0021-972X/91/7306-1158$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society
Vol. 73, No. 6 Printed in U.S.A.
GENETIC BASIS OF ENDOCRINE DISEASE 1 Molecular Genetics of Insulin Resistant Diabetes Mellitus SIMEON I. TAYLOR, ALESSANDRO CAMA, DOMENICO ACCILI, FABRIZIO BARBETTI, EIICHI IMANO, HIROKO KADOWAKI, AND TAKASHI KADOWAKI Diabetes Branch (S.I.T., A.C., D.A., F.B.), National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland 20892; Third Department of Internal Medicine (T.K.), Faculty of Medicine, University of Tokyo, Tokyo, Japan; Institutes for Diabetes Care and Research (H.K., T.K.), Asahi Life Foundation, Tokyo, Japan; and First Department of Internal Medicine (E.I.), Osaka University Medical School, Osaka, Japan
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ENETIC factors are an important determinant of whether an individual will develop noninsulindependent diabetes mellitus (NIDDM). However, the pattern of inheritance of NIDDM is complex and not well understood. In fact, the genetic complexity led one author to refer to diabetes mellitus as a "geneticist's nightmare" (1). Nevertheless, the pathophysiology of the disease is becoming better understood. Most patients with NIDDM are resistant to the biological actions of insulin (2, 3). Prospective studies have demonstrated that insulin resistance is a prominent feature early in the natural history of the disease when the patients have impaired glucose tolerance, even before the time that they develop overt diabetes. Whether or not an insulin resistant patient develops NIDDM is determined by the capacity of the pancreatic /3-cell to secrete sufficient insulin to overcome the insulin resistance (2-4). In those patients who develop insulin deficiency in addition to insulin resistance, the level of plasma glucose rises and the patient develops diabetes mellitus. For several decades, the literature has been filled with a lively debate about whether insulin resistance or insulin deficiency is the more important defect in the pathophysiology of NIDDM (2-4). Indeed, it is possible that one mutation causes the insulin resistance while another mutation at a distinct genetic locus causes the defect in insulin secretion. Fortunately, recent advances in molecular genetics now make it possible to address these questions directly. Application of genetic mapping techniques has led to the identification of the mutations responsible for causing such diseases as Duchenne's muscular dystrophy, cystic fibrosis, and neuroflbromatosis. Received July 27,1991. Address correspondence and requests for reprints to: Simeon I. Taylor, M.D., Ph.D., National Institutes of Health, Building 10, Room 8S-243, Bethesda, Maryland 20892.
Thus far, this approach has been most fruitful when applied to genetic diseases with simple Mendelian inheritance patterns. However, it is now possible to apply the methods of modern molecular genetics to investigate diabetes mellitus. One of the principal obstacles to genetic analysis of NIDDM is the possibility that the syndrome is a heterogeneous collection of distinct genetic diseases. To overcome this obstacle, many investigations have focused upon specific subtypes of diabetes in the hope that this would reduce the genetic heterogeneity. For example, maturity onset-type diabetes of youth (MOD Y) is a form of diabetes that has an autosomal dominant pattern of inheritance. By applying a gene mapping strategy to one large family with MODY, it has been possible to map a mutation causing MODY to chromosome 20 within 8 centimorgans of the adenosine deaminase gene (5). However, most of the progress in identifying mutations associated with diabetes has derived from studies of candidate genes. Because of their obvious importance to insulin action, the genes encoding insulin and the insulin receptor are the two genes that have been most intensively investigated. Five distinct mutations have been identified in the insulin gene (6): two mutations that impair the proteolytic processing of proinsulin to insulin, and three mutations that reduce the affinity with which the insulin molecule binds to its receptor. The first mutations in the insulin receptor gene were identified in 1988 (7, 8). Since that time, more than 25 mutations have been identified (Fig. 1). Most of the mutations in the insulin receptor gene have been identified in patients with genetic syndromes associated with insulin resistance and acanthosis nigricans. Syndromes of insulin resistance associated with acanthosis nigricans Leprechaunism is a congenital syndrome associated with extreme insulin resistance (9). These patients have 1158
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GENETIC BASIS OF ENDOCRINE DISEASE
glucose intolerance despite having peak insulin levels that may be increased as much as 100-fold over the normal range. In addition to insulin resistance, these patients have multiple abnormalities, including intrauterine growth retardation and fasting hypoglycemia (Fig. 2A). They usually die within the first year of life. Thus far, all of the patients with leprechaunism who have been studied have had two mutant alleles of the insulin receptor gene (7, 9-12). Type A insulin resistance is defined by the triad of insulin resistance, acanthosis nigricans, and hyperandrogenism in the absence of obesity or lipoatrophy (Fig. 2B) (9,13). Both acanthosis nigricans and hyperandrogenism correlate with hyperinsulinemia so that it has been proposed that they are caused by "toxic" effects of insulin upon the skin and the ovaries, respectively. Because these patients have defects in the function of their insulin receptors, it has been proposed that the toxic effects of insulin are not mediated by insulin receptors, but by receptors for homologous peptides such as insulin-like growth factor-I/somatomedin C (9). Several patients with type A insulin resistance have been reported to have either two mutant alleles (10,14,15) or one mutant allele Missense Mutations in Insulin Receptor Gene
A
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(16-19) of the insulin receptor gene. In general, the patients with one mutant allele appear to be less insulin resistant than the patients with two mutant alleles. The Rabson-Mendenhall syndrome is defined by the presence of several clinical features including extreme insulin resistance, acanthosis nigricans, abnormalities of teeth and nails, and pineal hyperplasia (9). From a clinical point of view, this syndrome appears to be intermediate in severity between leprechaunism and type A insulin resistance. At least one such patient has been studied who was a compound heterozygote with two different mutant alleles of the insulin receptor gene (10). Mutations in the insulin receptor gene The insulin receptor is located on the cell surface, and is responsible for mediating insulin action. It consists of two a- and two /3-subunits. The a-subunit is entirely extracellular, and contains the insulin binding site (Fig. 1). The /3-subunit anchors the receptor in the plasma membrane, and possesses tyrosine-specific protein kinase activity. When insulin binds to the extracellular domain, this activates the tyrosine kinase to phosphoPremature Termination Mutations in Insulin Receptor Gene
B
Excns
a i
(Impaired transport to cell surface; . Asn 15 -» Lys decreased binding affinity) 28
Phs-68,89 Cys-rlch (155-312)
• - V a l - > Ala ••Gly3 1 _>Arg I - His
209
I * Leu
233
Phe-88,89 Trp
- > Arg (impaired transport to cell surface) - > Pro (Decreased binding)
Cys-435 Cys-468 Cys-524
1
- * Val (Impaired transport to cell surface) (Increased binding affinity; •Lys460-»Glu decreased pH sensitivity) r A s n 4 6 2 - * S e r (Decreased pH sensitivity)
,s
.•Arg
(Uncleaved receptor; decreased binding affinity)
4
ys-
H
W
(AG
6
H
' A"
Cys-468 H — H
—> GG (Defective splice acceptor; Intron 4) (Truncated receptor; fusion protein)
Cys-524
Exon 11 • (718-729) ^
Tyr-1158,1162,1163
Val 9 8 5
-»Met -»Leu
Arg
—> Gin
•1 * '
1008
- » Val
*«\\Gly Tyr-1328,1334 I
,i\Ala -»Thr »\1135 ^ _ , •', Ala
Decreased tyrosine kinase
(Truncated receptor)
12
• Arg
•Pro
—> Stop
—
Extracellular
Extracellular
Tyr-972 ATP-Blndlng | (1003-1030)
(Decreased mRNA levels)
382
Gin Exon 11 (718-729)
Stop
Cys-rlch (155-312)
- G l y 3 6 8 - > Arg •-Phe
a
"^Stop
(Decreased mRNA levels)
(Truncated receptor; fusion protein)
Tyr-972 H ATP-Blndlng • (1003-1030) •
iS_ 17 ~
Tyr-1158,1162,1163 Q
^2-
Tyr-1328,1334 Q
P
1000 Arg - > Stop
(Decreased mRNA levels)
22
Pf
(Truncated receptor; fusion protein)
—» Glu
\W 1 5 S -»i1e
FIG. 1. Mutations in the insulin receptor gene in insulin-resistant patients. A, Structural map of the insulin receptor. Key structural landmarks of the insulin receptor are identified at the left of the drawings. Phe88, Phe89, and the cysteine-rich domain have all been implicated as playing a role in the insulin binding domain. Cys435, Cys468, and Cys524 are candidates to contribute sulfhydryl groups for formation of the disulfide bonds between adjacent a-subunits. Exon 11 is an exon which has been described to undergo variable splicing. The five tyrosine residues which are sites of autophosphorylation are indicated (Tyr1158, Tyr1162, Tyr1163, Tyr1328, and Tyr1334). Tyr972 is the fourth amino acid residue in the Asn-Pro-Glu-Tyr motif that has been suggested to play a necessary role in receptor endocytosis. In addition, evidence has been obtained suggesting that Tyr972 plays a role in determining the specificity of the receptor tyrosine kinase for biologically important protein substrates. The consensus sequence for an ATP binding domain is located between amino acid residues 1003-1030. A, Locations of all of the reported missense mutations are indicated in the right half of the drawing of the receptor. B, Locations of the 22 exons of the insulin receptor gene are indicated in the middle of the receptor cartoon. In addition, all of the published mutations causing premature chain termination are indicated on the right side of the cartoon.
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TAYLOR ET AL.
FlG. 2. Photograph of patients with leprechaunism and type A extreme insulin resistance. A, Photograph of leprechaun/Minn-1 (9, 12). This child was small for gestational age, weighing only 1.3 kg at birth. In addition, she had multiple phenotypic abnormalities including low-set ears and a depressed nasal bridge. B, Photograph of patient A-5 (9, 14). This photograph illustrates acanthosis nigricans on the back of her neck. The hirsutism noted on her back reflects the hyperandrogenism due to increased ovarian production of testosterone.
rylate tyrosine residues in the receptor, and also to phosphorylate other cellular proteins. A large body of evidence supports the hypothesis that activation of the tyrosine kinase is responsible for triggering the biological actions of insulin upon the target cell. Multiple different mutations have been identified in the insulin receptor genes of insulin resistant patients (7-12, 14-25). In several instances, the same mutation has been identified in two apparently unrelated patients {e.g. Refs. 10 and 15). However, most of the published mutations have been identified in only one single kindred (7-12,14-25). Some patients have been reported to have
JCE & M • 1991 Vol 73 • No 6
two mutant alleles of the insulin receptor gene. Patients from consanguineous pedigrees were homozygous for a single mutation (10, 11, 14); other patients were compound heterozygotes, each having inherited two distinct mutant alleles of the insulin receptor gene (7,10,12,15). Finally some patients with type A insulin resistance are heterozygous for a single mutation (16-19) while the second allele of the insulin receptor gene appears to be normal. Mutations in the insulin receptor gene can be classified in five classes: Class 1. Impaired receptor biosynthesis. Some mutations lead to a decrease in the level of insulin receptor mRNA. In some cases, the mutations have been mapped outside of the regions of the gene that encode the amino acid sequence of the receptor protein (12, 20). Although not proven definitively, it is likely that these mutations are located in regulatory domains of the gene and decrease the rate at which the gene is transcribed. In other patients, the level of insulin receptor messenger RNA is decreased by premature chain termination mutations (10, 12, 15, 21). Premature chain termination can be caused by several different types of mutations: nonsense mutations (10, 12, 15), mutations at intron-exon junctions that impair splicing of mRNA (21), and deletion mutations that shift the reading frame (22, 23). Class 2. Impaired transport of receptors to the cell surface. The insulin receptor gene encodes a precursor molecule that undergoes multiple posttranslational processing steps within the endoplasmic reticulum and Golgi (9): N-linked glycosylation; formation of intra- and intersubunit disulfide bonds; proteolytic cleavage into two subunits; O-linked glycosylation; and acylation. Some mutations interfere with the folding of the receptor into its normal conformation. Consequently, the receptors are not transported efficiently through the endoplasmic reticulum and Golgi to the plasma membrane. This type of mutation leads to a reduction in the number of receptors on the cell surface. In addition, because the mutations prevent the receptor from folding into its normal conformation, class 2 mutations may also reduce the affinity of insulin binding (24) or inhibit the activation of the receptor tyrosine kinase (25). All of the published mutations in this class (Lys15, Arg209, and Val382) have been mapped to the N-terminal half of the a-subunit (Fig. 1). Class 3. Decreased affinity of insulin binding. Mutations can decrease the affinity with which insulin binds to its receptor. In principle, this would be expected to cause a rightward shift in the dose-response curve for insulin action—i.e. to decrease the sensitivity with which the target cell responds to insulin without causing a decrease in the maximal responsiveness. The first mutation reported to decrease the affinity of insulin binding
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GENETIC BASIS OF ENDOCRINE DISEASE mapped to amino acid residue 735, the last amino acid in the Arg-Lys-Arg-Arg motif at the proteolytic cleavage site between the a- and /3-subunits (8). This mutation (substitution of serine for Arg735) inhibits cleavage of the precursor into two subunits, and distorts the conformation of the receptor so that the affinity of insulin binding is reduced. As noted above, the Lys15-mutation impairs transport of the receptor to the cell surface. However, a small percentage of the mutant receptors undergo normal posttranslational processing and are eventually transported to the cell surface. Nevertheless, even the receptors on the cell surface are impaired in their function in that the Lys16-mutant receptor has a 5-fold reduction in binding affinity (24). Class 4. Impaired tyrosine kinase activity. When insulin binds to the receptor, this activates the receptor tyrosine kinase activity. Many mutations have been identified in the tyrosine kinase domain that inhibit receptor tyrosine kinase activity (Fig. 1). This fact is among the compelling arguments that tyrosine kinase activity plays a necessary role in mediating the metabolic actions of insulin in man in vivo. The Val1008-mutation is the prototype of this type of mutation (16). In this mutation, valine is substituted for Gly1008, the third glycine in the Gly-X-Gly-X-X-Gly motif characteristic of the ATPbinding sites of all kinase enzymes. Since ATP is the phosphate donor in the reaction catalyzed by receptor tyrosine kinases, it is easy to understand that a mutation in the ATP binding site would inactivate tyrosine kinase activity. One other feature of mutations in the tyrosine kinase domain deserves emphasis: these mutation appear to cause insulin resistance in a dominant fashion (1619, 26). Since the insulin receptor has an 0L2P2 structure, it is possible to have a hybrid form (a^wtftnut) in addition to the symmetrical a2Wwt)2 and «2(/3mut)2 forms. It appears that only the symmetrical wild type receptor 2] is active as a tyrosine kinase. Both the hybrid ut and the symmetrical a2(j8mut)2 receptors are inactive as tyrosine kinase enzymes. This appears to be the molecular explanation of the dominant pattern of inheritance observed with mutations in the tyrosine kinase domain. Class 5. Accelerated receptor degradation. Insulin binding to the receptor triggers endocytosis of the insulin-receptor complex. Internalized receptors are located in endosomes with the insulin binding site oriented on the inside of the vesicle. Endosomal proton pumps generate an acidic pH inside the endosome (pH « 5.5). The acidic pH promotes dissociation of insulin from the receptor. Some mutations {e.g. Glu460 and Ser462) impair the ability of acidic pH to release insulin from the receptor (7, 9,10, 27). Subsequent to internalization, there are at least two
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pathways available to the receptor: recycling back to the plasma membrane for reutilization us. degradation within the lysosome. Mutations that desensitize the receptor to acidic pH are associated with an impaired recycling pathway, and consequently preferential targeting of the receptor toward lysosomal degradation (27). This accelerates the rate of receptor degradation, as a result of which there is a decrease in the number of receptors on the cell surface. Role of receptor defects in NIDDM Many patients with mutations in the insulin receptor gene, especially those with two mutant alleles, are extremely resistant to insulin, requiring therapy with as much as several thousand units of insulin per day (9,14). However, some patients, particularly those who are heterozygous for a single mutant allele, have more modest degrees of insulin resistance, comparable to that which is observed in patients with impaired glucose tolerance or NIDDM (7,9,19,23, 28). Moreover, like most patients with NIDDM, some of these patients are obese (19, 23). It is possible that mutations in the insulin receptor gene provide a genetic predisposition that exacerbates the effect of obesity to induce insulin resistance. Theoretical calculations have led to a prediction that approximately 0.1-1% of the total population might have at least one mutant allele of the insulin receptor gene (9). In a recent study of 30 Caucasian patients with NIDDM, two patients («7%) were found to have variant amino acid sequences in the tyrosine kinase domain of the insulin receptor (29). One of these variant sequences (substitution of methionine for Val985) was also detected in 1 of 13 control subjects. Furthermore, when the Met985substituted receptor was expressed in transfected cells, the variant receptor appeared to function normally (Moller, D., personal communication). Therefore, it is likely that the Met985-variant is a normal polymorphism without functional significance. However, a second amino acid substitution (replacement of Lys1068 by glutamic acid) was also identified in a patient with NIDDM (29). It is not yet known whether the Glu1068-substitution impairs receptor tyrosine kinase activity. In contrast, in studies of three insulin-resistant Pima Indians, a racial group with a prevalence of NIDDM in excess of 50%, no mutations were detected in the insulin receptor gene (30, 31). The earliest clinical investigations of the insulin receptor gene relied upon classical cloning techniques of recombinant DNA technology to identify mutations (7, 8, 14, 30). This approach required a significant investment of labor (« 1-man yr) to study each individual patient. Use of the polymerase chain reaction effected a 10-fold reduction in the effort required to study each
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TAYLOR ET AL.
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patient (14, 21, 31). Very recently, methods have been developed to allow for screening of relatively large numbers of patients to identify those with mutations in their insulin receptor genes. For example, one study analyzed single-stranded conformational polymorphisms to screen 43 individuals (29). We have used the technique of denaturing gradient gel electrophoresis to screen patients, and have shown that this rapid method has an approximately 90% sensitivity to detect mutations (Barbetti, F., Gejman, P. V., Taylor, S. I., etai, manuscript submitted). Thus, it is now possible to carry out studies on sufficiently large numbers of patients to obtain a reliable estimate of the prevalence of mutations in the insulin receptor gene among patients with NIDDM, and also in the general population. Conclusion Studies of the insulin receptor gene provide an excellent example of the power of modern molecular genetics in identifying mutations in genes that encode proteins required for normal insulin action and normal glucose homeostasis. By continuing to apply these techniques to relevant genes, it will be possible to obtain a better understanding of the causes of diabetes mellitus. It is to be hoped that improved understanding will eventually bear fruit by leading to novel approaches to treatment and prevention of diabetes.
Acknowledgments We are grateful to Drs. Jesse Roth and Phillip Gorden for helpful discussions and to Dr. Marc Reitman for critical reading of the manuscript. Partial research support was provided from the American Diabetes Association and the Juvenile Diabetes Foundation.
References 1. Neel JV. Diabetes mellitus—a geneticist's nightmare. In: The genetics of diabetes mellitus (Creutzfeldt W, Kobberling J, Neel JV, eds). New York: Springer-Verlag, 1976;p 1. 2. Reaven GM. Role of insulin resistance in human disease. Diabetes. 1988;37:1595-1607. 3. DeFronzo RA. Lilly lecture 1987. The triumvirate: beta-cell, muscle, liver. A collusion responsible for NIDDM. Diabetes. 1988;37:667-687. 4. Porte Jr D. Banting lecture 1990. Beta-cells in type II diabetes mellitus. Diabetes. 1991;40:166-180. 5. Bell GI, Xiang KS, Newman MV, Wu SH, Wright LG, Fajans SS, Spielman RS, Cox NJ. Gene for non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young subtype) is linked to DNA polymorphism on human chromosome 20q. Proc Natl Acad Sci USA. 1991;88:1484-1488. 6. Steiner DF, Tager HS, Chan SJ, Nanjo K, Sanke T, Rubenstein AH. Lessons learned from molecular biology of insulin-gene mutations. Diabetes Care. 1990; 13:600-609. 7. Kadowaki T, Bevins CL, Cama A, et al. Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance. Science. 1988;240:787-790.
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8. Yoshimasa Y, Seino S, Whittaker J, et al. Insulin-resistant diabetes due to a point mutation that prevents insulin proreceptor processing. Science. 1988;240:784-787. 9. Taylor SI, Kadowaki T, Kadowaki H, Accili D, Cama A, McKeon C. Mutations in the insulin receptor gene in insulin resistant patients. Diabetes Care. 1990;13:257-279. 10. Kadowaki T, Kadowaki H, Rechler MM, et al. Five mutant alleles of the insulin receptor gene in patients with genetic forms of insulin resistance. J Clin Invest. 1990;86:254-264. 11. Klinkhamer M, Groen NA, van der Zon GCM, Lindhout D, Sandkuyl LA, Krans HM, Moller W, Maassen JA. A leucine-to-proline mutation in the insulin receptor in a family with insulin resistance. EMBO J. 1989;8:2503-2507. 12. Kadowaki T, Kadowaki H, Taylor SI. A nonsense mutation causing decreased levels of insulin receptor mRNA: Detection by a simplified technique for direct sequencing of genomic DNA amplified by polymerase chain reaction. Proc Natl Acad Sci USA. 1990;87:658662. 13. Kahn CR, Flier JS, Bar RS, et al. The syndromes of insulin resistance and acanthosis nigricans. Insulin-receptor disorders in man. N Engl J Med. 1976;294:739-745. 14. Accili D, Frapier C, Mosthaf L, et al. A mutation in the insulin receptor gene that impairs transport of the receptor to the plasma membrane and causes insulin resistant diabetes. EMBO J. 1989;8:2509-2517. 15. Kusari J, Takata Y, Hatada E, Freidenberg G, Kolterman O, Olefsky JM. Insulin resistance and diabetes due to different mutations in the tyrosine kinase domain of both insulin receptor gene alleles. J Biol Chem. 1991;266:5260-5267 16. Odawara M, Kadowaki T, Yamamoto R, et al. Human diabetes associated with a mutation in the tyrosine kinase domain of the insulin receptor. Science. 1989;245:66-68. 17. Moller DE, Flier JS. Detection of an alteration in the insulinreceptor gene in a patient with insulin resistance, acanthosis nigricans, and the polycystic ovary syndrome (type A insulin resistance). N Engl J Med. 1988;319:1526-1529. 18. Moller DE, Yokota A, White M, Pazianos AG, Flier JS. A naturally occurring mutation of insulin receptor Ala1134 impairs tyrosine kinase function and is associated with dominantly inherited insulin resistance. J Biol Chem. 1990;265:14979-85. 19. Cama A, Sierra ML, Ottini L, Kadowaki T, Gorden P, ImperatoMcGinley J, Taylor SI. A mutation in the tyrosine kinase domain of the insulin receptor associated with insulin resistance in an obese woman. J Clin Endocrinol Metab. 1991;73:894-901. 20. Imano E, Kadowaki H, Kadowaki T, et al. Two patients with insulin resistance due to decreased levels of insulin receptor mRNA. Diabetes. 1991;40:548-557. 21. Kadowaki T, Kadowaki H, Ando A, Kaburagi Y, Quin JD, MacCuish A, Taylor SI, Yazaki Y, Kasuga M. Two mutant alleles of the insulin receptor gene in insulin resistant patients. Prog and Abstracts of 73rd Annual Meeting of the Endocrine Soc. 1991; p. 310 (Abstract 1119). 22. Taira M, Taira M, Hashimoto N, et al. Human diabetes associated with a deletion of the tyrosine kinase domain of the insulin receptor. Science. 1989;245:63-66. 23. Shimada F, Taira M, Suzuki Y, et al. Insulin-resistant diabetes associated with partial deletion of insulin-receptor gene. Lancet. 1990:335:1179-1181. 24. Kadowaki T, Kadowaki H, Accili D, Taylor SI. Substitution of lysine for asparagine-15 in the human insulin receptor impairs intracellular transport of the receptor to the cell surface and decreases the affinity of insulin binding. J Biol Chem. 1990;265:19143-19150. 25. Accili D, Mosthaf L, Ullrich A, Taylor SI. A mutation in the extracellular domain of the insulin receptor impairs the ability of insulin to stimulate receptor autophosphorylation. J Biol Chem. 1991;266:434-439. 26. Treadway JL, Morrison BD, Soos MA, et al. Transdominant in-
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GENETIC BASIS OF ENDOCRINE DISEASE hibition of tyrosine kinase activity in mutant insulin/insulin-like growth factor I hybrid receptors. Proc Natl Acad Sci USA. 1991;88:214-218. 27. Kadowaki H, Kadowaki T, Cama A, et al. Mutagenesis of lysine460 in the human insulin receptor: effects upon receptor recycling and site-site interactions among binding sites. J Biol Chem. 1990;265:21285-21296. 28. Lekanne Deprez RH, Potter van Loon BJ, et al. Individuals with only one allele for a functional insulin receptor have a tendency to hyperinsulinaemia but not to hyperglycaemia. Diabetologia.
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1989;32:740-744. 29. O'Rahilly S, Choi WH, Patel P, Turner RC, Flier JS, Moller DE. Detection of mutations in insulin-receptor gene in NIDDM patients by analysis of single-stranded conformation polymorphisms. Diabetes. 1991;40:777-82. 30. Cama A, Patterson A, Kadowaki T, et al. The amino acid sequence of the insulin receptor is normal in an insulin resistant Pima Indian. J Clin Endocrinol Metab. 1990;70:1155-1161. 31. Moller DE, Yokota A, Flier JS. Normal insulin-receptor cDNA sequence in Pima Indians with NIDDM. Diabetes. 1989;38:14961500.
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Vol. 73, No. 6 Printed in U.S.A.
GENETIC BASIS OF ENDOCRINE DISEASE 1 Molecular Genetics of Insulin Resistant Diabetes Mellitus SIMEON I. TAYLOR, ALESSANDRO CAMA, DOMENICO ACCILI, FABRIZIO BARBETTI, EIICHI IMANO, HIROKO KADOWAKI, AND TAKASHI KADOWAKI Diabetes Branch (S.I.T., A.C., D.A., F.B.), National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland 20892; Third Department of Internal Medicine (T.K.), Faculty of Medicine, University of Tokyo, Tokyo, Japan; Institutes for Diabetes Care and Research (H.K., T.K.), Asahi Life Foundation, Tokyo, Japan; and First Department of Internal Medicine (E.I.), Osaka University Medical School, Osaka, Japan
G
ENETIC factors are an important determinant of whether an individual will develop noninsulindependent diabetes mellitus (NIDDM). However, the pattern of inheritance of NIDDM is complex and not well understood. In fact, the genetic complexity led one author to refer to diabetes mellitus as a "geneticist's nightmare" (1). Nevertheless, the pathophysiology of the disease is becoming better understood. Most patients with NIDDM are resistant to the biological actions of insulin (2, 3). Prospective studies have demonstrated that insulin resistance is a prominent feature early in the natural history of the disease when the patients have impaired glucose tolerance, even before the time that they develop overt diabetes. Whether or not an insulin resistant patient develops NIDDM is determined by the capacity of the pancreatic /3-cell to secrete sufficient insulin to overcome the insulin resistance (2-4). In those patients who develop insulin deficiency in addition to insulin resistance, the level of plasma glucose rises and the patient develops diabetes mellitus. For several decades, the literature has been filled with a lively debate about whether insulin resistance or insulin deficiency is the more important defect in the pathophysiology of NIDDM (2-4). Indeed, it is possible that one mutation causes the insulin resistance while another mutation at a distinct genetic locus causes the defect in insulin secretion. Fortunately, recent advances in molecular genetics now make it possible to address these questions directly. Application of genetic mapping techniques has led to the identification of the mutations responsible for causing such diseases as Duchenne's muscular dystrophy, cystic fibrosis, and neuroflbromatosis. Received July 27,1991. Address correspondence and requests for reprints to: Simeon I. Taylor, M.D., Ph.D., National Institutes of Health, Building 10, Room 8S-243, Bethesda, Maryland 20892.
Thus far, this approach has been most fruitful when applied to genetic diseases with simple Mendelian inheritance patterns. However, it is now possible to apply the methods of modern molecular genetics to investigate diabetes mellitus. One of the principal obstacles to genetic analysis of NIDDM is the possibility that the syndrome is a heterogeneous collection of distinct genetic diseases. To overcome this obstacle, many investigations have focused upon specific subtypes of diabetes in the hope that this would reduce the genetic heterogeneity. For example, maturity onset-type diabetes of youth (MOD Y) is a form of diabetes that has an autosomal dominant pattern of inheritance. By applying a gene mapping strategy to one large family with MODY, it has been possible to map a mutation causing MODY to chromosome 20 within 8 centimorgans of the adenosine deaminase gene (5). However, most of the progress in identifying mutations associated with diabetes has derived from studies of candidate genes. Because of their obvious importance to insulin action, the genes encoding insulin and the insulin receptor are the two genes that have been most intensively investigated. Five distinct mutations have been identified in the insulin gene (6): two mutations that impair the proteolytic processing of proinsulin to insulin, and three mutations that reduce the affinity with which the insulin molecule binds to its receptor. The first mutations in the insulin receptor gene were identified in 1988 (7, 8). Since that time, more than 25 mutations have been identified (Fig. 1). Most of the mutations in the insulin receptor gene have been identified in patients with genetic syndromes associated with insulin resistance and acanthosis nigricans. Syndromes of insulin resistance associated with acanthosis nigricans Leprechaunism is a congenital syndrome associated with extreme insulin resistance (9). These patients have 1158
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GENETIC BASIS OF ENDOCRINE DISEASE
glucose intolerance despite having peak insulin levels that may be increased as much as 100-fold over the normal range. In addition to insulin resistance, these patients have multiple abnormalities, including intrauterine growth retardation and fasting hypoglycemia (Fig. 2A). They usually die within the first year of life. Thus far, all of the patients with leprechaunism who have been studied have had two mutant alleles of the insulin receptor gene (7, 9-12). Type A insulin resistance is defined by the triad of insulin resistance, acanthosis nigricans, and hyperandrogenism in the absence of obesity or lipoatrophy (Fig. 2B) (9,13). Both acanthosis nigricans and hyperandrogenism correlate with hyperinsulinemia so that it has been proposed that they are caused by "toxic" effects of insulin upon the skin and the ovaries, respectively. Because these patients have defects in the function of their insulin receptors, it has been proposed that the toxic effects of insulin are not mediated by insulin receptors, but by receptors for homologous peptides such as insulin-like growth factor-I/somatomedin C (9). Several patients with type A insulin resistance have been reported to have either two mutant alleles (10,14,15) or one mutant allele Missense Mutations in Insulin Receptor Gene
A
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(16-19) of the insulin receptor gene. In general, the patients with one mutant allele appear to be less insulin resistant than the patients with two mutant alleles. The Rabson-Mendenhall syndrome is defined by the presence of several clinical features including extreme insulin resistance, acanthosis nigricans, abnormalities of teeth and nails, and pineal hyperplasia (9). From a clinical point of view, this syndrome appears to be intermediate in severity between leprechaunism and type A insulin resistance. At least one such patient has been studied who was a compound heterozygote with two different mutant alleles of the insulin receptor gene (10). Mutations in the insulin receptor gene The insulin receptor is located on the cell surface, and is responsible for mediating insulin action. It consists of two a- and two /3-subunits. The a-subunit is entirely extracellular, and contains the insulin binding site (Fig. 1). The /3-subunit anchors the receptor in the plasma membrane, and possesses tyrosine-specific protein kinase activity. When insulin binds to the extracellular domain, this activates the tyrosine kinase to phosphoPremature Termination Mutations in Insulin Receptor Gene
B
Excns
a i
(Impaired transport to cell surface; . Asn 15 -» Lys decreased binding affinity) 28
Phs-68,89 Cys-rlch (155-312)
• - V a l - > Ala ••Gly3 1 _>Arg I - His
209
I * Leu
233
Phe-88,89 Trp
- > Arg (impaired transport to cell surface) - > Pro (Decreased binding)
Cys-435 Cys-468 Cys-524
1
- * Val (Impaired transport to cell surface) (Increased binding affinity; •Lys460-»Glu decreased pH sensitivity) r A s n 4 6 2 - * S e r (Decreased pH sensitivity)
,s
.•Arg
(Uncleaved receptor; decreased binding affinity)
4
ys-
H
W
(AG
6
H
' A"
Cys-468 H — H
—> GG (Defective splice acceptor; Intron 4) (Truncated receptor; fusion protein)
Cys-524
Exon 11 • (718-729) ^
Tyr-1158,1162,1163
Val 9 8 5
-»Met -»Leu
Arg
—> Gin
•1 * '
1008
- » Val
*«\\Gly Tyr-1328,1334 I
,i\Ala -»Thr »\1135 ^ _ , •', Ala
Decreased tyrosine kinase
(Truncated receptor)
12
• Arg
•Pro
—> Stop
—
Extracellular
Extracellular
Tyr-972 ATP-Blndlng | (1003-1030)
(Decreased mRNA levels)
382
Gin Exon 11 (718-729)
Stop
Cys-rlch (155-312)
- G l y 3 6 8 - > Arg •-Phe
a
"^Stop
(Decreased mRNA levels)
(Truncated receptor; fusion protein)
Tyr-972 H ATP-Blndlng • (1003-1030) •
iS_ 17 ~
Tyr-1158,1162,1163 Q
^2-
Tyr-1328,1334 Q
P
1000 Arg - > Stop
(Decreased mRNA levels)
22
Pf
(Truncated receptor; fusion protein)
—» Glu
\W 1 5 S -»i1e
FIG. 1. Mutations in the insulin receptor gene in insulin-resistant patients. A, Structural map of the insulin receptor. Key structural landmarks of the insulin receptor are identified at the left of the drawings. Phe88, Phe89, and the cysteine-rich domain have all been implicated as playing a role in the insulin binding domain. Cys435, Cys468, and Cys524 are candidates to contribute sulfhydryl groups for formation of the disulfide bonds between adjacent a-subunits. Exon 11 is an exon which has been described to undergo variable splicing. The five tyrosine residues which are sites of autophosphorylation are indicated (Tyr1158, Tyr1162, Tyr1163, Tyr1328, and Tyr1334). Tyr972 is the fourth amino acid residue in the Asn-Pro-Glu-Tyr motif that has been suggested to play a necessary role in receptor endocytosis. In addition, evidence has been obtained suggesting that Tyr972 plays a role in determining the specificity of the receptor tyrosine kinase for biologically important protein substrates. The consensus sequence for an ATP binding domain is located between amino acid residues 1003-1030. A, Locations of all of the reported missense mutations are indicated in the right half of the drawing of the receptor. B, Locations of the 22 exons of the insulin receptor gene are indicated in the middle of the receptor cartoon. In addition, all of the published mutations causing premature chain termination are indicated on the right side of the cartoon.
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TAYLOR ET AL.
FlG. 2. Photograph of patients with leprechaunism and type A extreme insulin resistance. A, Photograph of leprechaun/Minn-1 (9, 12). This child was small for gestational age, weighing only 1.3 kg at birth. In addition, she had multiple phenotypic abnormalities including low-set ears and a depressed nasal bridge. B, Photograph of patient A-5 (9, 14). This photograph illustrates acanthosis nigricans on the back of her neck. The hirsutism noted on her back reflects the hyperandrogenism due to increased ovarian production of testosterone.
rylate tyrosine residues in the receptor, and also to phosphorylate other cellular proteins. A large body of evidence supports the hypothesis that activation of the tyrosine kinase is responsible for triggering the biological actions of insulin upon the target cell. Multiple different mutations have been identified in the insulin receptor genes of insulin resistant patients (7-12, 14-25). In several instances, the same mutation has been identified in two apparently unrelated patients {e.g. Refs. 10 and 15). However, most of the published mutations have been identified in only one single kindred (7-12,14-25). Some patients have been reported to have
JCE & M • 1991 Vol 73 • No 6
two mutant alleles of the insulin receptor gene. Patients from consanguineous pedigrees were homozygous for a single mutation (10, 11, 14); other patients were compound heterozygotes, each having inherited two distinct mutant alleles of the insulin receptor gene (7,10,12,15). Finally some patients with type A insulin resistance are heterozygous for a single mutation (16-19) while the second allele of the insulin receptor gene appears to be normal. Mutations in the insulin receptor gene can be classified in five classes: Class 1. Impaired receptor biosynthesis. Some mutations lead to a decrease in the level of insulin receptor mRNA. In some cases, the mutations have been mapped outside of the regions of the gene that encode the amino acid sequence of the receptor protein (12, 20). Although not proven definitively, it is likely that these mutations are located in regulatory domains of the gene and decrease the rate at which the gene is transcribed. In other patients, the level of insulin receptor messenger RNA is decreased by premature chain termination mutations (10, 12, 15, 21). Premature chain termination can be caused by several different types of mutations: nonsense mutations (10, 12, 15), mutations at intron-exon junctions that impair splicing of mRNA (21), and deletion mutations that shift the reading frame (22, 23). Class 2. Impaired transport of receptors to the cell surface. The insulin receptor gene encodes a precursor molecule that undergoes multiple posttranslational processing steps within the endoplasmic reticulum and Golgi (9): N-linked glycosylation; formation of intra- and intersubunit disulfide bonds; proteolytic cleavage into two subunits; O-linked glycosylation; and acylation. Some mutations interfere with the folding of the receptor into its normal conformation. Consequently, the receptors are not transported efficiently through the endoplasmic reticulum and Golgi to the plasma membrane. This type of mutation leads to a reduction in the number of receptors on the cell surface. In addition, because the mutations prevent the receptor from folding into its normal conformation, class 2 mutations may also reduce the affinity of insulin binding (24) or inhibit the activation of the receptor tyrosine kinase (25). All of the published mutations in this class (Lys15, Arg209, and Val382) have been mapped to the N-terminal half of the a-subunit (Fig. 1). Class 3. Decreased affinity of insulin binding. Mutations can decrease the affinity with which insulin binds to its receptor. In principle, this would be expected to cause a rightward shift in the dose-response curve for insulin action—i.e. to decrease the sensitivity with which the target cell responds to insulin without causing a decrease in the maximal responsiveness. The first mutation reported to decrease the affinity of insulin binding
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GENETIC BASIS OF ENDOCRINE DISEASE mapped to amino acid residue 735, the last amino acid in the Arg-Lys-Arg-Arg motif at the proteolytic cleavage site between the a- and /3-subunits (8). This mutation (substitution of serine for Arg735) inhibits cleavage of the precursor into two subunits, and distorts the conformation of the receptor so that the affinity of insulin binding is reduced. As noted above, the Lys15-mutation impairs transport of the receptor to the cell surface. However, a small percentage of the mutant receptors undergo normal posttranslational processing and are eventually transported to the cell surface. Nevertheless, even the receptors on the cell surface are impaired in their function in that the Lys16-mutant receptor has a 5-fold reduction in binding affinity (24). Class 4. Impaired tyrosine kinase activity. When insulin binds to the receptor, this activates the receptor tyrosine kinase activity. Many mutations have been identified in the tyrosine kinase domain that inhibit receptor tyrosine kinase activity (Fig. 1). This fact is among the compelling arguments that tyrosine kinase activity plays a necessary role in mediating the metabolic actions of insulin in man in vivo. The Val1008-mutation is the prototype of this type of mutation (16). In this mutation, valine is substituted for Gly1008, the third glycine in the Gly-X-Gly-X-X-Gly motif characteristic of the ATPbinding sites of all kinase enzymes. Since ATP is the phosphate donor in the reaction catalyzed by receptor tyrosine kinases, it is easy to understand that a mutation in the ATP binding site would inactivate tyrosine kinase activity. One other feature of mutations in the tyrosine kinase domain deserves emphasis: these mutation appear to cause insulin resistance in a dominant fashion (1619, 26). Since the insulin receptor has an 0L2P2 structure, it is possible to have a hybrid form (a^wtftnut) in addition to the symmetrical a2Wwt)2 and «2(/3mut)2 forms. It appears that only the symmetrical wild type receptor 2] is active as a tyrosine kinase. Both the hybrid ut and the symmetrical a2(j8mut)2 receptors are inactive as tyrosine kinase enzymes. This appears to be the molecular explanation of the dominant pattern of inheritance observed with mutations in the tyrosine kinase domain. Class 5. Accelerated receptor degradation. Insulin binding to the receptor triggers endocytosis of the insulin-receptor complex. Internalized receptors are located in endosomes with the insulin binding site oriented on the inside of the vesicle. Endosomal proton pumps generate an acidic pH inside the endosome (pH « 5.5). The acidic pH promotes dissociation of insulin from the receptor. Some mutations {e.g. Glu460 and Ser462) impair the ability of acidic pH to release insulin from the receptor (7, 9,10, 27). Subsequent to internalization, there are at least two
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pathways available to the receptor: recycling back to the plasma membrane for reutilization us. degradation within the lysosome. Mutations that desensitize the receptor to acidic pH are associated with an impaired recycling pathway, and consequently preferential targeting of the receptor toward lysosomal degradation (27). This accelerates the rate of receptor degradation, as a result of which there is a decrease in the number of receptors on the cell surface. Role of receptor defects in NIDDM Many patients with mutations in the insulin receptor gene, especially those with two mutant alleles, are extremely resistant to insulin, requiring therapy with as much as several thousand units of insulin per day (9,14). However, some patients, particularly those who are heterozygous for a single mutant allele, have more modest degrees of insulin resistance, comparable to that which is observed in patients with impaired glucose tolerance or NIDDM (7,9,19,23, 28). Moreover, like most patients with NIDDM, some of these patients are obese (19, 23). It is possible that mutations in the insulin receptor gene provide a genetic predisposition that exacerbates the effect of obesity to induce insulin resistance. Theoretical calculations have led to a prediction that approximately 0.1-1% of the total population might have at least one mutant allele of the insulin receptor gene (9). In a recent study of 30 Caucasian patients with NIDDM, two patients («7%) were found to have variant amino acid sequences in the tyrosine kinase domain of the insulin receptor (29). One of these variant sequences (substitution of methionine for Val985) was also detected in 1 of 13 control subjects. Furthermore, when the Met985substituted receptor was expressed in transfected cells, the variant receptor appeared to function normally (Moller, D., personal communication). Therefore, it is likely that the Met985-variant is a normal polymorphism without functional significance. However, a second amino acid substitution (replacement of Lys1068 by glutamic acid) was also identified in a patient with NIDDM (29). It is not yet known whether the Glu1068-substitution impairs receptor tyrosine kinase activity. In contrast, in studies of three insulin-resistant Pima Indians, a racial group with a prevalence of NIDDM in excess of 50%, no mutations were detected in the insulin receptor gene (30, 31). The earliest clinical investigations of the insulin receptor gene relied upon classical cloning techniques of recombinant DNA technology to identify mutations (7, 8, 14, 30). This approach required a significant investment of labor (« 1-man yr) to study each individual patient. Use of the polymerase chain reaction effected a 10-fold reduction in the effort required to study each
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TAYLOR ET AL.
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patient (14, 21, 31). Very recently, methods have been developed to allow for screening of relatively large numbers of patients to identify those with mutations in their insulin receptor genes. For example, one study analyzed single-stranded conformational polymorphisms to screen 43 individuals (29). We have used the technique of denaturing gradient gel electrophoresis to screen patients, and have shown that this rapid method has an approximately 90% sensitivity to detect mutations (Barbetti, F., Gejman, P. V., Taylor, S. I., etai, manuscript submitted). Thus, it is now possible to carry out studies on sufficiently large numbers of patients to obtain a reliable estimate of the prevalence of mutations in the insulin receptor gene among patients with NIDDM, and also in the general population. Conclusion Studies of the insulin receptor gene provide an excellent example of the power of modern molecular genetics in identifying mutations in genes that encode proteins required for normal insulin action and normal glucose homeostasis. By continuing to apply these techniques to relevant genes, it will be possible to obtain a better understanding of the causes of diabetes mellitus. It is to be hoped that improved understanding will eventually bear fruit by leading to novel approaches to treatment and prevention of diabetes.
Acknowledgments We are grateful to Drs. Jesse Roth and Phillip Gorden for helpful discussions and to Dr. Marc Reitman for critical reading of the manuscript. Partial research support was provided from the American Diabetes Association and the Juvenile Diabetes Foundation.
References 1. Neel JV. Diabetes mellitus—a geneticist's nightmare. In: The genetics of diabetes mellitus (Creutzfeldt W, Kobberling J, Neel JV, eds). New York: Springer-Verlag, 1976;p 1. 2. Reaven GM. Role of insulin resistance in human disease. Diabetes. 1988;37:1595-1607. 3. DeFronzo RA. Lilly lecture 1987. The triumvirate: beta-cell, muscle, liver. A collusion responsible for NIDDM. Diabetes. 1988;37:667-687. 4. Porte Jr D. Banting lecture 1990. Beta-cells in type II diabetes mellitus. Diabetes. 1991;40:166-180. 5. Bell GI, Xiang KS, Newman MV, Wu SH, Wright LG, Fajans SS, Spielman RS, Cox NJ. Gene for non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young subtype) is linked to DNA polymorphism on human chromosome 20q. Proc Natl Acad Sci USA. 1991;88:1484-1488. 6. Steiner DF, Tager HS, Chan SJ, Nanjo K, Sanke T, Rubenstein AH. Lessons learned from molecular biology of insulin-gene mutations. Diabetes Care. 1990; 13:600-609. 7. Kadowaki T, Bevins CL, Cama A, et al. Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance. Science. 1988;240:787-790.
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8. Yoshimasa Y, Seino S, Whittaker J, et al. Insulin-resistant diabetes due to a point mutation that prevents insulin proreceptor processing. Science. 1988;240:784-787. 9. Taylor SI, Kadowaki T, Kadowaki H, Accili D, Cama A, McKeon C. Mutations in the insulin receptor gene in insulin resistant patients. Diabetes Care. 1990;13:257-279. 10. Kadowaki T, Kadowaki H, Rechler MM, et al. Five mutant alleles of the insulin receptor gene in patients with genetic forms of insulin resistance. J Clin Invest. 1990;86:254-264. 11. Klinkhamer M, Groen NA, van der Zon GCM, Lindhout D, Sandkuyl LA, Krans HM, Moller W, Maassen JA. A leucine-to-proline mutation in the insulin receptor in a family with insulin resistance. EMBO J. 1989;8:2503-2507. 12. Kadowaki T, Kadowaki H, Taylor SI. A nonsense mutation causing decreased levels of insulin receptor mRNA: Detection by a simplified technique for direct sequencing of genomic DNA amplified by polymerase chain reaction. Proc Natl Acad Sci USA. 1990;87:658662. 13. Kahn CR, Flier JS, Bar RS, et al. The syndromes of insulin resistance and acanthosis nigricans. Insulin-receptor disorders in man. N Engl J Med. 1976;294:739-745. 14. Accili D, Frapier C, Mosthaf L, et al. A mutation in the insulin receptor gene that impairs transport of the receptor to the plasma membrane and causes insulin resistant diabetes. EMBO J. 1989;8:2509-2517. 15. Kusari J, Takata Y, Hatada E, Freidenberg G, Kolterman O, Olefsky JM. Insulin resistance and diabetes due to different mutations in the tyrosine kinase domain of both insulin receptor gene alleles. J Biol Chem. 1991;266:5260-5267 16. Odawara M, Kadowaki T, Yamamoto R, et al. Human diabetes associated with a mutation in the tyrosine kinase domain of the insulin receptor. Science. 1989;245:66-68. 17. Moller DE, Flier JS. Detection of an alteration in the insulinreceptor gene in a patient with insulin resistance, acanthosis nigricans, and the polycystic ovary syndrome (type A insulin resistance). N Engl J Med. 1988;319:1526-1529. 18. Moller DE, Yokota A, White M, Pazianos AG, Flier JS. A naturally occurring mutation of insulin receptor Ala1134 impairs tyrosine kinase function and is associated with dominantly inherited insulin resistance. J Biol Chem. 1990;265:14979-85. 19. Cama A, Sierra ML, Ottini L, Kadowaki T, Gorden P, ImperatoMcGinley J, Taylor SI. A mutation in the tyrosine kinase domain of the insulin receptor associated with insulin resistance in an obese woman. J Clin Endocrinol Metab. 1991;73:894-901. 20. Imano E, Kadowaki H, Kadowaki T, et al. Two patients with insulin resistance due to decreased levels of insulin receptor mRNA. Diabetes. 1991;40:548-557. 21. Kadowaki T, Kadowaki H, Ando A, Kaburagi Y, Quin JD, MacCuish A, Taylor SI, Yazaki Y, Kasuga M. Two mutant alleles of the insulin receptor gene in insulin resistant patients. Prog and Abstracts of 73rd Annual Meeting of the Endocrine Soc. 1991; p. 310 (Abstract 1119). 22. Taira M, Taira M, Hashimoto N, et al. Human diabetes associated with a deletion of the tyrosine kinase domain of the insulin receptor. Science. 1989;245:63-66. 23. Shimada F, Taira M, Suzuki Y, et al. Insulin-resistant diabetes associated with partial deletion of insulin-receptor gene. Lancet. 1990:335:1179-1181. 24. Kadowaki T, Kadowaki H, Accili D, Taylor SI. Substitution of lysine for asparagine-15 in the human insulin receptor impairs intracellular transport of the receptor to the cell surface and decreases the affinity of insulin binding. J Biol Chem. 1990;265:19143-19150. 25. Accili D, Mosthaf L, Ullrich A, Taylor SI. A mutation in the extracellular domain of the insulin receptor impairs the ability of insulin to stimulate receptor autophosphorylation. J Biol Chem. 1991;266:434-439. 26. Treadway JL, Morrison BD, Soos MA, et al. Transdominant in-
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GENETIC BASIS OF ENDOCRINE DISEASE hibition of tyrosine kinase activity in mutant insulin/insulin-like growth factor I hybrid receptors. Proc Natl Acad Sci USA. 1991;88:214-218. 27. Kadowaki H, Kadowaki T, Cama A, et al. Mutagenesis of lysine460 in the human insulin receptor: effects upon receptor recycling and site-site interactions among binding sites. J Biol Chem. 1990;265:21285-21296. 28. Lekanne Deprez RH, Potter van Loon BJ, et al. Individuals with only one allele for a functional insulin receptor have a tendency to hyperinsulinaemia but not to hyperglycaemia. Diabetologia.
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1989;32:740-744. 29. O'Rahilly S, Choi WH, Patel P, Turner RC, Flier JS, Moller DE. Detection of mutations in insulin-receptor gene in NIDDM patients by analysis of single-stranded conformation polymorphisms. Diabetes. 1991;40:777-82. 30. Cama A, Patterson A, Kadowaki T, et al. The amino acid sequence of the insulin receptor is normal in an insulin resistant Pima Indian. J Clin Endocrinol Metab. 1990;70:1155-1161. 31. Moller DE, Yokota A, Flier JS. Normal insulin-receptor cDNA sequence in Pima Indians with NIDDM. Diabetes. 1989;38:14961500.
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