Detection of Mutations in Insulin Receptor Gene by Denaturing Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis (DGGE) has been used to screen for mutations in the insulin receptor gene. Each of the 22 exons was amplified by the polymerase chain reaction (PCR). For each exon, one of the two PCR primers contained a guanine-cytosine (GC) clamp at its 5′ end. The DNA was analyzed by electrophoresis through a polyacrylamide gel containing a gradient of denaturants. Two geometries for the gels were compared; the gradient of denaturants was oriented either parallel or perpendicular to the electric field. The sensitivity of the technique was evaluated by determining whether DGGE succeeded in detecting known mutations and polymorphisms in the insulin receptor gene. With parallel gels, 12 of 16 sequence variants were detected. The use of perpendicular gels increased the sensitivity of detection so that all 16 sequence variants were successfully detected when DNA was analyzed by a combination of perpendicular and parallel gels. Furthermore, DGGE was used to investigate a patient with leprechaunism whose insulin receptor genes had not previously been studied. Two mutant alleles were identified in this patient. The allele inherited from the father had a mutation substituting alanine for Val-28; in the allele inherited from the mother, arginine was substituted for Gly-366.

[1]  D. Lindhout,et al.  An Arg for Gly substitution at position 31 in the insulin receptor, linked to insulin resistance, inhibits receptor processing and transport. , 1992, The Journal of biological chemistry.

[2]  M. de la Luz Sierra,et al.  A mutation in the tyrosine kinase domain of the insulin receptor associated with insulin resistance in an obese woman. , 1991, The Journal of clinical endocrinology and metabolism.

[3]  S. O’Rahilly,et al.  Detection of Mutations in Insulin-Receptor Gene in NIDDM Patients by Analysis of Single-Stranded Conformation Polymorphisms , 1991, Diabetes.

[4]  Simeon I. Taylor,et al.  Two Patients With Insulin Resistance Due to Decreased Levels of Insulin-Receptor mRNA , 1991, Diabetes.

[5]  Jerrold,et al.  Insulin resistance and diabetes due to different mutations in the tyrosine kinase domain of both insulin receptor gene alleles. , 1991, The Journal of biological chemistry.

[6]  A. Ullrich,et al.  A mutation in the extracellular domain of the insulin receptor impairs the ability of insulin to stimulate receptor autophosphorylation. , 1991, The Journal of biological chemistry.

[7]  S. Lillioja,et al.  Normal Coding Sequence of Insulin Gene in Pima Indians and Nauruans, Two Groups With Highest Prevalence of Type II Diabetes , 1991, Diabetes.

[8]  Simeon I. Taylor,et al.  Mutagenesis of lysine 460 in the human insulin receptor. Effects upon receptor recycling and cooperative interactions among binding sites. , 1990, The Journal of biological chemistry.

[9]  E. Gershon,et al.  Mutations of the Gs alpha-subunit gene in Albright hereditary osteodystrophy detected by denaturing gradient gel electrophoresis. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[10]  J. Flier,et al.  A naturally occurring mutation of insulin receptor alanine 1134 impairs tyrosine kinase function and is associated with dominantly inherited insulin resistance. , 1990, The Journal of biological chemistry.

[11]  C. Caskey,et al.  Molecular scanning methods of mutation detection. , 1990, The Journal of biological chemistry.

[12]  Simeon I. Taylor,et al.  Five mutant alleles of the insulin receptor gene in patients with genetic forms of insulin resistance. , 1990, The Journal of clinical investigation.

[13]  Y. Ebina,et al.  Insulin-resistant diabetes associated with partial deletion of insulin-receptor gene , 1990, The Lancet.

[14]  D. Accili,et al.  Mutations in Insulin-Receptor Gene in Insulin-Resistant Patients , 1990, Diabetes Care.

[15]  S. Cai,et al.  Identification of the multiple beta-thalassemia mutations by denaturing gradient gel electrophoresis. , 1990, The Journal of clinical investigation.

[16]  A. L. Dudley,et al.  The rat insulin receptor: primary structure and conservation of tissue-specific alternative messenger RNA splicing. , 1990, Molecular endocrinology.

[17]  Simeon I. Taylor,et al.  A nonsense mutation causing decreased levels of insulin receptor mRNA: detection by a simplified technique for direct sequencing of genomic DNA amplified by the polymerase chain reaction. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[18]  M. Lane,et al.  Substrate phosphorylation catalyzed by the insulin receptor tyrosine kinase. Kinetic correlation to autophosphorylation of specific sites in the beta subunit. , 1989, The Journal of biological chemistry.

[19]  P. Shier,et al.  Primary structure of a putative receptor for a ligand of the insulin family. , 1989, The Journal of biological chemistry.

[20]  L. Sandkuyl,et al.  A leucine‐to‐proline mutation in the insulin receptor in a family with insulin resistance. , 1989, The EMBO journal.

[21]  E. Lander,et al.  A mutation in the insulin receptor gene that impairs transport of the receptor to the plasma membrane and causes insulin‐resistant diabetes. , 1989, The EMBO journal.

[22]  al. et,et al.  Human diabetes associated with a mutation in the tyrosine kinase domain of the insulin receptor. , 1989, Science.

[23]  C. Kahn,et al.  Insulin receptor messenger ribonucleic acid sequence alterations detected by ribonuclease cleavage in patients with syndromes of insulin resistance. , 1989, The Journal of clinical endocrinology and metabolism.

[24]  T. Sekiya,et al.  Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[25]  V. Sheffield,et al.  Mutation Detection by PCR, GC-Clamps, and Denaturing Gradient Gel Electrophoresis , 1989 .

[26]  Y. Ebina,et al.  Human diabetes associated with a deletion of the tyrosine kinase domain of the insulin receptor. , 1989, Science.

[27]  V. Sheffield,et al.  Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[28]  J. Flier,et al.  Detection of an alteration in the insulin-receptor gene in a patient with insulin resistance, acanthosis nigricans, and the polycystic ovary syndrome (type A insulin resistance). , 1988, The New England journal of medicine.

[29]  T. Sasaoka,et al.  Insulin resistance by unprocessed insulin proreceptors point mutation at the cleavage site. , 1988, Biochemical and biophysical research communications.

[30]  Simeon I. Taylor,et al.  Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance. , 1988, Science.

[31]  A. Kosaki,et al.  Insulin-resistant diabetes due to a point mutation that prevents insulin proreceptor processing. , 1988, Science.

[32]  W R Taylor,et al.  On the tertiary structure of the extracellular domains of the epidermal growth factor and insulin receptors. , 1987, Biochimica et biophysica acta.

[33]  L. Lerman,et al.  Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis. , 1987, Methods in enzymology.

[34]  R. Myers,et al.  Detection and localization of single base changes by denaturing gradient gel electrophoresis. , 1987, Methods in enzymology.

[35]  C Collins,et al.  Insulin‐like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. , 1986, The EMBO journal.

[36]  P. H. Seeburg,et al.  Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes , 1985, Nature.

[37]  P. Seeburg,et al.  Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells , 1984, Nature.