Mutations have been Identified In the Insulin receptor (IR) gene in patients who are insensitive to Insulin action. We studied an extremely Insulin resistant patient whose Insulin binding to Epstein-Barr virus (EBV) transformed lymphocytes was severely reduced. Transmembrane signalling, evaluated as insulin receptor autophosphorylation, was normal. The patient's IR was Immunoprecipitated normally by AbP6, a polycional antibody directed to the β subunit. However, there was an ∼50% decrease In the affinity of IR immunoprecipitation by a monoclonal antibody (MA-10) directed against the α subunlt. These observations suggested that there was likely to be a mutation In the patient's Insulin receptor that caused misfolding of the IR α subunit. Analysis of gene structure by Southern blotting experiments did not reveal any major deletion In the IR gene of the proband. Northern blot analysis showed a normal level of expression of IR gene. We applied denaturing gradlent gel electrophoresis (DGGE) as well as direct sequence analysis to study the 22 exons of IR gene amplified by polymerase chain reaction (PCR) using the proband's genomic DNA as a template. We Identified a new missense mutation substituting leucine (CTG) for proline (CCG) in homozygous state at codon 193 In exon 3. Both parents are heterozygous for the Leu 193 mutation. The Leu193 mutation was not detected In any of 75 normal subjects (150 chromosomes), Indicating that it is not a common sequence variant of the Insulin receptor. In addition, during the course of screening the patient's DNA with perpendicular DGGE, we Identified two previously unreported silent substitutions in exon 9. We conclude that Leu193 mutation is likely to be responsible for causing the patient's disease.

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