Gene Diagnosis Of DMD And SMA By Multiplex Ligation-dependent Probe Amplification Combined With DNA/cDNA Sequencing

Objective: To perform gene diagnosis for the families of the Duchenne muscular dystrophy and Spinal muscular atrophy using multiplex ligation-dependent probe amplification technique (MLPA) combined with DNA and cDNA sequencing. Moreover, to study the clinical applicable value of MLPA technique in gene diagnosis and Prenatal diagnosis of the DMD and SMA,which is in order to creating a reliable prenatal diagnosis technical platform.Methods: Genomic DNA of the peripheral blood and fetal amniotic fluid cells was extracted from the pedigrees' members with DMD and SMA. RNA was extracted from the bioptic muscle of the DMD pateients and was reversed transcription to cDNA. Gene diagnosis was performed for these DMD and SMA pedigrees' members using SALSA MLPA kit P034 /P035 and SALSA MLPA kit P021 /P060 respectively. There were 96 individuals accepted the DMD gene diagnosis, including 41 patients, 29 possible carrier and 6 healthy men in 32 DMD families. 91 individuals including 22 probands and 39 carriers were detected the SMA gene in 22 families.The mutations were detected by MLPA combined with DNA and/or cDNA sequence technique in DMD and combined with DNA sequencing and PCR-RFLP in SMA. Simultaneously, the diagnostic effectiveness of the methods used in detecting DMD and SMA gene mutation was evaluated. Prenatal gene diagnosis was successfully performed for 2 fetuses with risk of DMD and 1 fetu with risk of SMA in three families .Results: DMD gene mutations were screened by MLPA technique in 39 male and 2 female patients, showing the analysis results as follows:exons deletion were found in 27(69.23%,27/39) patients ; exons duplication were found in 3(7.69%,3/39)patients;No exon deletion or duplification could be detected in 9(23.08%,9/39)patients. Lower MLPA signals of exon 47-50 from one female patient and exon 35 from another female patient were observed. MLPA pictures of the two female patients were similar to that of carrier and the signals of the involved exons were apparently lower than ones of healthy women. Among 29 possible carrier 20 were detected to be DMD carriers, including 18 exon deletion and 2 exon duplication carriers. Moreover MLPA analysis and PCR-RFLP plus DNA sequencing showed that 16 patients with SMA presented homozygous deletion of exon7 and exon 8 of SMN1. The other 6 patients were SMN1 exon7 homozygous deletion. All the 39 parents were detected to be heterozygotes of deletion of SMN1 exon 7. Their average signal of SMN1 exon7 was lower 30~50% than the average of the control group. Two DMD carrier's pregnant women were performed prenatal gene diagnosis.One of their fetuses was DMD carrier and another was found no exon deletion. One fetus of SMA carrier's pregnant woman was the SMN1 exon7 and exon8 homozygous deletion. The result of prenatal gene diagnosis was confirmed after birth or abortion of the fetuses.Conclution:MLPA provides a simple,rapid and reliable method of simultaneously detecting homozygous,heterozygous deletions mutation in one reaction for all exons of DMD and SMA gene. Combined with DNA and cDNA seqeuencing, it could applied into clinical gene diagnosis and prenatal diagnosis for patients and fetuses with DMD and SMA.