Antenatal Gene Detection Of Duehenne Muscular Dystrophy With High Risk Family

Objective:Duehenne Muscular Dystrophy is a kind of frequently reported lethal disease involved of neuromuscular disorder, with X-linked inheritance. The incidence of this disease is 1/3500 in male. The most distinctive feature of Duchenne muscular dystrophy is a progressive proximal muscular dystrophy with characteristic pseudohypertrophy of the calves. The bulbar (extraocular) muscles are spared but the myocardium is affected. There is massive elevation of creatine kinase levels in the blood, myopathic changes by electromyography, and myofiber degeneration with fibrosis and fatty infiltration on muscle biopsy. The onset of Duchenne muscular dystrophy usually occurs before age 3 years, and the victim is chairridden by age 12 and dead by age 20 because of cardiopulmonary syndrome, which threatens male health greatly. There is no efficient therapy for this disease at present. One-third of DMD victims are caused by neonatal mutation, and the others are inherited from parents. So antenatal gene diagnosis of this desease is an important measure to decrease the populational frequency of victims, and it helps to increase the populational quality.Mapping and molecular genetic studies indicate that the huge gene that encodes dystrophin (also symbolized DMD) is the nosogenetic gene. It is located at Xp21.l-21.3, and covered 1% of X chromosome of about 2400kb, which is one of the longest genes in human genome. This gene is consisted of 79 exons and 78 introns. However, the total length of its exons is about 14kb, and the introns cover the most portion of this gene. The introns of dystrophin are fragile, and 92% of the chain breakpoint among dystrophin mutation can take place randomly in all introns, with hot points found in different race and nationality. Gene deletion is the most frequent type of mutation found in dystrophin, which cover 55%～65% of all mutations; 5%~ 10% of dystrophin mutation are gene duplication.In this study, DMD fetal victims with gene deletion were identified using 18 sets of primers based on mPCR after gender determining of the fetuses, and then the detailed deletion sites were verified. The experiments were optimized by recombination of primer sets. For DMD victims with non-deletion, gene diagnosis was performed using 5 sets of (CA)n primers based on STR-PCR method. Our aim is to establish an easy-performing and reliable protocol of gene diagnosis or antenatal gene diagnosis for DMD victims, which will provide a base of carrying out genetic counseling and preventing of DMD.Materials and methodsMaterials1 Clinical case: Four DMD victims exhibited typical clinical features and thus were diagnosed based on electromyogram(EMG), serum myocard oenzyme of creatine kinase and muscle biopsy.2 Samples: Five milliliter of venous blood from typical DMD victims and their sibs extracted. For antenatal gene diagnosis, 20 ml of amniotic fluid of mid-stage fetuses was obtained by amniocentesis, and 2 ml of umbilic blood was also obtained from late-stage fetuses. Genomic DNA was extracted and purified from all the sample. 3 Primers: One set primer of SYR, 18 sets primer of Dystrophin exon and other 5 set of (CA)n primers were designed by Primer5.0 and performed alignment searching of NCBI database to ensure its specificity. Finally all these primers were synthesized by Shanghai chemical industry biological engineering and technical service company limited.Methods:Firstly, gender determining of the fetuses was judged by specific PCR using the set primer of SYR. Then DMD victims with dystrophin deletion were screened by mPCR using 18 set primer of dystrophin. Once the delation of a DMD victim was confirmed, the same deletion in the fetal sample of victin's family was virified by single PCR to detect whether the fetus was another victin or not. For female fetus or male fetus without deletion, linkage analysis based on STR-PCR of specific marker among DMD victim and fetus was performed to judge the suffering risk of fetus.Establishment of the stable systemic PCR1 PCR analysis of SYR: In a 1x PCR buffer containing 50ng genomic DNA, 200umol/ L dNTP, 0.2u mol/L Primers, and 2u r-Taq polymerase, the specific PCR of SYR was carried out, with total volume of 25uL.2 mPCR analysis of dystrophin: Similar with above, the 1x PCR buffer contains 50ng genomic DNA, 200umol/ L dNTP, 0.2u mol/L of each set primers, and 3u r-Taq polymerase, and the final total volume was 15uL. Eighteen set primers were divide into four groups. 3 STR-PCR analysis: Also like above, the 1x PCR buffer contains 50ng genomic DNA, 200 umol/L dNTP, 0.2u mol/L of each set primers, and 3u r-Taq polymerase, and the final total volume was 25uL.After the final cycle of PCR, all the products were separated in 3% agarose gels or 12% non- degradative polyacrylamide gels before dying with SYRB Green. The images were scored by GelDoc 2000 and analyzed with QuantityOne (Bio-Rad).ResultsFour samples were tested for gender determining by PCR analysis of SYR, among which 2 samples and one of the twins were female, and the other twin was male. The result was consistent with that of karyotype analysis for amniotic cells. and complete same sex after fetus postnatal.. The further results of mPCR analysis for the four fetus samples showed two samples were positive of dystrophin deletion. Deletion of exon 50 and 51 were found in one sample, and exon 43 in the other sample of the female twin. However, the other twin was not a sufferer.At same time, carrying out detection for male fetus of twins's dystrophin gene exon 43,but not founding deletion of male fetus's dystrophin gene exon 43.All the deletion of exon was found in the hot point of exon 44 to 52, which consistent with the results of other researches. Another two DMD patients, mPCR of 18 sets dystrophin exons's primes do not detect exon deletion, for non-deletion type DMD patients. Four fetuses of the 3 core family (including two non-deletion type DMD's high risk familys,and one deletion type DMD's high risk twins familys)of DMD obtained the same maternal haplotype of DMD victims, and 3 male fetuses were potential sufferers except one female twin who was confirmed as a carrier of DMD. Moreover, the 3 mothers of each DMD victims were virified as heterozygote, and no dystrophin deletion was detected in the other members of each family.,strictly following heredity rule.ConclusionsIntegration analysis of gender determining, mPCR and STR-PCR of dystrophin is a powerful method for antenatal gene diagnosis of DMD. It can be take as a fast and efficient technique for clinical diagnosis, which efficiently provide important information for antenatal gene diagnosis and genetic counseling. Most important of all, this method is feasible for sporadic cases of DMD, and exhibits its superiority.