The Application Of MLPA In Gene Diagnosis Of Single Gene Disorders DMD/BMD And SMA

Background Both of DMD/BMD and SMA are common lethal single gene disorders. There are no effective treatments at present, so it's essential to detect the disease genes, screen carriers and carry out prenatal diagnosis for avoiding the birth of children suffering from diseases. Traditional methods used in DMD/SMA gene diagnosis, including 21 exons PCR in hot-spot, Real-time PCR and genetic linkage analysis, have certain limits.21 exons PCR in hot-spot can not detect heterozygous deletion and duplication mutations. Real-time PCR, quantify gene copy number as it can, is usually applied to test carriers with known mutation sites found in patients, for the difficulties in covering the whole DMD gene. And it hasn't been widely used in clinic because of large working amount. linkage analysis is limited on the patients to screen carriers and is prone to misdiagnosis for incapability of distinguishing sporadic cases. PCR-RFLP, the conventional SMA gene diagnosis method, is restricted to detect the patients with homozygous deletions of SMN1 exon7 and can not test carriers. Based on the defects of methods mentioned above and clinical demand, it's urgent for us to establish an efficient, sensitive and accurate carriers test platform which is also adapted to clinical large sample screening and not independent from patients'information, so to perfect DMD/BMD and SMA gene diagnosis systems. Objective To establish MLPA test platform for gene diagnosis of single gene disorders (DMD/BMD, SMA), and to improve the detection rate of patients and carriers, and to direct reproduction.Methods①There are eighteen DMD families with patients' information and eleven DMD families with out patients'information. Eighteen DMD patients had been tested by 21 exons PCR in hot-spot with a diagnostic rate of 66.7%(12/18), and could be determined X risk chromosome by genetic linkage analysis. Five patients with known deletion mutations,28 female carriers and one fetal in 29 DMD families were detected using MLPA. The gender of fetal was determined SRY gene defection in prenatal diagnosis. The deletion/duplication carriers, which were found by MLPA, were confirmed by Real-time PCR further.②Four SMA patients and twelve suspected carriers in eight SMA families were tested by MLPA. Homozygous absence of SMN1 exon7 had been detected from four SMA patients by PCR-RFLP.Results DMD:①Deletion of known sites and adjacent sites was detected by MLPA in Five DMD patients, indicating MLPA has a wide detection range and is also precise and reliable.②Eighteen DMD patients had been detected by 21 exons PCR in deletion hot-spot with a diagnostic rate of 66.7%(12/18), while the diagnostic rate increased to 83.3%(15/18) after the application of MLPA, by which we detected duplication mutations in three DMD families found no deletions before. We can supply informations for eighteen DMD families with patients by combining use of MLPA, mutation screening in deletion hot-spot and genetic linkage analysis, the diagnostic rate reaches 100%(18/18).③DMD carriers can be screened by linkage analysis only in the presence of the patients while MLPA test is independent of that. Eleven DMD female carriers were found by MLPA (five carriers coming from DMD families with no patients'information), and were further confirmed using Real time quantitative PCR. The diagnostic rate of DMD carriers is 50% (11/22).④The fetal with SRY negative did not inherit the defective X chromosome from mother and was excluded from carrier.⑤According to linkage analysis results, pathogenic X chromosome of five DMD patients in families 6-10 were all inherited from their mothers. But it was found by MLPA that the genotypes of the five mothers were normal, suggesting these were five sporadic cases. The incidence rate of new mutations is 35.7%(5/14). Carriers screening based only on linkage analysis might result in misdiagnosis sometimes, so the disease-causing gene of suspected carriers have to be detected directly in fertility regulation.SMA:①MLPA results of four SMA patients suggested:patient 1, SMN1 was homozygous absent; patient 2, one copy of SMN1 was absent and the other one conversed into SMN2; patient 3 and 4, SMN1 conversed in one allele and SMN1 exon7 only conversed in the other. MLPA could discriminate between SMN1 deletion and conversion to SMN2. Its results are more detailed than these of PCR-RFLP.②PCR-RFLP could not screen SMA carriers. Copy numbers of SMN1 and SMN2 in twelve SMA carriers were relatively quantified by MLPA, which could supply informations for their reproduction. The diagnostic rate of SMA carriers is 100%(12/12).③We had found eight SMN genotypes in this study.Conclusion MLPA is an efficient, sensitive, accurate and cost-effective method for DMD/BMD, SMA molecular diagnosis. Its application is independent from the information of patients. MLPA could relatively quantify the gene copy number efficiently, detect carrier with genetic defects and improve the diagnostic rate of patients.