A small intraexonic deletion within the dystrophin gene suggests a possible mechanism of mutagenesis

A small intraexonic deletion within the dystrophin gene suggests a possible mechanism of mutagenesis
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  Abstract A case of Duchenne muscular dystrophy is de-scribed with an unusual mutation consisting of a 17-bpdeletion within exon 47 of the dystrophin gene. The se-quences on either side of the deletion have a high degreeof intrastrand base complementarity. It is hypothesisedthat the mechanism generating the deletion may havebeen the formation of a hairpin loop structure in a singlestrand of DNA followed by enzymatic degradation at un-paired regions within the loop. Introduction Duchenne muscular dystrophy (DMD) is an X-linked re-cessive disorder affecting approximately 1 in 3500 new-born males. It is a severe muscle wasting disease and pa-tients rarely survive beyond the age of 20 years (Emery1991). It is caused by mutations in the dystrophin gene lo-cated on the X chromosome at Xp21. Approximately 65%of cases have large intragenic deletions of many thou-sands of bases and the remainder have other mutationssuch as partial gene duplications or point mutations. Onlya small minority of cases are caused by microdeletions of a single base or a few bases (Table 1).Different mechanisms must be responsible for the gen-eration of the different types of mutation, but there is asyet no detailed knowledge of the molecular events in-volved. However, the presence of repeat sequences closeto each other has been implicated in the generation of  small deletions in both dystrophin and other genes (Al-bertini et al. 1982; Kunkel 1990; Krawczak and Cooper1991). Materials and methods SubjectThe proband presented at 5 years of age with a waddling gait anddifficulty climbing steps. He was positive for Gowers’ sign and hisserum creatine kinase level was grossly raised at 17000 IU/l (nor-mal levels = 25–195 IU/l), consistent with a diagnosis of DMD.There was also a family history of the disease. The family pedigreeis shown in Fig.1.DNA analysisDeletion analysis of the proband was performed using PCR multi-plex amplification of the 18 most commonly deleted dystrophinexons, as described by Abbs et al. (1991). Subsequent mutation analysis in other family members was performed using a multi-plex reaction including primers for exons 43, 44, 47, 50 and 60(Fig.1) . For sequence analysis, genomic DNA samples from the proband and a control were amplified with primers 47F and 47R (Abbs etal. 1991) for 30 cycles. The amplification products were purifiedusing the Wizard system (Promega) and directly sequenced usingprimer 47F end-labelled with γ  -[ 32 P]ATP(3000 Ci/mmol) and theAmplitaq cycle sequencing kit (Perkin-Elmer).Computer-aided molecular modellingThe MFOLD program (Zuker and Stiegler 1981; Jaeger et al.1989; Zucker 1989) of the Wisconsin Genetics software package(Devereux et al. 1984) was used to identify single-stranded DNAconfigurations potentially involved in the deletion mechanism.The MFOLD algorithm identifies possible secondary structures ac-cording to the thermodynamics parameters involved in hydrogenbonding and RNA hairpin loop folding. It should be noted thatMFOLD scores one hydrogen bond for mismatched GT base pairs. DavidO. Robinson · DavidJ. Bunyan ·HenryA. Gabb · I.Karen Temple · ShuC. Yau A small intraexonic deletion within the dystrophin gene suggests a possible mechanism of mutagenesis Hum Genet (1997) 99:658–662©Springer-Verlag1997Received: 4 January 1997 / Revised: 21 January 1997 ORIGINAL INVESTIGATION D.O. Robinson (  ) · D.J. BunyanWessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury SP2 8BJ, UKTel.: +441722 336262 ext. 4080; Fax: +441722 338095H.A. GabbImperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, UKI.K. TempleWessex Clinical Genetics Service, Princess Anne Hospital, Southampton S016 5YA, UKS.C. YauPaediatric Research Unit, Division of Medical and Molecular Genetics, United Medical and Dental Schools of Guy’s and St. Thomas’, Guy’s Tower, Guy’s Hospital, London SE1 9RT, UK  The sequence used in the analysis is shown below and includes thedistal 23 bases of intron 46 and the proximal 59 bases of exon 47.1GTTGTTGCATTTGTCTGTTTCAGTTACTGGTGGAA-GAGTT41 GCCCCTGCGC CAGGGAATTC TCAAACAATT AAATG-AAACT81 GGMFOLD was allowed to fold this structure freely without con-straints or biasing parameters. Results PCR analysis of exon 47 of the dystrophin gene in theproband resulted in a PCR product which was slightlysmaller than that in the normal controls (Fig.1). Themother of the proband was heterozygous for the deletion.The proband’s maternal aunt and his phenotypically nor-mal maternal uncle and maternal grandfather had only theband of normal size. Sequence analysis identified a 17-bpdeletion of bases 6982–6998, bases 6983–6999 or bases6984–7000 within exon 47 of the dystrophin gene (Fig.2).Use of the MFOLD prediction algorithm to identifypotential configurations of the single-stranded DNA sequence including the deleted bases and surroundingDNA showed a variety of theoretically stable secondarystructures. Two likely structures in which both deletionbreakpoints are situated in unpaired regions are shown inFig.3. Discussion The proband has a 17-bp deletion within exon 47 of thedystrophin gene. This causes a frameshift mutation which is likely to cause DMD rather than the milder Beckerform of the disease (Monaco et al. 1988) and is consistentwith the proband’s phenotype. The proband’s mother alsocarries the deletion but her sister does not (Fig.1).This mutation can be easily identified in both affectedmales and female carriers by the presence of a product of unique size following PCR amplification of exon 47. Thesize of this deletion is unusual in the dystrophin gene and 659 Table 1 Small deletions in thedystrophin gene causingDuchenne (DMD) or Beckermuscular dystrophy (BMD) .(  IMD Phenotype intermediatein nature between DMD andBMD,  N-term N-terminal do-main of the dystrophin protein,  R rod domain,  H  hinge region, CYS  cystein-rich domain, C-term C-terminal domain, 3 ′    UTR 3 ′ untranslated region) a This deletion could also beA2438–G2439 b Described by Prior et al.(1993) to be C2568/69/70 butthis is at variance with the cur-rent Genome Data Base dys-trophin gene sequence c This deletion could also beA2670–G2673 or G2671–A2674 d This deletion could also beG3158-22–T3161 or G3158-20–A3163 e This deletion could also beG3669–G3676 f  This deletion could also be G6983–A6999 or A6984–G7000Size ofNucleotidesExonProteinPhenotypeReferencedeletion (bp)deleteddomain1A263/4/5/62N-termIMDPrior et al. (1995)2A382–A3833N-termIMDRoberts et al. (1994)7T393–G394 + 53N-termDMDTuffery et al. (1996)1C640/16N-termDMDTolun et al. (1994)1C7246N-termDMDRoberts et al. (1994)1C8526N-termDMDKavaslar et al. (1995)1A983/4/58N-termDMDPrior et al. (1995)11A989–A9998N-termDMDRoberts et al. (1994)1T2181/216R3DMDPrior et al. (1994)2A2436–G2437 a 18R4DMDTuffery et al. (1996)52G2532–T258319R4DMDMatsuo et al. (1990)1C2565/6/7 b 19R4DMDPrior et al. (1993)4G2669–A2672 c 20R4, R5DMDBarbieri et al. (1996)26G3158–T3162 d 23R6DMDBarbieri et al. (1996)1G3542/3/425R7IMDNigro et al. (1994)8G3668–T3675 e 26R8DMDNigro et al. (1994)13T4239–A425129R9DMDPrior et al. (1995)1C640843R16DMDKneppers et al. (1993)5T6643–G6646 + 144R16DMDSaad et al. (1992)12A6644–G6646 + 944R16DMDKneppers et al. (1995)17A6982–C6998 f  47R18DMDThis study1G8290/1/2/3/455R22DMDBarbieri et al. (1996)1C9297/861H4DMDPrior et al. (1995)1C9440/1/265CYSDMDTsukamoto et al. (1994)4T9679–T968265CYSDMDRoberts et al. (1994)10G10060–G1006968C-termDMDPrior et al. (1995)1T1019469C-termDMDPrior et al. (1995)1T1066274C-termIMDLenk et al. (1993)1T1066274C-termDMDRoberts et al. (1992)1C10683/4/574C-termBMDRoberts et al. (1994)1 + 7T10691/274C-termDMDLenk et al. (1993)+ A10694–C106991A10770/175C-termDMDPrior et al. (1995)13G11283–G11295–3 ′ UTRIMDNigro et al. (1994)  its detection in females is, paradoxically, considerablyeasier than detection of the majority of dystrophin dele-tions which consist of many thousands of bases, includeone or more exons and have intronic breakpoints.There is a paucity of documented cases of such smalldeletions (arbitrarily designated in this report as 1–100bases in length) in the dystrophin gene, although they arenot uncommon in human genes (Krawczak and Cooper1991). Only 15 cases of deletions of more than one baseand less than 100 bases of the dystrophin gene have beenpreviously reported (Table 1).The majority of deletions presented in Table 1 (17 outof 33) are of a single base. It is of interest to note that 8 of these 17 are deletions of cytosine, despite cytosines ac-counting for less than 20% of the dystrophin coding se- quence. However, Krawczak and Cooper (1991) in a study of small mutations in other genes did not find an excess of cytosine deletions in 16 single base deletions. Similarly, asearch of the mutation databases ( mg/hgmd0.html) of four genes (the factor VIII, factor IX,CFTR and adenomatous polyposis coli genes) showed noexcess of cytosine deletions (20 out of 101 single basedeletions were deletions of cytosine). Of the 17 singlebase dystrophin deletions listed in Table 1, 12 occurred insequences where the same nucleotide was repeated morethan once, suggesting nascent chain slippage during DNAreplication as a causative mechanism (Kunkel and Soni1988). Only 16 cases involve deletions of more than onebase. None of the deletions involves a multiple of threebases, however, such deletions are expected to cause dis-ease only rarely because they do not disrupt the readingframe.Krawczak and Cooper (1991) have studied the srcinof mutations in genes other than dystrophin and suggestthat small mutations are generated by mispairing duringDNA replication and that the length of the single-stranded 660 Fig.1 Pedigree of the family studied and multiplex PCR analysis,showing the agarose gel electrophoresis of products of multiplexPCR amplification of exons 44 (top) , 43, 50, 47 and 60 of the dys-trophin gene. [ 47D Band representing exon 47 with the 17-bp dele-tion, track 1 II.1, track 2 II.2, track 3 II.3, track 4 I.1, track 5 III.1(proband). Samples were unavailable from individuals I.2, I.3 andI.4] 1 2 3 4 547D Fig.2 Sequence of the 17-bp deletion in exon 47 of the dystrophingene plus flanking sequences ( 1, 2 and 3 the three possible deletedsegments, bold type deleted bases) Fig.3a, b The two secondary structures predicted by MFOLDwhich are most likely to result in the 17-bp deletion seen in theproband. The deleted segment is represented in bold type . Intronicsequence is represented in lower case  replication fork, 1000–2000 bases, may impose a physicallimit on the size of small deletions. This suggests that dif-ferent mechanisms exist for the generation of small andlarge deletions. With regard to the dystrophin gene, largedeletions of many thousands of bases are known to occurpreferentially in two deletion hot spots between exons 42and 60 and 5 ′ of exon 20 (Darras et al. 1988; Malhotra etal. 1988; Den Dunnen et al. 1989; Gillard et al. 1989;Koenig et al. 1989). There is no indication from the fewdocumented cases of small deletions that they also occurpreferentially in these areas.Albertini et al. (1982) and Kunkel (1990) have sug-gested that repeat sequences close to each other may gen-erate mispairing during DNA replication, resulting insmall deletions. A study of the breakpoints of a number of deletions of less than 20 bases in genes other than dys-trophin has found them often to be flanked by short directrepeats (Krawczak and Cooper 1991). Similarly, Saad etal. (1992) have reported a 5-bp dystrophin gene deletionwith TCT sequences closely flanking it on either side. The17-bp deletion in the present study is also flanked by re-peat sequences. There are three possible pairs of break-points deducible from the sequence shown in Fig.2. Inpossibility 1, the deletion is flanked by an AGG invertedrepeat and possibilities 2 and 3 are both closely flanked byGGAA repeats. However, possibility 1 would not be ex-pected to be the result of slippage because the flanking re-peat is inverted and in possibilities 2 and 3 slippage wouldbe expected to result in the deletion of 18 bases instead of 17. It therefore seems unlikely that replication slippagecaused by repeat sequences flanking the deletion has gen-erated the mutation in this case.However, the sequence environment on either side of the deletion is of interest because it exhibits a high degreeof intrastrand complementarity (Fig.3) and this may pre-dispose to the formation of a hairpin loop during DNAreplication when DNA becomes single stranded. In theabsence of constraints, there are many ways in which asingle-stranded sequence of this size may fold. However,in this case the deleted sequence is known. Also, deletionbreakpoints in general are known to srcinate in unpairedregions of loops because they are prone to enzymatic at- tack (Ripley 1982; DasGupta et al. 1987), therefore MFOLD folding patterns in which the cleavage of the 17-bp seg-ment begins and ends in a single-stranded region are themost feasible. Two structures proposed by the MFOLD al-gorithm which satisfy these criteria are shown in Fig.3.The proposed deletion breakpoints lie within the unpairedregion of the hairpin loop. They are also between bases at the start of unpaired regions which are thought to be under greater physical strain (Gralla and Crothers 1973; Borer etal. 1974). In both cases there is a GC base pair at the baseof the loop. This is known to have a strong stabilising ef-fect (Uhlenbeck et al. 1973). Both structures predict that the 17-bp deletion spans dystrophin bases 6984–7000 (pos- sibility 3 in Fig.2).Matsuo et al. (1992) reported the presence of in-trastrand complementarity in all of the 23 dystrophin ex-ons which they examined (exons 3–7, 10, 12, 17, 19, 22,36, 37, 41, 44, 45, 47–51, 53–55) and proposed that thismay be an essential feature of exons because loop struc-ture formation is necessary for the correct splicing of pri-mary RNA transcripts. In the deletion presented here, theregion of high autocomplementarity included the last 23bases of intron 46 and the following 59 bases of exon 47.We analysed the same number of bases at intron/exonboundaries for several other exons of the dystrophin geneusing the MFOLD algorithm. A degree of autocomple- mentarity was found in all sequences studied but, although the i46/e47 sequence showed a slightly higher than aver-age score, this difference was not statistically significant(data not shown).In conclusion, we have described a unique 17-bp dele-tion within the dystrophin gene. The presence of in-trastrand complementarity in the region of the deletion re-sulting in loop formation during DNA replication may bethe causative mechanism of the mutation. However, thereis no evidence to show that such deletions are more likelyto occur at this position within the gene. References Abbs S, Yau SC, Clark S, Mathew CG, Bobrow M (1991) A con-venient multiplex PCR system for the detection of dystrophingene deletions: a comparative analysis with cDNA hybridisa-tion shows mistypings by both methods. J Med Genet 28:304–311Albertini AM, Hofer M, Calos MP, Miller JH (1982) On the for-mation of spontaneous deletions: the importance of short se- quence homologies in the generation of large deletions. 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