AMPLIFICATION AND ANALYSIS OF PROMOTER REGION OF INSULIN-RECEPTOR GENE IN A PATIENT WITH LEPRECHAUNISM ASSOCIATED WITH SEVERE INSULIN-RESISTANCE

A patient with leprechaunism associated with severe insulin resistance was studied to identify the molecular and genetic basis for insulin r esistance. Insulin binding and surface labeling of transformed lymphoc ytes prepared from the patient showed a significantly decreased insuli n receptor number on the cell surface. Southern blot analysis of the i nsulin receptor gene showed no evidence of large insertions or deletio ns. Furthermore, direct sequencing of all 22 exons and exon-intron jun ctions of the insulin receptor gene failed to show any missense mutati ons, nonsense mutations, or mutations at exon-intron junctions. Howeve r, Northern blot analysis indicated significantly decreased insulin re ceptor mRNA expression in the patient's cells. Moreover, restriction e ndonuclease digestion of the amplified cDNA suggested that the express ion levels of one allele were less efficient than the other. These fin dings suggested that the regulatory region of the insulin receptor gen e might have abnormalities. Therefore, we examined the 5' flanking reg ion of the insulin receptor gene. Southern blot analysis showed no maj or deletions or insertions between positions -1,823 and -2 relative to the translation initiation site. A 5' flanking region of the insulin receptor gene spanning positions -881 similar to +7 was amplified by p olymerase chain reaction (PCR) and introduced into a reporter plasmid carrying the human growth hormone (hGH) gene. The nucleotide sequence of the amplified fragment showed two polymorphic sites at positions -6 03 and -500 in the patient, as well as in normal subjects. No other ab normal sequence was found in the patient. Promoter activity measured b y hGH expression in transfected mouse L. cells was not influenced by t he polymorphism at position -603 located in a cluster of GC boxes. The se results indicated that the 5' flanking promoter region of the insul in receptor gene could be analyzed with the use of PCR, and that the d ecreased expression of insulin receptor gene in a leprechaun patient w as not due to alteration of the nucleotide sequence in the examined pr omoter region, but possibly in another regulatory region of the insuli n receptor gene. Copyright (C) 1995 by W.B. Saunders Company