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1、 ORIGINAL ARTICLE Cloning and sequencing of junction fragment with exons 45-54 deletion of dystrophin gene ZHONG Min，PAN Su-yue，LU Bing-xun，LI Wei （Department of Neurology，Nanfang Hospital，Southern Medical University，Guangzhou，Guangdong，510515 P . R . China） Corresponding author：PAN Su-yue，Email：pan
2、suyue82yahoo . com . cn 【Abstract】 ObjectiveTo study the mechanisms of dystrophin gene deletion，the junction fragment with exons 45-54 deletion were cloned and sequenced. MethodsA Duchenne muscular dystrophy（DMD）patient with exons 45-54 deletion has been substantiated by PCR amplification of the exo
3、ns. Then we used a PCR-based genome-walking method for localizing the breakpoints in introns 44 and 54. At last，the deletion-junction fragment was directly amplified by PCR approach with forward and reverse primers annealing to a DNA sequence as close as possible to the breakpoints in introns 45 and
4、 54. The sequencing result of the deletion-junction fragment was compared with the normal intronic sequences. ResultsA total of 2716 bp sequence containing the junction fragment was obtained. The 5 breakpoint was located in LINE/ L1 element of intron 44 and close to a matrix attachment region（MAR） .
5、 The 3 breakpoint was located in the mi- nor potential MAR with topoisomerasecleavage sites around. Beside the 3 breakpoint there was a 6 bp palindromic sequence. A 4 bp microhomologous sequence（AGAG）was in the joint of the deletion-junction fragment. Conclusion The nonhomologous recombination cause
6、d by L1 repeated element，topoisomerasecleavage sites，MARs and the non- homologous end joining of microhomologous sequence may be the important factors in this huge gene fragment deletion. 【Key words】 Duchenne muscular dystrophy； junction fragment； gene deletion INTRODUCTION Dystrophin gene mutation
7、is the molecular pathological ba- sis for pseudohypertrophic muscular dystrophy，a lethal X- linked recessive disease. Dystrophin gene mutation give pri- ority to deletion which accounts for 55%-65% in all the mutants. It is the principal method in exploring the mecha- nism of dystrophin gene deletio
8、n to know the position of local breakpoint of gene deletion and features of sequence contain- ing junction fragment by cloning and sequencing dystrophin gene deletion-junction fragment. At present the data of sequence containing dystrophin gene deletion-junction fragment are comparatively scarce. In
9、 order to further study the mechanism of dystrophin gene deletion，we use PCR to fulfill the cloning and sequencing of long fragment gene deletion-junction fragment in exons 45- 54，and give an analysis of breakpoint and gene deletion- junction fragment. MATERIALS AND METHODS Patient data A 9-year-old
10、 male Duchenne muscular dystrophy（DMD） patient without family history of DMD was substantiated with exons 45-54 deletion of dystrophin gene. Reagents Taq Plus DNA polymerase and dNTP were purchased from Shanghai Sangon Biological Engineering Technology & Ser- vice Co.，Ltd. LA Taq DNA polymerase in a
11、mplifying long fragment from TaKaRa Biotechnology（Dalian）Co.，Ltd. Agarose was imported from Biowest Agarose，Hispanagar Company，Spain. Other reagents were home made analyti- cally pure. Primer design This experiment involves 24 pairs of exon primers，of which 18 pairs are designed referring to literat
12、ure of Cham- berlain，et al . 1and Beggs，et al .2，while the other 6 pairs designed referring to literature of Abbs，et al . 3and Prior，et al . 4and obtained from Leiden Muscular Dystro- phy Pages（http： / / www.dmd.nl） . The experiment involves 27 pairs of intron primers consisting of 17 pairs of intro
13、n 44 and 10 pairs of intron 54. The design was fulfilled by primer on-line design program（http： / / frodo.wi.mit.edu/ cgi-bin/ primer3/ primer3-www. cgi） . The design principle of intron 44 forward primer is to design 5 pairs of primer，which di- vides the whole intron sequence into 6 tested regions
14、with the length about 40 kb roughly on average. One pair of primer was designed every 3 kb sequence in the confirmed break- point localized region. The design principle of intron 54 for- ward primer is to directly design 1 pair of primer every 3 kb sequence. Both primer designs are limited with thei
15、r PCR sequence length within 301-400 bp. In order to improve the specificity of cloned gene deletion-junction fragment，we re- designed 1 pair of matched-pair primer close to the break- point of intron 44 and intron 54（D5-F： 5- GTTATCTTATG- GATGCACGGTTTTG-3，D5-R：5-AGATTCTTCAGAGAT- CATGGATGGA-3） to be
16、 used in long fragment PCR amplifi- cation of joint fragment. All the primers mentioned above were synthesized by Shanghai Invitrogen Biotechnology Co.， Ltd. Cloning of exons 45-54 deletion-junction fragment Genomic DNA of the DMD patient mentioned above was 831中华医学遗传学杂志 2006 年 4 月第 23 卷第 2 期Chin J
17、Med Genet，April 2006，Vol. 23，No. 2 extracted and prepared by routine phenol-chloroform meth- ods. The patient was confirmed to have exon deletion in exon 45 after primarily screening by PCR on 18 pairs of exon primers，and confirmed to have exons 45-54 deletion after adding PCR on exons 46，49，53-56 p
18、rimers. PCR condi- tion：at 96 for 2 min； at 94 for 30 s， annealing（the concrete temperature was determined by Tm value of each prime）for 30 s， at 72 for 1 min， 35 cycles； at 72 for 10 min. The appropriate position of the breakpoint in introns was found by using designed intron primers. If one pair o
19、f in- trons was amplificated into normal positive result while the other pair negative result in the neighboring 2 pairs of intron primers，we could judge the appropriate position of the breakpoint in introns. PCR condition is the same as above paragraph. Long fragment PCR amplification was directly
20、conducted on junction fragment of primers D5-F and D5-R. PCR con- dition：at 96 for 2 min； at 94 for 30 s， at 54 for 30 s， at 72 for 3 min， 35 cycles； at 72 for 10 min. Sequencing of gene deletion-junction fragment Sequencing after purifying successfully PCR production. The sequencing was performed b
21、y Shanghai Sangon Biologi- cal Engineering Technology & Service Co.，Ltd by de- terming double sequence of PCR production. Analysis of features of junction fragment and breakpoint sequence The determined sequence and normal introns sequence were compared to analyse the molecular features of 100 bp se
22、quence both sides of 3 breakpoint and 5 breakpoint. Analysis of intron sequence repeat was adopted by Repeat- Masker program（http： / / www. repeatmasker. org/ cgi-bin/ WEBRepeatMasker） ，matrix attachment regions（ MARs） analysis by MAR-Wiz program （http： / / www.futuresoft.org/ MAR-Wiz） . RESULTS Pro
23、ved by PCR reaction of intron primer，5 breakpoint was appropriately located in about 178 kb site in intron 44， and 3 breakpoint was appropriately located in about 21 kb site in intron 54. The production fragment of about 2.7 kb in length was amplified from long fragment PCR reaction in gene deletion
24、-junction fragment（Fig.1） . A total of 2716 bp sequence containing the junction frag- ment was obtained，which was proved to be dystrophin gene fragment by comparing the similarity with http： / / www. ncbi.nlm. nih. gov/ genome/ seq/ HsBlast. htm with the total deletion length about 402 kb. The 5 bre
25、akpoint was located in 176998-177190 sites of LINE/ L1 of intron 44，and 3 breakpoint located in the unique sequence. MARs analysis showed that the 5 breakpoint was happen to be located in the reverse stream from a MAR 1897 bp of intron 44. The 3 breakpoint was not close to MAR of intron 5 4 ，but it
26、was Fig.1The agarose gel electrophoresis of the PCR amplified product of junction fragment.M： DL15000 marker；lane 1：the PCR amplified product of about 2.7 kb from a DMD patient；lane 2：normal control located in the minor potential region possibly formed by in- tron 54. Topoisomerasecleavage sites wer
27、e not found in forward and reverse sequences from 5 breakpoint. Topoiso- merasecleavage sites were found in 20230 and 20373 sites in normal chain and in 9869 and 9879 sites of abnormal chain of intron 54 near 3 breakpoint. At the same time， beside the breakpoint in intron 54 there was a 6 bp palin-
28、dromic sequence. No extensive homogeneity was found in gene deletion conjuncted sequence in this case，but 4 bp microhomologous sequence（AGAG）was in the joint of the deletion-junction fragment（Fig.2 and Fig.3） . DISCUSSION Because of the previous complicated methods and lower success rate in cloning
29、dystrophin gene deletion-junction fragment，the present data of junction fragment sequence are not so much. Since cDNA probe was used to find junction fragment under the condition of intron unknown in dystrophin gene，there are only about 50 cases of data of junction frag- ment sequence in literatures
30、 at home and abroad. But since the first dystrophin gene deletion-junction fragment was suc- cessfully cloned with reversed PCR by Pan，et al . 5and Sheny，et al . 6 at home，there are only 2 cases of se- quence analysis of junction fragment at home. At present dystrophin gene sequence of about 2500 kb
31、 in length，the largest gene of the human being，has been basically defi- nite，which has provided us with foundation to find out breakpoint sites in introns using PCR walking method. This is the first time in China to find the approximate gene break- point sites by PCR walking method，and perform PCR a
32、m- plification and sequencing of gene deletion-junction fragment by successfully adopting a pair of primers in forward stream and reverse stream near breakpoints.Experiments have proved this method，with higher feasibility and reliable re- sults，as a new promising accurate method for the carrier test
33、. The previous research results have proved，though there are differences in deletion length and sites，most dystrophin gene deletions are located in central deletion potential region containing exons 40-50 and minor potential region containing intron 7，which also represent for the recombination poten
34、tial region of meiosis 7 . 5 breakpoint of this case was located 931中华医学遗传学杂志 2006 年 4 月第 23 卷第 2 期Chin J Med Genet，April 2006，Vol. 23，No. 2 Fig.2The sequence of deletion-junction fragment from a DMD patient with exons 45-54 deletion. 2a： the forward sequence； 2b： the reverse sequence. Arrows indica
35、te the 4 bp microhomologous sequenceFig.3Nucleotide sequence spanning the deletion junction，with the corresponding normal intron sequences.Numbers in brack- ets indicate the breakpoint position with respect to the first nucleotide of the intron（I44，I54） . The sequence in the box is LINE/ L1 repeated
36、 element. The shaded char- acter is microhomologous sequence. Arrows indicate palindromic sequence in intron 44 in central deletion potential region. Intron 44 is the biggest intron in dystrophin gene，whose deletion fre- quency is the highest due to the effect of its length 8. Study by Sirioni，et al
37、 . 9showed that breakpoints in intron 44 might spread its sequence with the length of 248 kb，and no aggregating trend in certain sites. But by analyzing the fea- tures of breakpoints and 100 bp forward and reverse se- quence，we still could find some features of gene deletion breakpoint in this case，
38、i.e.，5 breakpoint was located in LINE/ L1 element of intron 44 and close to a MAR. The 3 breakpoint was not close to MAR in intron 54，but MAR- Wiz analysis showed the site in intron 54 might form a minor potential MAR with topoisomerasecleavage sites around. Now sequence repeat and correlation with
39、topoisomerase cleavage sites might be some factors involved in DNA dou- ble-strand fragmentation 6， 10. For example，L1 repeated el- ement is one of the important sequence repeats in human genome. Studies have shown that L1 repeated element would often make gene recombined by many kinds of mecha- nis
40、m 11，so it is an important reason for the instability of in- trons. The nonhomologous recombination caused by L1 repeated element in intron 54 and one unique sequence in intron 54 might be an important factor in inducing this huge gene frag- ment deletion with the length reaching 402 kb. Although pr
41、esent literatures have not proved there might be significant correlation between MARs and gene breakpoints，partial breakpoints have been actually found to aggregate in neigh- boring MARs 10. MARs are believed to be the region close- ly related to DNA metabolism process，such as duplication， repair， t
42、ranscription， relaxationoftorsionaltension， etc . 12. So we have reasons to hypothesize two MARs might form in intron 44 and larger potential of intron 54，like gal- lows of chromosome which forms chromatin loop by pulling the two elements in the distant breakpoint in physical space， which is an impo
43、rtant reason for ultra-long gene deletion fragmentation. This is in accordance with the fact that repeat element is liable to form hairpin loop structure that induces nonhomologous recombination. When DNA double strand happens to be fragmented，cells can have two kinds of repair with the mechanism be
44、ing ho- mologous combination and nonhomologous end joining. Now most dystrophin gene deletions are found to have repair by nonhomologous end joining. Nonhomologous end joining of- ten manifests junction features with three kinds of character- istic breakpoint：insertion of 1-5 bp nucleotide element，2
45、-4 bp microhomologous sequence and short element repeat in different lengths. Nonhomologous end joining takes an cen- tral role in the process of nonhomologous gene recombina- tion，which is one of the major ways to manifest cells active- ly adapting in DNA double strand fragmentation 13. Be- sides，D
46、NA fragmentation joint or its nearby palindromic se- quence could promote this junction 14. A 4 bp microhomol- ogous sequence（AGAG）was found in the joint of the dele- tion-junction fragment to this case. Meanwhile，we also found a 6 bp palindromic sequence was in the joint of frag- mentation in intro
47、n 54，which might be another important factor to cause the liability of fragmentation recombination in 041中华医学遗传学杂志 2006 年 4 月第 23 卷第 2 期Chin J Med Genet，April 2006，Vol. 23，No. 2 that region. FUNDING/ SUPPORT： This work was supported by the Guangdong Natural Science Foundation（Proj. No. 001032） REFER
48、ENCES 1Chamberlain JS，Gibbs RA，Ranier JE，et al . Multiplex PCR for the di- agnosis of Duchenne muscular dystrophy. In：PCR protocols：a guide to methods and applications. San Diego：Academic Press，1990. 272-281. 2Beggs AH，Koenig M，Boyce FM，et al . Detection of 98% of DMD/ BMD gene deletions by polymera
49、se chain reaction. Hum Genet， 1990，86:45-48. 3Abbs S，Yau SC，Clark S，et al . A convenient multiplex PCR system for the detection of dystrophin gene deletions：a comparative analysis with cD- NA hybridisation shows mistypings by both methods. J Med Genet， 1991， 28:304-311. 4Prior TW，Wenger GD，Papp AC，et al . Rapid DNA haplotyping using a multiplex heteroduplex approach：application to Duchenne muscular dystro- phy carrier testing. Hum Mutat， 1995，5:263-268. 5Pan SY，Zhang C，Liu ZL，et al . Cloning and sequencing of junction