MLPA Coupled With DHPLC For Rapid Mutation Detection Of The Causative Gene In DMD

IntroductionDuchenne muscular dystrophy (DMD) is a devastating X-linked recessive dystrophinopathy, characterized by rapidly progressive degeneration and necrosis of proximal muscles and calf pseudo-hypertrophy. DMD is caused by the changes in the structure and function of dystrophin protein with a world-wide incidence of 1 in 3500 live male birth. Female are usually carriers. To date, no effective treatment is available.The dystrophin gene is the causative gene for DMD, which is located on Xp21.2, consists of 79 exons and encodes the dystrophin protein. The large size of the dystrophin gene is liable to a high incidence of de-novo mutations and ectopic breakpoints. In specific, approximately 55～65% of DMD cases are associated with one or more exons deletions clustered in 2 hotspot regions in 5' terminus and around exon 44～53 of the gene. And about 5%～10% of DMD mutations are associated with duplications, whereas the remaining cases may be caused by point mutations and small insertions/deletions sporadically.Due to the large size and complex mutation types of the dystrophin gene, the common multiplex polymerase chain reaction (mPCR) cannot meet the need of gene diagnosis and genetic counseling. Therefore, it is necessary to develop an effective and comprehensive strategy for molecular diagnosis in DMD. Recently, a new technique, multiplex ligation-dependent probe amplification (MLPA), has been depicted as a first screening that allows the detection of large genomic rearrangements (deletions/ duplications) in the dystrophin gene by simultaneous amplification of up to 40 target sequences. Additionally, denaturing high-performance liquid chromatography (DHPLC) has been widely used in gene diagnosis of genetic disorders. As an effective complement to MLPA, DHPLC is capable of rapidly analyzing unknown single nucleotide polymorphisms (SNPs) and mutations. In this study, we described a new application combining MLPA with DHPLC, aimed to develop a comprehensive and rapid strategy, which will be valuable in improving the availability of molecular testing for DMD patients.Materials and Methods1. Materials44 unrelated male DMD patients at the age of 2～10 years old, from the Pediatric Department, the 2nd Affiliated Hospital of China Medical University, entered the study. 20 unaffected males and 50～100 unaffected females without a DMD family history were recruited in the study as the controls. Genomic DNA was extracted from peripheral blood samples of the patients and controls using a standard proteinase K digestion and phenol-choloroform extraction procedure. All patients were negative for mPCR analysis with 12 pairs of primers that referred to parts of the deletion hotspots. MLPA DMD KIT (SALSA MLPA KIT P034/035 DMD/BMD) was purchased from the MRC-Holland (Amsterdam, Nertherlands). The main facilities included UNO II 48 PCR thermal cycler (Biometra, German), Beckman CEQ-8000 genetic analytic system (Beckman, USA) and WAVE^? nucleotide fragment analysis system (Transgenomic, USA).2. Methods(1) Detection of deletions/duplications in the dystrophin gene by MLPAMLPA was performed using the MLPA DMD KIT. MLPA probes are divided into two probemixes, P034 and P035, including probes for each exon sequence. Briefly, 50～500 ng of genomic DNA in a volume of 5μL Tris-EDTA was denatured (98℃,5 min), cooled and mixed with MLPA P034 or P035 probemix. The mixture was heated to 95℃for 5 min and then incubated at 60℃overnight for probe hybridization. After 16 h, ligation was performed with Ligase-65 enzyme at 54℃for 15 min and Ligase-65 enzyme was inactivated at 98℃for 5 min. Then PCR was performed with the specific SALSA FAM PCR primers. Amplification products were run and analyzed by capillary electrophoresis on the Beckman CEQ-8000 genetic analytic system. The peaks obtained after capillary electrophoresis were analyzed by Fragment Analysis software and processed by Microsoft Excel Template. Three unaffected males were included in the analysis as controls. Any result with obvious single-exon deletion was validated by further PCR and sequencing.(2) Detection of small mutations in the dystrophin gene by DHPLC and sequencingPatients negative for MLPA analysis in the dystrophin gene were subjected to further DHPLC screening. Exons were separately amplified with primers designed to cover each of the 79 exons and their flanking sequences. Unpurified PCR amplicons from patients were mixed with those from male controls (1:1) and then denatured, followed by cooling slowly down in a thermal cycler.DHPLC was performed on the WAVE^? system. The pre-treated amplicons were processed at the optimal separation gradient and temperature determined by the WAVEMARKER 4.1 software. If a PCR amplicon presents a chromatogram different from the control (wild type) in shape or retention time, the corresponding exon was directly sequenced to identify the position and the type of the mutations using the Beckman CEQ8000 DNA genetic analytic system. The National Center for Biotechnology database of genetic variation (dbSNP) was queried to identify the existence of the common SNPs, or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was employed to identify the unreported SNPs. Results1. MLPADeletions of one or more exons resulted in total absence of the corresponding MLPA products. Changes in exon copy number were identified in 11 out of the 44 cases (27.3%). 4 cases showed single-exon deletion and the other 7 cases multiple exon deletions, but no duplication was identified in all patients. All cases with single-exon deletion were confirmed in the uniplex PCR except the deletion of exon 47 in patient 13, on which the sequence analysis revealed a small new deletion (c.6808₆811 del) that was predicted to produce a premature stop codon (p.Leu2270MetfsX9).2. DHPLC and sequencingOnly 45 exons covering two deletion hotspots and the 3'UTR region were amplified because of the lack of DNA specimen. A total of 4 disease-causing mutations, including 3 unreported before, were identified by detecting the heteroduplexes on DHPLC and sequencing the appropriate exons. Two frameshift mutations, c.1976₁980del and c.4959₄960insA, would lead to the premature stop codons (p.Ser661ValfsX58 and p.Serl654LysfsX5, respectively). The other two were nonsense mutations (c.8656C>T and C.8608C>T) that were predicted to result in p.R2886X and p.R2870X, respectively. The remaining genetic variants were 2 missense mutations and 7 intronic variations.Conclusion1. Superior to mPCR, MLPA, a simple and rapid new mutation detection technique with high sensitivity. Considered as the first-line in molecular diagnosis for DMD, MLPA can easily, rapidly and semi-quantitatively screen for copy number changes of all exons in the dystrophin gene.2. As a complement to MLPA, DHPLC is very accurate and sensitive in detecting small mutations of the dystrophin gene.3. A combination of MLPA and DHPLC could provide a comprehensive and effective strategy for screening DMD causative mutation and increasing the pick-up rate, which will be very informative to the routine and prenatal diagnosis and genetic counseling of DMD.