SUBSTITUTION OF LEU FOR PRO-193 IN THE INSULIN-RECEPTOR IN A PATIENT WITH A GENETIC FORM OF SEVERE INSULIN-RESISTANCE

Mutations have been identified in the insulin receptor (IR) gene in pa tients who are insensitive to insulin action. We studied an extremely insulin resistant patient whose insulin binding to Epstein - Barr viru s (EBV) transformed lymphocytes was severely reduced. Transmembrane si gnalling, evaluated as insulin receptor autophosphorylation, was norma l. The patient's IR was immunoprecipitated normally by AbP6, a polyclo nal antibody directed to the beta subunit. However, there was an almos t-equal-to 50% decrease in the affinity of IR immunoprecipitation by a monoclonal antibody (MA-10) directed against the alpha subunit. These observations suggested that there was likely to be a mutation in the patient's insulin receptor that caused misfolding of the IR alpha subu nit. Analysis of gene structure by Southern blotting experiments did n ot reveal any major deletion in the IR gene of the proband. Northern b lot analysis showed a normal level of expression of IR gene. We applie d denaturing gradient gel electrophoresis (DGGE) as well as direct seq uence analysis to study the 22 exons of IR gene amplified by polymeras e chain reaction (PCR) using the proband's genomic DNA as a template. We identified a new missense mutation substituting leucine (CTG) for p roline (CCG) in homozygous state at codon 193 in exon 3. Both parents are heterozygous for the Leu193 mutation. The Leu193 mutation was not detected in any of 75 normal subjects (150 chromosomes), indicating th at it is not a common sequence variant of the insulin receptor. In add ition, during the course of screening the patient's DNA with perpendic ular DGGE, we identified two previously unreported silent substitution s in exon 9. We conclude that Leu193 mutation is likely to be responsi ble for causing the patient's disease.