Detection and characterization of new genetic mutations in individuals heterozygous for lactate dehydrogenase-B(H) deficiency using DNA conformation polymorphism analysis and silver staining.
Human lactate dehydrogenase (LDH)--B(H) mutant genes were analyzed by polymerase chain reaction (PCR) and DNA conformation polymorphism. We used polyacrylamide gradient gel and silver staining procedures for DCP analysis, and observed abnormal migration patterns in individuals heterozygous for the LDH-B deficiency. Subsequent sequence determination of the mutant alleles consistently resulted in detection of three single base substitutions (transversions), viz., a C to A at residue "35" (GCG, Ala-->GAG, Glu), a T to G at residue "172" (TTT, Phe-->GTT, Val), and an A to T at residue "176" (ATG, Met-->TTG, Leu). Furthermore, mismatched PCR or amplification refractory mutation system was developed for the rapid screening and confirmation of these mutations. These amino acid replacements may cause conformational changes in neighboring residues; this probably affects the active site arrangement and results in the loss of enzyme activity.
Maekawa
M
M
Department of Laboratory Medicine, Hamamatsu University School of Medicine, Japan.
Sudo
K
K
Kitajima
M
M
Matsuura
Y
Y
Li
S S
SS
Kanno
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D004252:DNA Mutational Analysis / Q000379:methods*; D015252:Deoxyribonucleases, Type II Site-Specific; D004591:Electrophoresis, Polyacrylamide Gel; D006580:Genetic Carrier Screening; D006801:Humans; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000172:deficiency* / Q000235:genetics*; D008322:Mammals; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D016622:Silver Staining; D013045:Species Specificity
D007527:Isoenzymes; D009692:Nucleic Acid Heteroduplexes; D004247:DNA; D007770:L-Lactate Dehydrogenase; D015252:Deoxyribonucleases, Type II Site-Specific; C032229:GANTC-specific type II deoxyribonucleases