| ObjectiveDuchenne muscular dystrophy ( DMD) is a common, lethal, chromosome X - linked inherited neuromuscular disease, which is caused by mutations in the dystrophin gene, affects approximately 1 in 3,500 live male births. Its typical clinical manifestations are progressive muscular dystrophy, muscular weakness and calf hypertrophy. The onset of weakness usually occurs between 2 and 3 years of age, but it may be delayed. The affected children are usually wheelchair bound by 7 and 10 years of age and most die in their late teens or early twenties from the heart or lung complication. It not only influence the health of adolescent male seriously, but also bring the heavy burden of mind and economy to the patients' family, but at present there is no effective method to cure it. One third of all cases arise from a new mutation, the others from inheritance, so it can provide effective information to prenatal diagnosis by the detection of the carriers.Dystrophin gene locus at XP21, which is very large (containing 79 exons and 78 introns) and has an usually high mutation rate 55% to 65% of all cases of DMD result from partial gene deletion. Because of the clustering of deletions in two " hot spot" areas, Chamberlain designed 9 pair primers to detect deletions in male patients by using PCR technique, which can detect 80% of the deletions, Beggs added the other 9 pair primers which make the detection rate increase to more than 98%.In nowadays the detection rate of the carriers with the deletion of dystrophin gene can reach to 98% abroad by using fluorescence Q - PCR, but the cost is expensive, so it is difficult to use it in clinical diagnosis. In our country Q -PCR was used to detect the carriers, but the reports were few, so its reliability need to be verified.First we used mPCR to detect the deletion of dystrophin gene, second Q -PCR was used to identify the carrier status in a female relative of deletions, Our object was to establish a simple and reliable method to examine the detection of dystrophin gene in patients and carriers with DMD to lay a foundation for prenatal diagnosis.Materials and MethodsGenomic DNA was prepared from peripheral blood of affected probands, their mothers and control groups using a salt/chloroform procedure. The concentrations of genomic DNA were measured by UV spectrophotometry and adjusted to a working concentration of 25ng/|xl.The deletion analysis was performed by mPCR using 18 pairs of primers. Amplifications were performed in 25 l volumes containing 100ng genomic DNA, 0. 2 mol/L of each primer, 1 x buffer, 200mmol/L dNTPs and 1 unit of Taq DNA polymerase in the system of Al, A2 and A3, 2. 5 units in B system. PCR products (10 l) was mixed with 1 l of agarose gel loading dye and elec-trophoresed on 3% agarose gel at 110V for 2. 5 hours. Stained with ethidium bromide.Multiplex PCR reaction was done as follows, selected deleted and nondelet-ed exons in the same system, the reaction system was the same as A3 system. Followed by 21 cycles which was well within the exponential phase, PCR products (5 ui) was mixed with 1 jjj. of agarose gel loading dye and electrophoresed on 2% agarose gel at 80V for one hour. Stained with ethidium bromide. Then used the ID - muti software to analysis the scanogram. Dosage quotients for pairs of exons in a sample were calculated by dividing the ratio of the two exons' peak areas from the sample by the corresponding ratio from the controls. Thedosage quotients in the case of a deletion carrier will be 2-1.ResultsAmong the 48 patients, 25 had different deletions, the rate was 52%. The frequency of deletion in exon 49 and 50 was highest, which was 52% , exon 48 was 44% nextly. The exons of deletion mainly distributed in two hot areas; centre hot area and 5'end hot area. The frequency were 84% and 16% respective-The fragments' density of two exons were no different in ten controls, that is the rate of their peak areas approach 1-1. 8 carriers were identified in 15 mothers whose sons had been confirmed the deletion of dystrophin gene because the rate of the peak area in delete exon and control exon approach 2- 1; 7 of them were not carriers because the rate approach 1*1. 4 obligate carriers were all i-dentified carriers and 4 carriers were identified in 11 possible carriers.DiscussionDystrophin gene is one of the extremely largest size gene in human at present. It contains 79 exons and 78 introns. In these years a lot of researches manifest that the frequency of its mutation is high and its forms are all kinds of. Majority of the mutations are deletions (55% -65% ). The deletions in the dystrophin gene are non - randomly distribute in two regions, at the 5' terminus and in the distal half of the central rod domain around exons44 -50. The frequency of deletion in exon 48 is highest. mPCR was used to detect the deletion of dystrophin gene in 48 DMD patients, from which 25 patients were identified deletions in dystrophin gene. The frequency of deletion was 52%. 21 deletions distributed the centre hot deletion area, 4 distributed the 5' end hot deletion area. The results were the same as the other reports in the world. But in our research the frequency of deletion in exon 49 and 50 was highest, next was exon 48, which was different from the other reports. It may be related to the short of samples or the difference of the areas. We found the reaction condition of A system was rigor-ous, it was difficult to repeat the result of the same reaction and easy lead to false positive results, so we departed A system into three groups: A1,A2 and A3. Since then the results were stable. In our research the frequency of deletion in A3 group was higher (44% ) , so when detecting the deletions in DMD patients, we may use A3 group firstly, if didn' t find the deletions, using A1,A2 and B systems further, which can save reagents and suit to the application of clinical diagnosis.There are many techniques to detect the carriers with the deletion of dystro-phin gene, but they are all many or few limitations. In our research Q — PCR was used to identify the carriers. Four obligate carriers were all identified carriers and four carriers were identified in eleven possible carriers. Among the levels of CK in four obligate carriers, two were higher, one was normal, one was unknown; In six possible carriers, whose levels of CK were normal, three were manifested carriers by using Q-PCR. That showed that the method of Q - PCR was more sensitive than that of CK, what's more, it was simple and rapid, but it need to control the conditions rigorously, or it was easy to lead to false positive or false negative results. In order to reduce the errors in experiment, we took actions as follows: 1) assuring the original concentration of DNA in all samples were same; 2) because the sequence and length of every exon was different, the amount of produce was different in the same reactional conditions, so we selected the exon whose products' amount was approach to that of control; 3) assuring the reaction was in the period of the exponential in order to make the amount of the product can reflect the original copy. 4) assuring the thick of agarose gel in all sides were same.By using Q - PCR to detect the carriers with the deletion of dystrophin gene, 8 carriers were identified in 15 mothers whose sons had been confirmed the deletion of dystrophin gene, the frequency of positive was approach 2/3, which was consistent with the theory that 1/3 mutation in DMD patients was de novo mutations. It showed that combinating mPCR and Q - PCR can diagnosis patients and carriers in DMD families with the deletion of dystrophin gene effectively , which may provide significent information for prenatal diagnosis, but the phenomenon of germline mosaicism should be considered when answering for the...
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