Study Of DMD Gene Deletion Mutation And Using The Junction Fragment For Gene Diagnosis

DMD gene mutation is the molecular pathological basis for pseudohypertrophic muscular dystrophy.The large gene fragment deletion accounts for～65%in all the mutants.According to the reading-frame hypothesis,the frameshift mutations of DMD gene generally result in Duchenne muscular dystrophy(DMD),whereas the codon mutations result in Becker muscular dystrophy(BMD).Although the mechanism of DMD gene deletion is very complicated,it can be found that DMD gene with breaks and deletions are almost repaired by non-homologous end joining, which results in a new unique DNA sequence—DMD gene deletion junction fragment by the illegitimate recombination of the two ends of breakpoints in each case.Cloning and analyzing the sequence features of the junction fragments is useful for studying the mechanism of DMD gene deletion and recombination.We have also considered that using the PCR technique to amplify this unique DNA sequence could be apply to deletional DMD/BMD carrier detection and prenatal diagnosis.This study has 3 chapters:1.Analysis of DMD gene deletion and the sequences of 5 Chinese junction fragments;2.Using the junction fragment for deletional DMD/BMD carrier detection;3.Application and evaluation of the junction fragment for deletional DMD/BMD prenatal diagnosis.1.Analysis of DMD gene deletion and the sequences of 5 Chinese junction fragments (1)ObjectiveUsing the reading-frame rule to analyze the relationship between the genotype and the phenotype of the choosed deletional DMD/BMD patients.Sequencing the Chinese DMD gene deletion junction fragments to analyze some features of them and discuss the mechanism of DMD gene deletion and repair.(2)MethodsStudy objects:6 definite deletional DMD/BMD patients.Case 1,case 2,case 3, case 4 were affected by DMD.Case 5 and case 6 from a same family were affected by BMD.So actually there were 5 cases that had no blood relationship each other. The peripheral blood genomic DNA of these patients was extracted and prepared.Analysis of DMD gene deletion mutations by the reading-frame rule:Using exon primers for PCR detection,case 1 was substantiated with exons 46-50 deletion of DMD gene,and case 2 with exons 51-55 deletion,case 3 with exons 31-43 deletion, case 4 with exons 45-54 deletion,Case 5 and case 6 from the same family with exons 3-5 deletion.Then the 6 cases were analyzed by on line reading-frame program to know their changed types of open reading frame caused by the exons deletion.Cloning and sequencing of the junction fragments:We first designed the primer at intervals(often 3 kb sequence)in the corresponding introns that the DMD gene deletion breakpoints of the DMD/BMD cases were located in.After the breakpoints in introns of the 5 cases were localized by the PCR-based genome-walking method successfully,we designed 1 pair of matched-pair primer close to the upstream and the downstream breakpoints respectively to be used in long fragment PCR amplification of each junction fragment.Sequencing after purifying successfully PCR productions.Analysis of the sequence features of the junction fragments:The sequences both sides of 5' and 3' breakpoints of 5 forementioned Chinese junction fragments were analyzed by means of repetitive sequence,matrix attachment regions(MARs), palindromic sequence,the sequence TTTAAA and the repair means after gene deletion.(3)ResultsRelationship between the genotype and the phenotype of the 6 cases:Through the detection,the deletion mutations of case 1,case 2,case 3 and case 4 all belong to out-of-frame deletion which is the frameshift mutation changing the genetic information of open reading frame.The corresponding clinical phenotype is DMD. The deletion mutations of case 5 and case 6 from the same family belong to in-frame deletion which is the codon mutation maintaining open reading frame correct downstream the deletion.The corresponding clinical phenotype is BMD.Sequence features of 5 Chinese junction fragments:It was found that the 5' breakpoint of JUNCTION 1 was located in LINE/L1 element of intron 45 and close to a MAR,with a palindromic sequence nearby.It's 3' breakpoint was close to a MAR of intron 50.The 5' breakpoint of JUNCTION 2 had a palindromic sequence nearby.It's 3' breakpoint was located in LINE/L1 element of intron 55.The 5' breakpoint of JUNCTION 3 was located in LINE/L1 element of intron 30,with a palindromic sequence nearby.The 5' breakpoint of JUNCTION 4 was located in LINE/L1 element of intron 44 and close to a MAR.It's 3' breakpoint had a palindromic sequence beside.The 5' breakpoint of JUNCTION 5 was located in SINE/Alu element of intron 2.It's 3' breakpoint was close to a MAR of intron 5,with the sequence TTTAAA nearby.There wasn't extensive homogeneity in gene deletion conjuncted sequence in the 5 cases.A 5 bp microhomologous sequence was found in the joint of JUNCTION 1. The blunt end ligation was found in the joint of JUNCTION 2,with an A→G point mutation at 39 bp site upstream it's 5' breakpoint.A 7 bp inserted sequence was found in the joint of JUNCTION 3.A 4 bp microhomologous sequence was found in the joint of JUNCTION 4.A 26 bp inserted sequence was found in the joint of JUNCTION 5 which formed three 13 bp duplications around it,with an A→G point mutation at 20 bp site upstream it's 5' breakpoint.(4)ConclusionThe relationship between the genotype and the phenotype of the 6 pseudohypertrophic muscular dystrophy cases all fit with the reading-frame rule.4 cases of them are out-of-frame deletions leading to DMD.2 cases of them are in-frame deletions leading to BMD.Some secondary structures of DMD gene facilitate gene breakage and recombination.Overall,the sequences of 5 Chinese junction fragments of our study have all the molecular features including repeated sequence,MARs,palindromic sequence,the sequence TTTAAA,etc,which cause DMD gene deletion and recombination easily.Moreover,5 Chinese junction fragments are all repaired by non-homologous end joining whose characteristic junctions showing microhomologies,small insertions,duplications,or blunt end ligation.2.Using the junction fragment for deletional DMD/BMD carrier detection(1)ObjectiveOn the basis of DMD gene deletion junction fragment is the unique DNA sequence resulted from illegitimate recombination after the gene deletion,a novel approach is presented here for the detection of deletional DMD/BMD carriers with the junction fragments.(2)MethodsStudy objects:The female relations of 2 DMD/BMD families and a sporadic case's family.Family 1 was a BMD family.In this familyâ…¢₁ andâ…¢₄ were definite patients.The probandâ…¢₁(Case 5)had been substantiated with exons 3-5 deletion and its junction fragment(JUNCTION 5)had been cloned.â…¢₁'s motherâ…¡₂ and â…¢₄'s daughterâ…£₄ were definite carriers.â…¢₁'s younger sisterâ…¢₃ was a possible carrier.Family 2 was a DMD family.In this family the probandâ…¢₂(Case 3)had been substantiated with exons 31-43 deletion and its junction fragment(JUNCTION 3)had been cloned.â…¢₂'s motherâ…¡₃ was a definite carrier.Case 4 was a DMD sporadic case that had been substantiated with exons 45-54 deletion and its junction fragment(JUNCTION 4)had been cloned.His mother was to be excluded as a carrier. The peripheral blood genomic DNA of them was extracted and prepared.Carriers detection:On the basis of successful sequencing of the junction fragments,we redesigned the primers that amplified above JUNCTION 5, JUNCTION 3,JUNCTION 4 to elevate the achievement ratio of routine PCR amplifying the junction fragments by cutting short the PCR products length.Then 3 pairs of redesigned primer D4-F/R,D31-F/R2,D6-F/R were used to PCR amplify and sequence the possible junction fragments of the female objects of above 2 families and case 4's mother respectively.At the same time the junction fragments of the patients were amplified as control.(3)ResultsResults of family 1:The PCR amplifications of the junction fragments of 3 femalesâ…¢₃,â…¡₂,â…£₄ of family 1 were the same positive results as those of patientsâ…¢₁ andâ…¢₄.Meanwhile the sequences of the junction fragments ofâ…¢₃,â…¡₂,â…£₄ andâ…¢₁,â…¢₄ had no difference by sequencing comparison.Soâ…¢₃,â…¡₂,â…£₄ of family 1 were all diagnosed as female carriers of BMD.Results of family 2:The PCR amplification of the junction fragment of a femaleâ…¡₃ of family 2 was the same positive result as that of patientâ…¢₂.Meanwhile the sequences of the junction fragments ofâ…¡₃ andâ…¢₂ had no difference by sequencing comparison.Soâ…¡₃ of family 2 was diagnosed as female carrier of DMD.Results of case 4's mother:The PCR amplification of the junction fragment of case 4's mother was negative result.But the control of case 4 was positive result.So case 4's mother was excluded as female carrier of DMD.(4)ConclusionAfter the junction fragment of the DMD/BMD proband was cloned and sequenced,using the routine PCR technique to detect the female relation to be diagnosed having the junction fragment or not directly can achieve the aim of accurate carrier detection.The sequencing results indicate that the junction fragments of the patients and the carriers from the same families are identical,so generally the same family has the same genotype of DMD gene deletion despite the patients from the different families having the different deletions.3.Application and evaluation of the junction fragment for deletional DMD/BMD prenatal diagnosis(1)ObjectiveTo study the approach of using the junction fragment for deletional DMD/BMD prenatal diagnosis.It's advantage and disadvantage has been evaluated through the practice.(2)MethodsStudy objects:Afterâ…¢₃ of family 1 was diagnosed as female carrier,she was choosed as the study object of practising prenatal diagnosis under her agreement. Carrierâ…¢₃ was gravida 3.â…£₁ andâ…£₂ were 2 chorionic villi from her successive artificial abortion.â…£₃ was the fetus of tertigravida to be diagnosed.The chorionic villi at 11 weeks' gestation and the amniotic fluid at 20 weeks' gestation were taken respectively as the samples of prenatal diagnosis.The genomic DNA of these samples was extracted and prepared.The methods of gene diagnosis of the chorionic villi from artificial abortion:The redesigned primer D4-F/R was used to PCR amplify and sequence the possible junction fragments ofâ…£₁ andâ…£₂ respectively.At the same time the junction fragment of probandâ…¢₁ was amplified as control.The primer ex3-F/R was used to identifyâ…£₁ andâ…£₂ with corresponding exons deletion or not respectively.The primer of amplifying SRY gene was used to diagnose the sex ofâ…£₁ andâ…£₂ respectively.The methods of prenatal diagnosis:The redesigned primer D4-F/R was used to PCR amplify and sequence the possible junction fragments of the samples of the chorionic villi and the amniotic fluid at different gestation respectively.At the same time the junction fragment of probandâ…¢₁ was amplified as control.The primer ex3-F/R was used to amplify exon 3 of the 2 samples to identify them with exons deletion or not respectively,with the primer ex6-F/R being used to amplify the undeleted exon 6 as normal control.The primer of amplifying SRY gene was used to diagnose the sex of the 2 samples respectively.(3)ResultsResults of gene diagnosis of 2 chorionic villi fromâ…¢₃ 's artificial abortion of family 1:The PCR amplification of the junction fragment of the first chorionic villiâ…£₁ was the same positive result as that of probandâ…¢₁.The PCR amplification of exon 3 ofâ…£₁ was positive result without deletion.The sex diagnosis ofâ…£₁ was female.Meanwhile the sequences of the junction fragments ofâ…£₁ and probandâ…¢₁ of the family had no difference by sequencing comparison.So the chorionic villiâ…£₁ from carrierâ…¢₃'s first artificial abortion was diagnosed as female carrier of BMD.The PCR amplification of the junction fragment of the second chorionic villiâ…£₂ was negative result.The PCR amplification of exon 3 ofâ…£₂ was positive result without deletion.The sex diagnosis ofâ…£₂ was male.So the chorionic villiâ…£₂ from carrierâ…¢₃'s second artificial abortion was diagnosed as normal male fetus. Results of prenatal diagnosis of tertigravidaâ…¢₃ of family 1:The PCR amplification of the junction fragment of the chorionic villi sample genomic DNA prepared at 11 weeks' gestation was the same positive result as that of probandâ…¢₁. The PCR amplification of it's exon 3 was positive result without deletion.It's sex diagnosis was male.The contradictory outcome of DMD gene deletion junction fragment being detect but without corresponding exons deletion in a male fetus indicated that the chorionic villi sample had been contaminated by maternal cell.The PCR amplification of the junction fragment of the amniotic cell genomic DNA prepared at 20 weeks' gestation was the same positive result as that of probandâ…¢₁.The PCR amplification of it's exon 3 was negative result(It was checked by exon 4 which was also negative amplification),with corresponding exons deletion.It's sex diagnosis was male.Meanwhile the sequence of it's junction fragment and that of probandâ…¢₁ of the family had no difference by sequencing comparison.So fetusâ…£₃ was diagnosed as diseased male fetus with BMD and was instructed in induced abortion.The result was rechecked without mistake by the fetus tissue from the induced abortion.(4)ConclusionBy PCR amplifying the junction fragment and detecting the corresponding exons with deletion or not for deletional DMD/BMD prenatal diagnosis,the male fetus can be diagnosed accurately as diseased fetus or not.As well as the study sample can be identified effectively being contaminated by maternal cell or not.The disadvantage of this approach is laborious and time-consuming,and can only be applied to deletional DMD/BMD.Summarization(1)The 6 deletional DMD/BMD cases all fit with the reading-frame rule at the DNA level. (2)Our study has fulfilled the sequencing of 5 Chinese junction fragments, reporting the largest sequence data of DMD gene deletion junction fragments of east Asian people up to now.(3)Some secondary structures of DMD gene facilitate gene breakage and recombination.Overall,the sequences of the 5 Chinese junction fragments have all the molecular features including repeated sequence,MARs,palindromic sequence, the sequence TTTAAA,etc,which cause DMD gene deletion and recombination easily.(4)The 5 Chinese junction fragments are all repaired by non-homologous end joining whose characteristic junctions showing microhomologies,small insertions, duplications,or blunt end ligation.(5)The DMD gene deletion junction fragments of several patients and carriers from the same DMD/BMD families have been sequenced and analyzed for the first time.(6)Our study discovers that generally the same family has the same genotype of DMD gene deletion despite the patients from the different families having the different deletions.(7)Our study has used routine PCR amplifying the junction fragment for the carriers detection of 3 different deletional DMD/BMD families successfully.(8)The main difference between this approach and the common approaches of carrier detection at present is that it doesn't need quantitative analysis of DMD gene. The female object can be diagnosed as a carrier as long as she was detected in having the same junction fragment as the proband,with direct-viewing and accurateness.(9)Our study has applied the junction fragment to prenatal diagnosis for a female carrier of BMD for the first time,realizing the presumption that the junction fragment could be applied to deletional DMD/BMD prenatal diagnosis. (10)The disadvantage of the approach of using the junction fragment for gene diagnosis of DMD/BMD is that the genotype of the proband need to be investigated first,which is laborious and time-consuming,and can only be applied to deletional DMD/BMD.Innovation(1)5 new sequences of DMD gene deletion mutation have been cloned.(2)The approach based on routine PCR amplifying the junction fragment has been used for deletional DMD/BMD carrier detection and prenatal diagnosis for the first time.(3)The previous viewpoint that the routine PCR technique can't detect the female carriers of DMD/BMD has been broken through.(4)By PCR amplifying the junction fragment and detecting the corresponding exons with deletion or not for deletional DMD/BMD prenatal diagnosis,the male fetus can be diagnosed accurately as diseased fetus or not.As well as the study sample can be identified effectively being contaminated by maternal cell or not.(5)The approach has the feasibility of being applied to preimplantation genetic diagnosis,with a little check sample needed.