations. We also felt that relatively recent advances in automated sequencing using capillary gel electrophoresis directly loaded from microliter plates of RT-PCR reactions might make full sequence analysis of the 11 kb dystrophin coding region technically and economically feasible. Point Mutation Detection METHODS This study included 15 unrelated DMD patients (from different areas of the USA) tested negative for deletion mutation of the dystrophin gene by either multiplex PCR or Southern blot analysis. Muscle biopsies was the initial starting material. Sequence Analysis of Complete Coding Sequence From Muscle Biopsy RNA RNA was isolated from 25 mg of flash-frozen muscle biopsy using guanidium isothiocynanate homogenization, followed by precipitation of RNA through CsCl cushion [Ljunggren et al., 1995]. Total RNA (500 ng) was reverse transcribed into cDNA using oligo-dT and AMV reverse transcriptase. RT-PCR reactions were done using 36 sets of primers designed against the dystrophin coding sequence. Primer pairs were designed so as to amplify 330–535 bp fragments (Table I). Fragments typically overlapped by 50–100 bp. Common amplification conditions were used for all primer sets (948C for 10 min, 35 cycles of 948C for 30 sec, 558C for 30 sec, 728C for 2 min, and final extension of 728C for 10 min). RT-PCR products were checked on 1.5% agarose gels, then purified using QIAquick PCR purification kits (Qiagen, BD Biosciences Clontech, Mountain View, CA), and sequenced by automated capillary sequencing using 8 capillary bed machine using protocols recommended by the manufacturer (Beckman-Coulter CEQ2000, Fullerton, CA). Sequencing reactions were prepared in 20 ml reaction using the DTS sequencing kits (Dye Terminator Cycle sequencing Kits) (Beckman-Coulter). Each RT-PCR product was sequenced in both directions, for a total of 72 sequencing reactions. Diluted templates were heated at 968C for 1 min as a hot start followed by 35 cycles of 968C for 20 sec, 508C for 20 sec, and 608C for 4 min. Sequencing PCR products were purified using CENTRI-SEP as described by the supplier (Princeton, Princeton, NJ), vacuum dried for 30– 45 min and then resuspended in 40 ml of deionized formamide. Samples were run on Beckman-Coulter CEQ2000 8 channel capillary electrophoresis and sequence data were compared to the normal published dystrophin mRNA sequence. Nucleotide changes were confirmed in genomic DNA of each patient by amplification of the mutation-containing exon, using DNA purified from peripheral blood or muscle biopsy [Pegoraro et al., 1995]. In Mutation-Negative Patients We sequenced the promoter region (535 bp) of the dystrophin gene amplified from genomic DNA. This PCR product covers from 433 bp from the transcription start site, through þ95 bp in the first exon. Quantitation of Dystrophin mRNA for Mutation-Negative Patients We used low cycle number co-amplification of multiple RTPCR products (QMF-PCR) [Zhou and Hoffman, 1994; Hamed et al., 2005]. Briefly, three PCR products were designed which could be co-amplified in a single reaction: dystrophin 50 (244 bp, 1,355–1,589 bp); dystrophin 30 (250 bp, 9,362–9,612 bp); ryanodine receptor (247 bp, 11,989–11,236 bp), their RT-PCR products differed by only 3 bp each. The forward primer of each PCR primer was synthesized with an infrared dye (IRD700, LiCor Inc) (LiCor Bioscience, Lincoln, NE). Patients’ RNA was reverse transcribed and random-primed cDNA corresponding to approximately 20 ng of mRNA was subjected to 25 cycles using all three sets of labeled RT-PCR primers (dystrophin 50 , dystrophin 30 , and ryanodine receptor). A small aliquot of each reaction was then resolved on 6% acrylamide gels on a LiCor 45 TABLE I. Primer Pairs Used to Amplify the Complete Coding Sequence of Dystrophin cDNA Primer name Primer sequence 433F þ95R 44F 21F 428R 305F 726R 621F 1137R 1030F 1431R 1355F 1729R 1682F 2030R 1985F 2342R 2300F 2648R 2603F 2952R 2909F 3256R 3207F 3187F 3556R 3507F 3487F 3857R 3859R 3813F 4160R 4114F 4464R 4418F 4782R 4740F 5090R 5043F 5393R 5348F 5700R 5656F 6006R 5959F 6308R 6262F 6237F 6624R 6578F 6927R 6883F 7232R 7177F 7526R 7481F 7811R 7762F 8112R 8068F 8038F 8413R 8360F 8706R 8660F Gaagatctagaacagtggatacataacaaatgcatg Ttctccgaaggtaattgcctccccagatctgagtcc CAATTACCTTCGGAGAAAAACG CTACAGGACTCAGATCTGGG GGGCATGAACTCTTGTGGAT GGGAAGCAGCATATTGAGAA ATGAGAGCATTCAAAGCCAG AGATTCTCCTGAGCTGGGTC GAGGTGGTGACATAAGCAGC TTCTCAACAGATCACGGTCA CCAATCAGCTTACTTCCCAA GAGGGGTACATGATGGATTTG GACCCTGACTTGTTCTTGTT CAACATAAGGTGCTTCAAGA CGCTTTTAAAACGGCCAGTT CAAAATGAAATGTTATCAAG TCTTCTTTTGGGGAGGTGGT ATGCTCAAGAGGAACTTCCA CGATCCACCGGCTGTTCAGT GATAGCATCAAACAAGCCTC GTAAAGGCCACAAAGTCTGC GGACAAGGACCCATGTTCCT CCGGCTAATTTCAGAGGGCG GCACCACTGTGAAAGAGATG AAGTGGGCTATACTATCTCAG CTTTATCTTCTGCCCACCTT CAATTCAGCCCAGTCTAAAC TTTAGTCAGTGATATTCAGAC GGAGTTTCACTTTCGCTTCT AAGGAGTTTCACTTTCGCTTC GAGCTAAAGAAGAGGCCCAA CCTCTGAATGTCGCATCAAA CTCTGAGGTGCTAGATTCAC CTTGTCAAATCAGATTGGAT GCTCAAAATGCCTCAGGAAGC GTCTTTATCACCATTTCCAC GTCTGAGTGAAGTGAAGTCT GGTGCACCTTCTGTTTCTCA CCTGGGGAAAGCTACTCAA GGCGTATGTCATTCAGTTCT GAAGACGTGCTTAAGCGTTT CCTCTTTGCAACAATTCTTT GAATGAAGACAATGAGGGTA GCTTGCCTACGCACTGCATT GGAATTGCAGAAGAAGAAAG GCTTAAAGAGATCTTCAAAG CAATGCTCCTGACCTCTGTG GCCCTATTAGAAGTGGAACAAC GCATGTTCCCAATTCTCAGG GAACAGTTTCTCAGAAAGAC GGTTCAAGTGGGATACTAGC ATGGTTGGAGGAAGCAGATA GCAGCAGATGATTTAACTGC TGGGCAGCTTGAAAAAAAGCT GAGTAGGAGAGGCTCCAATA CCTGACCTAGCTCCTGGACT GGGCAGCGGTAATGATCATC AGGATTTGGAACAGAGGCGTC GTCTGCCACTGGCGGAGGTC CACAGAAACCAAGCAGTTGG GGAGGGTCCCTATACAGTAG CATCAGCTCTTTTACTCCCTT GCTACCCGTAAGGAAAGGCT GGTGCCTGCCGGCTTAATTC CTGGTGTGGCTACAGCTGAA Product size 535 bp 385 bp 408 bp 422 bp 517 bp 402 bp 349 bp 351 bp 358 bp 349 bp 350 bp 380 bp 350 bp 370 bp 351 bp 373 bp 348 bp 351 bp 365 bp 351 bp 351 bp 353 bp 351 bp 350 bp 363 bp 388 bp 350 bp 350 bp 351 bp 331 bp 351 bp 346 bp 376 bp 347 bp 346 bp (Continued ) 46 Hamed and Hoffman TABLE I. (Continued ) Primer name Primer sequence Product size transcript, suggesting absence of splicing defect or some other type of loss-of-function mutation. DISCUSSION 9005R 8952F 9302R 9143F 9581R 9458F 9885R 9751F 10227R 10164F 10631R 10531F 10975R 10937F 11383R GGGTCTCATCTATTTTTCTC GGGAAAAATTGAACCTGCAC CGACGGCCACCTGCAGAAGC AAGGCACTTCGAGGAGAAAT TCAAGAGATCCAAGCAAAGG GACCATCCCAAAAATGACAGA GTTGAACTTGCCACTTGCTT GCTGCTGAATGTTTATGATA CAGCTTTGGCAGATGTCATA GTCCAATCATTGGATTCAGG AATGCTGGATTAACAAATGT TAGCAGGCTAGCAGAAATGG CTGTGACTCCAGCTGTTT CAAATCCTGGAAGACCACAA AAACCATGCGGGAATCAG 351 bp 439 bp 428 bp 477 bp 468 bp 445 bp 447 bp F, forward; R, reverse. IR2 automated sequencer (LiCor Bioscience) and the respective peaks were quantitated. Peak areas of infrared fluorescence for both dystrophin 50 and 30 were normalized to 100% in 10 controls showing normal dystrophin; the average normalization factor was then applied to each peak ratio in the experimental samples to drive corrected ratios. All samples were tested in duplicate reactions and gels. RESULTS Small Pathogenic Mutations in the Dystrophin Gene Detected by Complete Sequencing of the Coding Sequence We identified 10 small causative loss-of-function mutations distributed throughout the dystrophin gene (Fig. 1) in 67% of our patients (10/15) (Table II) including five stop codons (Gln625X, Ser757X, Glu1353X, Arg1967X, Gln2198X), a single nucleotide deletion (DA2882), intra-exon deletion of 11 bp between nucleotides 3245 and 3255 (Fig. 2), complete duplication of exon exon 2 (n ¼ 2) and exon 43 (n ¼ 1). The RT-PCR products containing that exon was of increased size corresponding to such duplications seen by direct sequencing (Figs. 3 and 4). Mutation-Negative DMD Patients Five DMD patients (5/15) tested negative for mutations in the promoter and whole coding sequence of the dystrophin. Clinical, laboratory, and biochemical features of each is shown in Table III. All showed faint and variable immunostaining results for both amino- and carboxyl-terminal dystrophin and alpha-sarcoglycan antibodies, yet showed no detectable dystrophin by immunoblot. QMF-PCR showed reductions in RNA levels of dystrophin ranging from 25% to 70% of normal levels. None showed preferential loss of the 30 end of the dystrophin Fig. 1. Localization of mutations identified in 10 Duchenne muscular dystrophy (DMD) patients through sequencing of the complete dystrophin coding sequence. Here, we present a new strategy for automated capillary gel sequence analysis that permits systematic scanning of the complete coding sequence of the dystrophin gene [Cohen et al., 1990; Huang et al., 1992]. We believe that our method can simplify the detection of small mutations in this enormous gene. The feasibility of capillary electrophoresis instruments for high-throughput genomic sequencing is further illustrated by the announcement of a whole human genome shotgun sequencing project facilitated by this technology. We identified causative mutations in 10 out of 15 patients studied. This suggests that the sensitivity of our method is 67%, which, to our knowledge, is the highest sensitivity reported to date. All our mutations are added on Leiden Muscular Dystrophy Pages, http://www.dmd.nl/. Many specific techniques have been addressed to reveal the causative mutation in Duchenne dystrophy. All were PCRbased to first localize a potential mutation by electrophoretic shift, RT-PCR products, or protein fragments with subsequent sequencing of the aberrant bands. Most are technically demanding, labor intensive, and in addition costly. However, even with application of specific precautions, many pathogenic mutations will go undetected. Prior et al.  screened 80% of the dystrophin coding region using amplification of individual exons from patient genomic DNA by heteroduplex analysis, followed by sequencing of aberrant bands. Twenty six small mutations were identified in 110 DMD patients, i.e., 25% sensitivity rate. Both Lenk et al.  and KeKou et al.  studied patients using SSCP analysis of exons corresponding to carboxyl-terminal region of the dystrophin protein; Lenk et al. found 6 point mutations in 26 patients, while Kekou et al. found 5 mutations in 31 DMD patients, both consistent with a similar 25% sensitivity rate as that reported by Prior et al. Roberts et al.  used CMC to identify seven point mutations in amplified exons, however the total number screened was not reported. Roest et al.  reported an application of the PTT to dystrophin RNA amplified from peripheral blood. A similar assay was used by Gardner et al.  and able to detect 12 mutations in 22 patients, i.e., 60% sensitivity rate. Both Whittock et al.  and TufferyGiraud et al.  used PTT, and both found 10 mutations in 10 non-deletion patients. A recently described technique, multiplex amplifiable probe hybridization (MAPH) [Armour et al., 2000] and modified by White et al.  was utilized for screening DMD gene. Only 37 samples out of 72 (i.e., 51%) were utilized and the 29 out of 39 (74%) checked for deletions and point mutations were tested positive for mutations by the use of this technique including small novel rearrangements especially small one or two exon duplications. MAPH involves the quantitative recovery of specifically designed probes following hybridization to immobilized genomic DNA. Engineered probes for each of the 79 exons of the DMD gene will then be analyzed using a 96-capillary sequencer. The authors suggested that this technique is simple, quick, and accurate based on existing technology (i.e., hybridization, PCR, and electrophoresis) and should allow easy implementation in routine diagnostic laboratories. Furthermore, they suggested that this methodology should be applicable to any genetic disease, it could be easily expandable to cover >200 probes and its characteristics should facilitate high-throughput screening. Unrelated 2 of the 15 patients sequenced (13.3%) showed the same novel duplication of exon 2 suggesting that this may be a recurring mutation of the dystrophin gene. Previous surveys of deletion and duplication mutations of the dystrophin gene have found that some introns appear to be ‘‘hot spots’’ for Point Mutation Detection 47 TABLE II. Small Pathogenic Mutations in the Dystrophin Gene Patient no. 1 2 3 4 5 6 7 8 9 10 a Nucleotide change Amino acid change Exon no. 6107C > Ta 240?_301þ?dup 6326?_6499þ?dup C2081T D A2882 240?_301þ?dup 3245_3255delGAAATTAGCCG 2478C > G 6800C > T 4265G > T Arg 1967X 41 2 43 16 20 2 23 18 45 29 Gln625X Ser757X Gln2198X Glu1353X Protein domain Rod (R15) N-terminus Rod (R 16) Rod (R 3) Rod (R 4,5) N-Terminus Rod (R 6) Rod (R 4) Rod (R 17) Rod (R 9) CPK IU/L Dystrophin results 19,180 25,000 9,200 22,000 20,000 38,340 16,000 10,000 25,000 9,600 0% 0% 1% of 427 kDa 0% 0% 3% of 427 kDa 0% 0% 0% 0% This mutation has been previously reported by Saad et al. . deletion/duplication, for example 33% of DMD/BMD deletion mutations show a breakpoint in intron 44 (119 of 362 deletion positive patients) [Koenig et al., 1987; den Dunnen et al., 1989]. This translates into 20% of all DMD/BMD (both deletion and non-deletion) patients having one breakpoint in this intron. Intron 44 is approximately 170 kb in size [den Dunnen et al., 1989], and has been shown to be a major replication termination site in the dystrophin gene [Verbovaia and Razin, 1997]. Pulse-field analyses of the breakpoints for deletions within intron 44 have shown that they are relatively widespread over intron 44, with 30% of the intron 44 deletions originating in a 25–40 kb region [Wapenaar et al., 1988]. Thus, there does not appear to be sequence specificity (hot spot for deletion) to intron 44, but instead a relative genomic instability. The mechanism for duplication and deletion hot spots are thought to be quite different. It is generally accepted that most duplication mutations occur due to tandemly arranged homologous sequences which result in mispairing during recombination, or slippage of the DNA replication machinery [Hu et al., 1990; Cooper and Krawczak, 1993; Galvagni et al., 1994]. If unequal cross-over during meiotic recombination were the mechanism that lead to exon 2 duplication, then we would expect to see reciprocal exon 2 deletion in some patients. To the contrary, deletion of exon 2 has never been reported in either DMD or BMD (Leiden muscular dystrophy pages). Taken Fig. 2. Detection of an 11 bp deletion of exon 23 by direct sequence analysis of cDNA. Shown are RT-PCR products from muscle biopsy RNA from DMD patient no. 7 and three normal controls (panel A). No difference in size is seen in the 350 bp RT-PCR product, however direct sequence analysis shows an intra-exon deletion of 11 bp (panel B). Note that the deletion seems to have been caused by recombination between a short repeat sequence (ATTTC) (dots) flanking the deleted region. together, this data suggests that there is a region of homology between intron 1 and 2 that leads to intragenic slippage or mispairing during replication, leading to the duplication of exon 2. Given the extremely large size (190 and 170 kb) of introns flanking exon 2, it is not surprising that a deletion or duplication of exon 2 by itself is a mutation that has been found more than once. Two cosmids have been sequenced which contain exon 2 (130 kb) and exon 3 (100 kb), however these two cosmid sequences do not overlap. Interestingly, White et al.  recently reported that duplication of exon 2 is the single most common duplication, occurring five times among his patients. However, no deletion of exon 2 alone has so far been reported (Leiden Muscular, Dystrophy Pages). We attributed the previously unreported duplication of exon 2 is due to a number of confounding technical issues: (1) exon 2 is a particularly small exon of only 64 bp. The early characterization of deletion and duplication mutations in DMD/BMD patients was done using Southern blot analysis of genomic DNA, with the dystrophin cDNA as a probe to detect exon-containing Fig. 3. Duplication of exon 2 is seen by RT-PCR and direct sequencing in two unrelated DMD patient muscle biopsy cDNAs. Shown are 350 bp RTPCR products from muscle biopsy cDNA (panel A), where two unrelated patients (no’s 1, 4) show an abnormally large amplicon relative to controls. Direct automated sequence analysis of patients showed a duplication of exon 2 (panel B). 46.5 50.0 0% Faint 26.2 30.5 0% Faint 66.8 61.0 0% Faint 27.1 30.2 0% Faint 69.2 72.8 1% of 417 kDa Faint 7,800 Negative 12,000 Positive (two maternal uncles) 12,000 Negative 1 3 3 10 12 13 14 15 Delayed developmental mile stones Onset at the age of 3 years Progressive proximal girdle weakness Calf hypertrophy Waddling gait Slow progressive lower extremity weakness, easy fatigue Calf hypertrophy Still ambulant Negative 35,000 Faint and variable Nice group of revertants 60 kDa and d10 Faint and variable 60 kDa and d10 Faint and variable 60 kDa and d10 Faint and variable Nice group of revertants 60 kDa and d10 Faint and variable 25,000 Positive family history (maternal lineage) 8 11 Onset at the age of 5 years Progressive proximal girdle weakness Still ambulant CK screening a-sarcoglycan Immunostaining Dystrophin CPK IU/L Family history Clinical presentation Age at biopsy restriction fragments [Koenig et al., 1987; den Dunnen et al., 1989]. Thus, only 64 bp hybridizes to exon 2 containing genomic restriction fragments, which leads to a relatively weak and variable hybridization signal on Southern blot. As a result, it was probably difficult to ascertain duplication mutations of exon 2, (2) the restriction enzyme typically used for study of the dystrophin gene in genomic DNA was HindIII. In HindIII digest, the genomic fragment containing exon 2 was very closely spaced with fragments containing exon 1 and 5 (exon 5 ¼ 3.1 kb, exon 1 ¼ 3.2 kb, exon 2 ¼ 3.25 kb), making discernment of dosage abnormalities of the weakly hybridizing exon 2 technically problematic, (3) duplications are inherently more difficult to detect than deletions in hemizygous males, as 2:1 dosage relative to control individuals must be observed, rather than the simple absence of a band. Thus, methods that are not strictly quantitative, such as PCR from genomic DNA, are not adequately sensitive for detecting duplications, and (4) the failure to see the duplication of exon 2 in early studies led to exclusion of exon 2 in multiplex PCR reactions, so exon 2 is not assayed in the now-standard 18 exon multiplex PCR [Chamberlain et al., 1988; Beggs et al., 1990]. We observed that patients with duplication mutations (exon 2 and 43) showed some common clinical and histological features, including (1) weakness was milder than typically seen in age-matched DMD patients; all were fully ambulant at 12 years of age, (2) their muscle biopsies showed a particularly high rate of ‘‘revertant fibers’’ (data not shown). Mutation analysis revealed that revertants occurred in DMD with identifiable deletions or duplications and in non-deletion patients. Revertants are most likely due to a second-site mutation occurring in a somatic cell allowing restoration of the translational reading frame of the dystrophin transcript [Hoffman et al., 1990; Klein et al., 1992]. A single report has found a correlation between the presence of revertant fibers and lessening clinical course [Nicholson et al., 1993], and it Pat no. Fig. 4. Duplication of exon 43 is seen by RT-PCR and direct sequencing in DMD patient muscle biopsy cDNAs. Shown are 350 bp RT-PCR products from muscle biopsy cDNA (panel A), where the patient (no. 3) show an abnormally large amplicon relative to controls. Shown also a low level on normal sized transcript in the patient’s cDNA. Direct automated sequence analysis of patients showed a duplication of exon 43 (panel B). TABLE III. Clinical and Molecular Features of Mutation-Negative DMD Patients Immuno-blot 50 30 Hamed and Hoffman RNA quantitation % of control 48 Point Mutation Detection would be interesting to determine if patients showing such a milder course might have the same duplication mutation as we identified here. Interestingly, we were able to detect a low level of normal size transcript in the cDNA of the patient with exon 43 duplication (Fig. 4). We also identified a series of novel stop codon mutations (Gln625X, Ser757X, Glu1353X, Arg1967X [Saad et al., 1993], Gln2198X). Although, it has been postulated that there should be a possible impact of premature translation termination on the steady level of mRNA, premature termination of translation destabilize the mRNA released by the polysomes [Baserga and Benz, 1988; Chelly et al., 1991; Culterson, 1999; Hentze and Kulozik, 1999]; It has also been claimed that premature stop codons that may result from nonsense or frame shift mutations can induce intranuclear instability of the transcript and impairs its transport to the cytoplasm [Takeshita et al., 1984; Humphires et al., 1994]. Although mutation detection obviously is critical for diagnosis, it may also be important for future therapeutic purposes. Recent reports have showed the potential use of read-through protein synthesis (Gentamicin) [Barton-Davis et al., 1999] and exon skipping (with antisense oligoribonucleotides) [van Deutekom et al., 2001] in the restoration of the reading frame of the dystrophin transcript. White et al.  suggested that single-exon duplications, in particular would make an ideal target for exon skipping. The presence of two targets will not double the efficiency but also should produce a normal transcript, leading to a wild-type protein [Barton-Davis et al., 1999; Wagner et al., 2001]. We also identified two small deletions (delA2882, del(11 bp) 3245-3255). The mechanism generating the deletion of 11 bp is likely slipped-mispairing at the replication fork between a 5 bp repeat due to the presence of repetitive DNA sequence in the vicinity (ATTTC) [Kunkel and Soni, 1988]. Although the whole dystrophin coding region and the 535 bp of the gene promoter were screened for pathogenic mutations, we failed to identify the disease causing mutation in 1/3 of patients. The clinical presentation and disease progression in such patients were consistent with DMD, as were serum CK levels. Interestingly, all five patients showed faint and variable immunostaining results for both amino- and carboxyl-terminal dystrophin and alpha-sarcoglycan antibodies, yet showed no detectable dystrophin by immunoblot. This data suggested that some residual dystrophin might be produced by these patients that was not detected by immunoblot studies. We suggested that such patients might have a problem with the 3.5 kb 30 UTR, polyA site or undetected stop codons. It is also possible that splicing defects could result in addition of intron sequence that could lead to preferential amplification of low level residual normal transcript skipping [Cooper and Krawczak, 1990]. Germline mosaicism has been reported elsewhere [Bakker et al., 1987; Wood and McGillivray, 1988] and would not necessarily be detectable by use of the available methods. Another less likely reason, is mutation in a gene that is involved in the regulation of dystrophin expression. Alternatively, we suggested that mutation negative patients may have reduction in dystrophin protein in muscle that is secondary to other detect; i.e., not ‘‘primary’’ ‘‘dystrophinopathy’’ and this has also been suggested by others [White et al., 2002]. Studying the steady state RNA levels in muscle biopsies of mutationnegative patients using QMF-PCR, to test the possibility of transcription abnormality, revealed that there was an equal levels of 50 and 30 RT-PCR products, indicating that there was no missed stop codon mutation or splicing error. However, all showed relatively mild reductions in dystrophin RNA, ranging from 25% to 60% of normal. While the reductions in RNA might suggest a promoter mutation or mRNA metabolism defect, we find it hard to rationalize the complete lack of dystrophin protein in those patients’, despite the persistence of apparently 49 normal RNA at 25%–60% levels, further studies are needed. 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