genetics of non-insulin-dependent (type-ii) diabetes mellitus

Annu. Rev. Med. 1996. 47:509–31 Copyright © 1996 by Annual Reviews Inc. All rights reserved

GENETICS OF NON-INSULIN-DEPENDENT (TYPE-II) DIABETES MELLITUS C. Ronald Kahn, M.D., David Vicent, M.D., and Alessandro Doria, M.D., Ph.D. Research Division, Joslin Diabetes Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215 KEY WORDS: insulin secretion, insulin resistance, β-cell, insulin receptor, IRS-1

ABSTRACT Both genetic and environmental factors contribute to the etiology of non-insulin-dependent diabetes. The genetic component is heterogeneous and in some patients is probably complex, involving multiple genes. Specific genetic defects have been identified for rare monogenic forms of NIDDM: maturity-onset diabetes of the young, or MODY (which is due to glucokinase mutations in about 40% of families), syndromes of extreme insulin resistance (which often involve the insulin receptor), and diabetes-deafness syndromes (with defects in mitochondrial genes). In contrast, the genes involved in common forms of NIDDM are still uncertain. Mutations have been extensively searched in genes regulating insulin signaling and secretion. Some evidence of involvement has been produced for insulin-receptor substrate-1, glycogen synthase, the glucagon receptor, a ras-related protein (Rad), histocompatibility antigens, PC-1, and fatty acid binding protein, but the contribution of these genes to NIDDM is probably small. Other candidate genes (e.g. insulin, insulin receptor, glucose transporters) have been excluded as major diabetogenes. New insights are expected in the near future from the systematic scanning of the genome for linkage with NIDDM.

INTRODUCTION Non-insulin-dependent diabetes mellitus (NIDDM), also referred to as type-II diabetes, is the most common of all metabolic disorders. NIDDM currently

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affects about 6–7% of the US population, with a cumulative risk of 17% by age 80 (1). The metabolic derangements created by chronic hyperglycemia, plus the strong association between NIDDM, obesity, hypertension, and hyperlipidemia, lead to an extensive list of long-term complications, including a high rate of cardiovascular death and amputation due to accelerated atherosclerosis, as well as the typical complications of diabetes such as retinopathy, nephropathy, and neuropathy. The economic burden created by diabetes and its complications has been estimated at over $120 billion annually in the United States alone. The clinical hallmark of diabetes is the presence of hyperglycemia in the fasting and/or postprandial state. Normal glucose homeostasis depends on four factors: the ability of tissues to take up glucose in response to insulin [insulin sensitivity (Si)]; the ability of cells to take up glucose in the absence of insulin [insulin-independent glucose uptake, sometimes referred to as glucose sensitivity (Sg)]; and the ability of the pancreatic β-cell to control this process by both rapid and sustained insulin secretion, i.e. first- and second-phase insulin secretion (2). Pathophysiologically, patients with NIDDM exhibit two defects: the first is a decrease in response of peripheral tissues to insulin, i.e. insulin resistance; the second is a failure of the β-cell to compensate for the insulin resistance by appropriately increasing insulin secretion. Although there continues to be debate about which of these defects is primary and which is secondary, most longitudinal studies have indicated that in populations with a high prevalence of NIDDM, such as offspring of diabetic parents, Pima Indians, and Mexican-Americans, insulin resistance occurs early in life and precedes any evidence of glucose intolerance, whereas the β-cell failure develops somewhat later, usually in association with impaired glucose tolerance (3–5) (Figure 1). However, the order of these defects can be clearly reversed in some patients with NIDDM, especially patients with the MODY (maturity-onset diabetes of the young) form of diabetes (see below), as well as in several animals with this disease. Indeed, it is likely that both insulin-resistance and insulin-secretory defects are genetically determined, as both exhibit family clustering (6–9), although in the offspring of NIDDM parents, low Si and low Sg are more predictive of subsequent development of NIDDM than is low insulin secretion (3).

The Genetics in NIDDM is Complex As with most common medical disorders, NIDDM is heterogeneous and the result of an interaction between genetic and environmental factors (1, 10). A strong genetic component is suggested by the remarkable clustering of NIDDM in families (11), by the high prevalence of NIDDM in certain ethnic groups (5, 12), and especially by the high concordance rate (50–95%) for NIDDM between monozygotic twins (13). The presence of strong environ-

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Figure 1 Model of the progressive pathogenesis of type-II diabetes (NIDDM). In most individuals there is a slow progression from normal to impaired glucose tolerance (IGT) to frank diabetes. This depends on interactions between genetic and environmental factors that act on both initiation and progression of the disease.

mental components is indicated by the differences in occurrence of NIDDM among genetically similar populations living in different areas, the many obvious links to lifestyle and diet, and ability of other disorders (such as obesity, pregnancy, surgical stress, steroids, etc) to create transient states of NIDDM in individuals who may bear genetic risk but who are otherwise normal. The genetic components of NIDDM appear to be complex. The ratio between risk of diabetes in relatives of NIDDM patients and risk of diabetes in the general population has been estimated to be about 10 for monozygotic twins, 3.5 for first-degree relatives, and 1.5 for second-degrees relatives (14). These figures fit well a polygenic disease. In the case of NIDDM, however, there are two types of polygenic inheritance that need to be considered. The first is that of a true polygenic disease, i.e. one in which a single individual exhibits a disorder due to simultaneous inheritance of two or more major genes interacting with several other loci, each having a small effect; such disorders fit mixed or multiplicative genetic models (14). In addition, NIDDM is also heterogeneous, i.e. in a given population not all individuals with NIDDM have the same genetic factors or primary cause. What is not certain, however, is whether NIDDM has two or three major forms and many minor forms, or whether it is truly a “Heinz-57" collection with no form representing more than 0–3% (Figure 2).

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Figure 2 Two models of the possible genetic heterogeneity of NIDDM. The “Heinz 57" model for NIDDM was initially proposed by M.P. Stern (University of Texas, San Antonio, TX).

Because of the complex inheritance and the interaction with environment, identifying the genes involved in the common forms of NIDDM is a formidable task (15). Linkage studies, a powerful tool in the case of monogenic disorders, may not be sensitive enough to detect the contribution of a locus that is neither necessary nor sufficient to produce NIDDM (16). Association studies may also be inadequate, as they necessarily rely on linkage disequilibrium between polymorphic markers and the susceptibility locus (17). Thus, the genes involved in the common form(s) of NIDDM are still uncertain. However, the genetic determinants of several relatively rare monogenic subtypes of NIDDM have now been identified. A tentative classification scheme emphasizing the genetics of NIDDM is presented in Table 1.

GENETICALLY CHARACTERIZED FORMS OF NIDDM Maturity-Onset Diabetes of the Young The best studied form of NIDDM from the point of view of genetics is maturity onset diabetes of the young (MODY). MODY is a relatively rare type of familial NIDDM, characterized by an early age of onset (childhood or adolescence) and appearance in at least three consecutive generations, suggesting an autosomal dominant mode of inheritance (18). This form probably accounts for 1–3% of NIDDM cases, although precise estimates of prevalence based on genotyping have not yet been performed. The presence of large families with multiple affected members has facilitated linkage studies of this disorder. Three different MODY genes have been mapped in different pedi-


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Table 1 Genetic classification of NIDDMa Genetically characterized forms of NIDDM MODY MODY1—linked to chromosome 20q MODY2—linked to glucokinase (7p13–15) MODY3—linked to chromosome 12q MODY4—others Defects in the insulin gene (11p15) Familial hyperproinsulinemia Mutant insulin molecules Defects in the insulin receptor (10p13) Leprechaunism Type-A syndrome of insulin resistance Rabson-Mendenhall syndrome Mutation in mitochondrial gene for tRNA-Leu Maternally inherited diabetes with neurosensory deafness MELAS syndrome Mutation in GLUT2 glucose transporter (3q26) (one case only) Genes involved in garden variety NIDDM Genes with some evidence for involvement HLA locus-DR4 (6p21–23)—elderly NIDDM only Glucagon receptor gene (17q25) Insulin receptor substrate-1 (2q36) Glycogen synthase (19q13) Intestinal fatty acid binding proteins (4q) Rad (16q22) Genes for which significant involvement has been ruled out Insulin gene (11p15) MODY genes (20q, 7p, 12q) ATP-sensitive K+ channel (21q22) Glucagon-like peptide-1 (GLP-1) receptor (6p21) GLUT2 (3q36) GLUT4 (17p13) Insulin receptor (19p13) a MODY, Maturity-onset diabetes of the young; Rad, Ras-related protein associated with diabetes; ATP, adenosine triphosphate.

grees, which together may account for up to 70% of affected individuals. The best characterized and most frequent of these is the glucokinase gene (7p13–15) (19, 20). In other kindreds, MODY maps to two as yet unidentified genes on chromosomes 20q and 12q (21, 22). These three forms have been referred to as MODY2, MODY1, and MODY3, respectively, based on the order of description in the literature.

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The glucokinase gene (MODY2) spans about 50 kilobases on chromosome 7p13–15 and consists of 12 exons (23, 24). Glucokinase is expressed both in the β-cell and the liver. Transcripts in the two tissues differ slightly because of alternative promoters and splicing of the 5′-end of the gene, but they code for proteins with similar characteristics (23). Glucokinase is also known as hexo kinase IV and serves to phosphorylate glucose to glucose-6-phosphate (Figure 3). As phosphorylation is necessary for glucose to activate insulin secretion in β-cells, glucokinase can be considered a major component of the glucose sensor, which mediates the fine tuning between insulin secretion and blood glucose level (25). The glucokinase locus was initially found to cosegregate with MODY in a large study of French families and one English pedigree (19, 20). Following these reports, numerous nonsense and missense mutations were identified in the glucokinase gene of affected members of MODY pedigrees (22, 26–28). Some mutations involve amino acids in the active site encoded by exons 7 and 8; others occur in domains that are distant from the active site but that probably result in conformational changes that alter glucose binding (29). Both types of missense mutations cause alterations in the kinetics of glucose phosphorylation, with reductions in Vmax or increases in Km, or both (29). Nonsense mutations result in the absence of active enzyme (29).

Figure 3 Schematic representation of a pancreatic β-cell. The numbers in parentheses indicate the approximate frequency of genetic mutation in NIDDM.


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The clinical correlate of glucokinase mutations is an impairment of insulin secretion, including inappropriately low fasting-insulin concentrations in relation to glycemic levels and decreased insulin response to continuous hyperglycemia (30). This results in a relatively mild form of diabetes or glucose intolerance, which usually becomes evident before age 25 and is often controlled by diet or sulphonylureas (31). Glucokinase mutations underlie the clinical picture of MODY in up to 50% of French cases (31) but appear to be less frequent in other countries. In the remainder of patients, other MODY genes exist (31). One of these, MODY1, has been mapped to a 13-centi-Morgan (cM) region on chromosome 20q12–q13.1 and cosegregates closely with diabetes in one large American family (21, 32). Thus far, however, this locus has not been implicated in any other MODY pedigrees (19, 20). The identity of the MODY1 locus is unknown, and no gene known to be involved in glucose metabolism has been identified so far in this region. Like the patients with glucokinase mutations, MODY1 is associated with low-basal and stimulated-insulin secretion. A third MODY locus (MODY3) has been recently mapped to a 7–cM region on chromosome 12q22–qter (22). Alterations at this locus could be responsible for up to 25% of MODY cases, especially those characterized by a more severe diabetic phenotype. Other as yet unidentified loci account for the remaining 25% of MODY cases.

Genetic Defects in the (Pro)Insulin Gene The insulin gene (11p15) was the first candidate gene that could be explored for a potential role in diabetes. However, since 1979, when the first mutation was found (33), a total of only six mutations have been identified [reviewed by Steiner et al (34)]. Two types of mutations in the insulin gene have been described. The first results in alterations in the primary sequence of the A- and B-chains, which affect the binding of the hormone to the receptor and ultimate bioactivity. These produce a phenotype characterized by high circulating insulin concentrations, modest hyperglycemia, and an increase in the half-life of the hormone (because much of insulin clearance is due to receptor-mediated uptake). Three families with such mutations were described between 1979 and 1990 (insulin Chicago, B25Phe→Leu; insulin Wakayama, A3Val→Leu; insulin Los Angeles, B24Phe→Ser). The second type of mutation results in an impairment of the processing of the proinsulin. Again, three such mutations have been described. These mutations have little effect on glucose homeostasis and are characterized by a high circulating concentration of proinsulin-like material. In two of these families this is due to a mutation at the proinsulin processing site (Arg 65His or Arg 65Leu), whereas in one family there is a mutation that converts the His at position 10 in the B-chain to Asp (35). This latter mutation appears to alter proinsulin processing by altering zinc binding

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and crystal formation in the β-cell granule (36). Interestingly, if B10Asp-insulin were normally processed, it would produce a super-insulin analogue with increased receptor binding and increased agonist activity (37). Because of the rarity of insulin gene mutations, almost all affected individuals are heterozygous and can usually be identified only by the presence of hyperinsulinemia or hyperproinsulinemia. The degree of impairment of glucose homeostasis is quite variable, but usually very mild, and depends on age, obesity, and possibly other interacting genetic factors (38, 39). It is not clear whether so few families with insulin mutations have been reported because of the very mild or nonexistent clinical picture, because of the lack of screening, or because of a low frequency of mutation at this site. The very low prevalence of these mutations in “garden-variety” diabetic patients, however, has been confirmed by direct sequencing of the insulin gene (40), or by testing for the presence of restriction fragment length polymorphisms (RFLPs) in groups of patients (41). Thus, it seems that mutations in the coding region of the insulin gene do not play a significant role in type-II diabetes mellitus.

Insulin Receptor Gene Mutations and Syndromes of Insulin Resistance The insulin receptor gene was the first major candidate gene in the insulin action pathway to be cloned, therefore it has been one of the most extensively studied. The gene consists of 22 exons spread over 150 kilobases and encodes the single-chain precursor of the insulin receptor tyrosine kinase. After dimerization and processing, the mature receptor consists of two α (insulin binding)and two β (tyrosine kinase)-subunits in a β-α-α-β heterotetramer (42, 43) (Figure 4). Molecular genetic analysis of the receptor has been possible only since 1983, but based on the clinical picture and insulin binding data, the first patients identified with insulin receptor mutations were described in 1975 (44). Over the past decade it has become clear that mutations in the insulin receptor account for the extreme insulin resistance found in a number of rare genetic syndromes associated with severe insulin resistance, such as leprechaunism, the Rabson-Mendenhall syndrome, and the type-A syndrome of insulin resistance, and may occur with a frequency of ∼1/300 alleles in the population at large (45, 46). At least 40 mutations have been identified in the structure of the insulin receptor in patients with severe insulin resistance (45–50) (Figure 4). Although these rare syndromes and NIDDM are both characterized by insulin resistance, patients with receptor mutations usually have much more severe resistance. Thus, in patients with receptor mutations, circulating insulin levels are 50–300 µU/ml in the fasting state and 400–2500 µU/ml after a glucose load, and in patients receiving insulin therapy, 10–100 times the normal insulin dose is often required. The clinical picture in each of the syndromes associated with insulin receptor mutations is usually quite distinct


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Figure 4 Schematic representation of the insulin receptor tyrosine kinase. The labeling on the left indicates the functional domains of the receptor. The labeling on the right gives a partial list of the naturally occurring mutations.

from that in NIDDM. In the most severe form, leprechaunism, there is intrauterine and neonatal growth retardation, as well as other developmental abnormalities and death in early infancy. This is almost always associated with mutations at both insulin receptor alleles (homozygosity or compound heterozygosity). In the Rabson-Mendenhall and type-A syndromes, patients are usually heterozygous for the mutation. Thus, there may be some mild developmental abnormalities, but the major features are severe insulin resistance, acanthosis nigricans, and hyperandrogenization with polycystic ovarian disease. The exact relationship between changes in the skin and ovary and insulin resistance are still poorly understood and point to the difficulty in predicting a clinical phenotype based simply on the known biochemisty of a protein. The specific site of mutation does not relate to the clinical syndrome. Thus, in all three disorders, mutations have been described throughout the receptor, including the insulin binding α-subunit, the kinase domain of the β-subunit, and even the processing site between the two subunits present in the insulin receptor precursor (Figure 4). Despite the relatively high frequency of insulin receptor mutations in individuals with syndromes of severe insulin resistance, screening of NIDDM patients in different populations for polymorphisms in the insulin receptor has revealed few mutations and probably no increase in prevalence over that which occurs in the nondiabetic population [51, 52]. Three groups have also reported

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normal complementary DNA sequences in patients with NIDDM [53–55], ruling out the possibility that subtle mutations in the coding region of the insulin receptor could be a frequent cause of NIDDM.

Mitochondrial Gene Mutations and NIDDM Between 3 and 5% of DNA in mammalian cells is contained in the mitochondrial genome. This DNA encodes a number of metabolic enzymes, as well as most tRNAs. Several facts have prompted investigators to search for alterations in mitochondrial DNA in patients with diabetes or impaired glucose intolerance. First, mitochondrial oxidative phosphorylation is involved in peripheral glucose metabolism, as well as in glucose stimulation of β-cell insulin secretion (56, 57) (Figure 3). Secondly, in NIDDM there is a slight maternal preference in inheritance that would be consistent with the fact that mitochondrial DNA is primarily maternally inherited (58). Third, a number of rare mitochondrial cytopathies have been identified that are associated with diabetes at some time during evolution of the disease (59). Finally, mitochondrial DNA is known to accumulate mutations with increasing age, a fact that could be consistent with the increasing prevalence of NIDDM with age (57). Over the past several years, deletions (60, 61), duplications (62, 63), and point mutations (58, 64–69) of the mitochondrial genome have been described in patients that have diabetes mellitus as part of complex encephalomyopathic syndromes. One particular mutation in the gene for tRNA-Leu at position 3243 affects oxidative phosphorylation (70) and has been found repeatedly in patients with diabetes mellitus and neurosensory deafness (58, 64–69). It is important to note, however, that the same mutation is observed in patients with the MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) with or without diabetes (61). Diabetic patients carrying this mutation exhibit impaired insulin secretion and normal insulin sensitivity (71, 72). Clinically, NIDDM patients with the tRNA-Leu mutation represent a distinct subtype of diabetes characterized by its strict maternal transmission and association with nerve deafness (67, 72). In a recent study, this mutation was observed in 0.8% of patients with NIDDM presenting to a large clinic in Japan, the highest prevalence among candidate genes screened to date (73).

GENES FOR “GARDEN VARIETY” NIDDM The Insulin Gene As mutations in the coding region of the insulin gene have not been found to be involved in common forms of NIDDM, it has been hypothesized that the observed impairment of insulin secretion in NIDDM might be related to ab-


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normalities in regulatory sequences of the gene. In this regard, considerable interest has been drawn to a complex polymorphism located 5′ of the insulin gene in the proximity of promoter/enhancer sequences, which could affect, at least theoretically, insulin gene expression (74). The polymorphism consists of a variable number of tandem repeats; these have been subdivided into three classes (I, II, and III) according to their length. Although an association has been consistently found between homozygosity for class-I alleles and IDDM (75, 76), this does not seem to be the case for NIDDM (75, 77). A rare 8-base pair (bp) insertion/deletion polymorphism has been described in the insulin gene promoter among African-Americans, but its contribution to NIDDM seems to be small, if any (78). Consistent with all these findings, any linkage between this chromosomal region (11p15) and NIDDM has been excluded (79). Thus, a role for insulin gene mutations in the etiology of garden variety NIDDM seems unlikely.

MODY Genes The major genetic loci that contribute to known causes of MODY do not appear to play a role in the common form(s) of NIDDM characterized by insulin resistance, late onset, and complex genetics. An association between some microsatellite alleles of glucokinase and late-onset NIDDM has been described in African Americans, Mauritian Creoles, and Japanese (80–82), but mutations altering glucokinase expression or structure have not been found in these NIDDM populations (83, 84). Attempts to demonstrate linkage between the glucokinase locus and NIDDM have also been negative in family studies from France, England, and Utah (85–87). Likewise, a thorough molecular screening of several European pedigrees failed to identify glucokinase mutations segregating with late-onset NIDDM (88). Similarly, the MODY1 locus on chromosome 20q does not appear to be linked to late-onset, garden-variety NIDDM (89).

IDDM Genes Insulin-dependent diabetes mellitus (IDDM) or type-I diabetes represents about 5–10% of all patients with diabetes. IDDM is well characterized as an autoimmune disorder with a strong association to the HLA major histocompatibility locus; it is also influenced by up to six minor genetic loci, including the insulin gene locus (90). IDDM may occur at any age and in later-onset cases may be rather slow to develop, thus mimicking NIDDM. Recent reports have suggested a shared genetic susceptibility between NIDDM and IDDM at the HLA locus, specifically DR4 (91, 92). This association seemed to be strongest in NIDDM patients who had survived to age 80. Additional studies will be needed to address whether or not these HLA data hold true in other

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populations, and to determine the importance of HLA to NIDDM etiology. However, there is no evidence that NIDDM is an autoimmune disorder.

Genes Implicated in Insulin Secretion In addition to glucokinase, two other proteins play a crucial role in the feedback between glycemia and insulin secretion: the GLUT2 glucose transporter (GLUT2, 3q26) (93, 94) and the adenosine triphosphate (ATP)-dependent K+channel of β-cell membranes (KATP-2, 21q22) (95) (Figure 3). GLUT2 is a liver- and β-cell-specific glucose transporter and a critical part of the β-cell glucose sensing apparatus, whereas KATP-2 is the effector arm of the feedback, as its inhibition by ATP generated by glucose metabolism leads to β-cell depolarization and insulin release (25). Both association and linkage studies have shown that these two genes are not major type-II diabetogenes (95–100). An association between a TaqI RFLP in the GLUT2 gene and NIDDM has been reported among British individuals (101), but this has not been confirmed in other populations (94–98). A mutation (Val197 to Ile) abolishing GLUT2 activity, and possibly causing diabetes, has been described in a single patient with NIDDM (102, 103). Allelic variation in GLUT2 resulting in substitution of Thr110 by Ile has also been found in screening studies, but it occurs with equal frequency in diabetic and control populations and has no detectable effect on function. Two other genes regulating insulin expression or secretion (Islet-1 on 5q12 and glucagon-like peptide-1 receptor on 6p21), also do not contribute to the common forms of NIDDM (75, 104-108). In contrast to these largely negative studies, positive results have been recently reported for the glucagon receptor gene (GCG-R, 17q25) (109, 110). A missense mutation (Gly40 to Ser), resulting in a lower affinity of the receptor for glucagon, is associated with late-onset NIDDM (110). However, the proportion of NIDDM patients carrying the mutation is very small (∼5% vs ∼1% in controls) (110). In addition, the role of this mutation in pathophysiology is unclear because a reduced affinity of the glucagon receptor could contribute to diabetes by impairing glucagon-stimulated insulin secretion, but it could just as well protect from hyperglycemia by blunting glucagon-stimulated glucose production from the liver.

Genes Implicated in Insulin Sensitivity Insulin action at the cellular level is a complex cascade that begins with the insulin receptor kinase and progresses to a broad range of final effects on glucose, lipid, and protein metabolism (reviewed in Reference 42) (Figure 5, Top). Although many families with severe insulin resistance and mutations in the insulin receptor gene have been described, as noted above, a major role of the insulin receptor locus (INSR, 19p13) in common forms of NIDDM is unlikely. Silent mutations and polymorphisms have been identified in the


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Figure 5 Models of insulin action and sites of insulin resistance. (Top) Genetic mutations affecting insulin signaling. (Bottom) Potential inhibitors of insulin action. The numbers in parentheses indicate the approximate prevalence. See text for description.

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insulin receptor gene, allowing linkage and association studies between RFLP markers and the diabetic phenotype (111). However, there is no evidence for a strong linkage with NIDDM (52, 112), and the results obtained in association studies in different populations have been inconclusive (51, 113–118). Thus, at present, the insulin receptor gene is not considered a primary genetic locus in common NIDDM. In addition to alterations in sequence, alterations in expression of the insulin receptor could also cause diabetes. Indeed, in all hyperinsulinemic states, including NIDDM, there is a decrease in insulin receptor number, but this is due to down-regulation of insulin receptors via increased rates of receptor degradation (119). In addition, there are two subtypes of insulin receptors that result from alternative splicing of the receptor messenger RNA (53, 120–122). This results in two forms of receptor, which differ by 12 amino acids near the C-terminus of the α (insulin-binding)-subunit. These two receptor isoforms exhibit subtle differences in binding affinity, tyrosine kinase activities, and internalization kinetics (123–127). Although differences in the expression of these two subtypes between NIDDM patients and nondiabetic subjects has been reported, the significance of this result is controversial because it has not been found in all studies and the functional differences between both types are small (128–131). The case might be different for insulin receptor substrate-1 (IRS-1) (2q36). IRS-1 is a 131-kDalton cytosolic protein and is the best characterized intracellular substrate of insulin-receptor tyrosine kinase activity (132, 133) (Figure 5, Top). Five amino acid substitutions have been identified in IRS-1 among Caucasian and Japanese individuals (134–136). Two of them (Gly972 to Arg972, Ala513 to Pro513), located in proximity with tyrosine phosphorylation motifs, have been found to be significantly associated with late-onset NIDDM in studies of Danish, Italian, and Japanese patients. In both European studies, the proportion of mutation carriers was ∼20% among NIDDM patients as compared to 6–7% among controls (134), whereas in the Japanese, the prevalence in both controls and diabetics was somewhat lower, but the difference was maintained. Although the pathophysiology and the relevance of these mutations in NIDDM etiology are unclear, preliminary studies of the 972 mutation suggest that it results in a modest reduction in insulin signaling (K Almind, O Pedersen, and CR Kahn, unpublished observation). In one study from Japan, both diabetics and nondiabetics with mutations in IRS-1 had about a 30% lower value for insulin-stimulated glucose uptake as compared to those in individuals with a normal IRS-1 sequence (137). Although IRS-1 mutations are not specifically associated with insulin resistance (134, 135), recent studies by Clausen et al (138) also suggest that IRS-1 gene variants may interact with obesity in the pathogenesis of common insulin-resistant disorders. On the other hand, an IRS-1


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association between NIDDM and IRS-1 polymorphism has not been found in other Caucasian populations (135, 136, 139; R Sigal, A Doria, J Warram, AS Krolewski, unpublished observation). Because genetically engineered, IRS-1deficient mice develop only impaired glucose tolerance, not frank diabetes, it seems likely that genetic variability in IRS-1 could only contribute modestly to development of NIDDM (140).

Glucose Transporters and Other Postreceptor Molecules Genetic studies of NIDDM have considered a number of genes coding for proteins involved in glucose metabolism. Variability in the insulin-dependent glucose transporter GLUT4 (17p13) does not seem to contribute to NIDDM. Similarly to other genes, mutations in GLUT4 promoter and coding sequence have been identified but are either extremely rare or equally frequent in NIDDM cases and nondiabetic controls (55, 141–143). An XbaI RFLP in an intron of the glycogen-synthase gene (GSY, 19q13) has been reported to be associated with familial NIDDM, impaired nonoxidative glucose metabolism, and hypertension in Finland (144). However, this association has not been confirmed in France, Japan, or the United States (145, 146; and AS Krolewski, personal communication). In addition, biologically active mutations in linkage disequilibrium with the XbaI marker have not been identified yet. On the basis of recent results of linkage studies, it has been hypothesized that the mutation responsible for the association with NIDDM may not be located in the GSY gene itself but in a flanking locus (147).

Random Genetic Loci Associated with NIDDM Recently, several laboratories have performed random genetic screens in search of diabetes-associated genes. Thus far, these have yielded only one potential candidate gene on chromosome 4q, which might be important in the regulation of insulin sensitivity (148). Linkage has been reported between a region on this chromosome and measures of insulin action (fasting insulin concentrations and maximal insulin-stimulated glucose uptake) in Pima Indians (148). In this population, a frequent amino acid substitution (Ala54 to Thr) in the intestinal fatty acid-binding protein gene (FABP2) on 4q has been observed and is associated with increased intestinal absorption of fatty acids and enhanced fat oxidation leading to insulin resistance (149). However, among either Pimas or Caucasians, variability at the FABP2 locus does not seem to specifically contribute to NIDDM (149, 150).

Possible Inhibitors of Insulin Action as Diabetogenes All of the potential diabetogenes related to insulin resistance described above have assumed that there is a genetic alteration in some normal component of the insulin action pathway that leads to insulin resistance. The alternative

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concept, namely that insulin resistance could be due to abnormal synthesis or release of an inhibitor of insulin action, is an old one but has only recently been explored in terms of the genetics of diabetes. At least four different proteins have been identified as potential inhibitors of insulin action, and at least two of these have been studied as potential diabetogenes (Figure 5, Bottom). pp63, also known as α-HS glycoprotein, was the first inhibitor of insulin action identified in extracts of rat liver (151, 152). In both rodents and humans, this protein is present at relatively high levels in the circulation where it might act on peripheral tissues involved in insulin action. pp63 has been shown to inhibit the insulin receptor tyrosine kinase and insulin-stimulated DNA synthesis. Thus far, however, there is no evidence for genetic alterations in expression, secretion, or action of this protein in NIDDM. Another circulating inhibitor of the insulin receptor kinase is the cytokine TNF-α. TNF-α may contribute to the insulin resistance of infection or stress, and it may also contribute to NIDDM, since TNF-α is hypersecreted by adipocytes in obesity (153, 154). Thus far, there have been no direct studies of the TNF gene in NIDDM, but it seems likely that in most individuals the defects in secretion are acquired and secondary to obesity rather than primary and genetic in nature. On the other hand, considerable evidence is now rapidly accumulating that obesity itself may be genetically determined, but there is little yet in the literature about the role of these obesity genes in human NIDDM. Recently, two cellular proteins have been identified that are also potential inhibitors of insulin action and for which there may be a more important genetic control of expression. The first protein identified is a novel member of the Ras superfamily termed Rad (Ras-related protein associated with diabetes) (155). Rad was identified by subtraction cloning between skeletal muscle of a type-II diabetic individual and a nondiabetic. Rad expression is increased in about 20% of individuals with NIDDM and has been shown in cultured cells to inhibit insulin-stimulated glucose uptake. Preliminary studies also suggest some association of rad gene markers with the development of NIDDM (156), although more studies in this area are clearly needed. The second member of this group is a cellular glycoprotein termed PC-1 (157). This protein also appears to be increased in its expression in cells and tissues of NIDDM patients and inhibits insulin action at the level of the receptor kinase. No studies of the genetics of this protein have yet been published.

FUTURE DIRECTIONS Despite the strong evidence for a role of genetics in NIDDM, it is clear that at present most of the genes contributing to this susceptibility have yet to be


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identified. Further studies need to expand the number of candidate genes studied and to use other novel methods such as polymerase chain reaction differential display, subtraction cloning, and use of systematic genome scanning to identify potential NIDDM genes. These will require well-characterized families and patients where the relative contribution of insulin secretion and insulin resistance is better understood. Ultimately, however, it seems likely that over the next decade most patients with NIDDM will be genetically classified, and a new era of prediction and attempts at prevention may begin. Any Annual Review chapter, as well as any article cited in an Annual Review chapter, may be purchased from the Annual Reviews Preprints and Reprints service. 1-800-347-8007; 415-259-5017; email: [email protected]

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