title: Detection of known base substitution mutations in human mitochondrial DNA of MERRF and MELAS by biochip technology.
abstract: We developed a DNA biochip specialized for detection of known base substitution mutations in mitochondrial DNA causing mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) and myoclonic epilepsy associated with ragged-red fibers (MERRF). A set of probes sharing a given allele-specific sequence with a single base substitution near the middle of the sequence was covalently immobilized. Cy5-labeled DNA targets were amplified from sample DNAs containing 31 potential MELAS and/or MERRF mutations by a multiplex PCR method. Detection parameters for the DNA biochip-based assay were accordingly optimized. Seven clinically confirmed patients with MELAS, 5 patients with MERRF, 1 suspected MERRF case and 25 healthy controls were tested using the DNA biochip. For discriminating of homoplasmic and heteroplasmic point mutations in mtDNA, a diagnostic factor based on the ratio between the hybridization signals from the reference and test targets with each probe was used. The results showed that all the cases with MELAS had a causal heteroplasmic A3243G tRNA(Leu(UUR)) mutation. In the MERRF patients, four cases were found to be a homoplasmic A8344G tRNA(Lys) mutation and one case was a heteroplasmic T8356C tRNA(Lys) mutation. None of the healthy controls carried the potential mutations. The results of the DNA biochip were completely consistent with those by DNA sequencing. Thus, the DNA biochip would potentially become a valuable tool in clinical specific screening of the mtDNA point mutations associated with MELAS and/or MERRF syndrome.
authors.affiliation: Key Lab of Gene Resource Utilization for Severe Hereditary Diseases of Ministry of Education of China, Anhui Medical University, Hefei 230032, China. weidongdu@hotmail.com
authors.forename: Weidong
authors.initials: W
authors.lastname: Du
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Gang
authors.initials: G
authors.lastname: Chen
authors.forename: Huimin
authors.initials: H
authors.lastname: Cao
authors.forename: Huayang
authors.initials: H
authors.lastname: Tang
authors.forename: Xianfa
authors.initials: X
authors.lastname: Tang
authors.forename: Qinghui
authors.initials: Q
authors.lastname: Jin
authors.forename: Zhongwu
authors.initials: Z
authors.lastname: Sun
authors.forename: Hui
authors.initials: H
authors.lastname: Zhao
authors.forename: Wenming
authors.initials: W
authors.lastname: Zhou
authors.forename: Sumin
authors.initials: S
authors.lastname: He
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Lv
authors.forename: Jianlong
authors.initials: J
authors.lastname: Zhao
authors.forename: Xuejun
authors.initials: X
authors.lastname: Zhang
mesh_terms: D020137:Base Pair Mismatch / Q000235:genetics*; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000295:instrumentation* / Q000379:methods; D004272:DNA, Mitochondrial / Q000032:analysis / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D017241:MELAS Syndrome / Q000175:diagnosis / Q000235:genetics*; D017243:MERRF Syndrome / Q000175:diagnosis / Q000235:genetics*; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation*
chemical_list: D004272:DNA, Mitochondrial