Insulin receptor messenger ribonucleic acid sequence alterations detected by ribonuclease cleavage in patients with syndromes of insulin resistance.
We used a ribonuclease cleavage assay to screen for insulin receptor mRNA sequence alterations in 12 patients with syndromes of severe insulin resistance. Uniformly labeled [32P]antisense RNA probes complementary to insulin receptor mRNA were prepared by an SP6 or T7 RNA polymerase transcription reaction. Four probes ranging in size from 670-1470 bases were used to examine the entire 4.2-kilobase receptor protein-coding region. Patient RNA samples were hybridized to individual probes in solution, and mismatched sequences were detected by susceptibility to cleavage by a mixture of RNAses A and T1. The method was validated with insulin receptor mRNAs from cells transfected with cDNA constructs bearing known point and deletion mutations. Alterations in the insulin receptor mRNA sequence of two patients were detected. A patient with the type A syndrome of severe insulin resistance (A2-Boston) had a mutation in the insulin receptor beta-subunit mRNA sequence that localized to the region coding for amino acid residues 1174-1211 near the tyrosine kinase domain. The second alteration was a sequence polymorphism in the insulin receptor alpha-subunit mRNA in a patient with lipoatropic diabetes (LA-2) that localized to a region within amino acids 268-272. Direct sequence analysis revealed that the ribonuclease cleavage sites in patients A2-Boston and LA-2 were due to distinct single base changes in the insulin receptor gene and mRNA. Additional insulin receptor mRNA sequence polymorphisms were also identified as mismatches between the labeled RNA probes used and mRNA from several cultured human cell types. This study demonstrates that ribonuclease cleavage can rapidly detect and localize insulin receptor mRNA sequence mutations and polymorphic variations as small as single base changes. Further analysis of insulin receptor mRNA sequence alterations identified in this way may elucidate a possible genetic basis for functional insulin receptor defects in patients with severe insulin resistance and can also reveal some insulin receptor sequence polymorphisms that occur in the population at large.
Goldstein
B J
BJ
Research Division, Joslin Diabetes Center, Boston, Massachusetts 02215.
Kahn
C R
CR
D000052:Acanthosis Nigricans / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000032:analysis; D005815:Genetic Code; D007333:Insulin Resistance; D009154:Mutation; D010446:Peptide Fragments / Q000032:analysis; D011110:Polymorphism, Genetic; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis*; D011972:Receptor, Insulin / Q000032:analysis / Q000235:genetics*; D012260:Ribonucleases
D010446:Peptide Fragments; D015347:RNA Probes; D012333:RNA, Messenger; D004247:DNA; D011972:Receptor, Insulin; D012260:Ribonucleases