Rapid screening method of abnormal insulin-receptor gene expression: allele-specific oligonucleotide hybridization by using silent polymorphisms.
An asymmetrical reduction in the levels of the insulin receptor mRNA transcribed from one allele was reported in some patients with severe insulin resistance and non-insulin-dependent diabetes mellitus (NIDDM). To detect this abnormality, we designed the less laborious method; Allele-specific oligonucleotide hybridization of the amplified mRNA (cDNA) by using silent polymorphisms in the insulin receptor gene (nucleotide positions at 1686 and 1698). The allelic frequencies of C-1686 and T-1686 were 0.63 and 0.37, respectively (0.60 and 0.40 in 10 normal subjects, and 0.67 and 0.33 in 20 NIDDMs; n.s.). Similarly, the allelic frequencies of A-1698 and G-1698 were 0.47 and 0.53, respectively (0.50 and 0.50 in the normal subjects, and 0.45, and 0.55 in the NIDDMs; n.s.). These results suggest that these two polymorphisms are very common in Japanese. Nineteen (64%) out of 30 cases are heterozygous at one or two position(s), suggesting that it is possible to distinguish the mRNA transcribed from each of two alleles of the insulin receptor gene with using allele-specific oligonucleotide hybridization. Although we successfully measured the ratio of mRNA expression from two alleles of the gene in 20 NIDDMs, there was no patient whose mRNA transcribed from one allele of the insulin receptor gene was extremely decreased. We showed that allele-specific oligonucleotide hybridization method is useful for the screening of abnormal insulin-receptor gene expression.
Imano
E
E
First Department of Medicine, Osaka University Medical School, Japan.
Kawamori
R
R
Iwama
N
N
Umayahara
Y
Y
Yamasaki
Y
Y
Kamada
T
T
D000483:Alleles; D001483:Base Sequence; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007333:Insulin Resistance / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization / Q000379:methods*; D011110:Polymorphism, Genetic; D012333:RNA, Messenger / Q000032:analysis; D011972:Receptor, Insulin / Q000235:genetics*
D012333:RNA, Messenger; D011972:Receptor, Insulin