NIDDM Associated With Mutation in Tyrosine Kinase Domain of Insulin Receptor Gene SERGIO COCOZZA, ANTONIO PORCELLINI, GABRIELE RICCARDI, ANTONELLA MONTICELLI, GIANLUIGI CONDORELLI, ASSIAMIRA FERRARA, LUIGI PIANESE, CLAUDIA MIELE, BRUNELLA CAPALDO, FRANCESCO BEGUINOT, AND STELIO VARRONE

A population of 103 patients with non-insulindependent diabetes mellitus (NIDDM) was screened for mutations in the tyrosine kinase domain of the insulin receptor gene. Patient genomic DNAs corresponding to exons 17-21 of the insulin receptor gene have been amplified by polymerase chain reaction and analyzed by denaturing gradient gel electrophoresis (DGGE). One patient was identified with an altered pattern of mobility of exon 20 in the DGGE assay. Direct sequence of amplified DNA showed a single nucleotide substitution in the codon 1152 (CGG — > CAG), resulting in the replacement of Arg with Gin. Two bands appeared in the sequence of exon 20 of the insulin receptor (nucleotide position 3584), indicating that this patient was heterozygous for the mutation. Insulin binding to intact erythrocytes from the patient was in the normal range. Although autophosphorylation of the purified insulin receptor also seemed normal, its kinase activity toward the exogenous substrate poly Glu:Tyr (4:1) was undetectable. This mutation may impair insulin receptor kinase and contribute to insulin resistance in this patient. Diabetes 41:521-26,1992

A

lthough the existence of a strong genetic component in the etiology of non-insulin-dependent diabetes mellitus (NIDDM) is well established (1,2), the nature of the genetic defect remains unknown. The candidate-gene approach could provide useful information to the identification of

From the Department of Cellular and Molecular Biology and Pathology, " L Califano," Centra di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Richerche; and the Medical Department, Institute of Internal Medicine and Metabolic Diseases, 2nd Faculty of Medicine, University of Naples, Italy. Address correspondence and reprint requests to Sergio Cocozza, Dipartimento di Biologia e Patologia Cellulare e Molecolare, dell'Universita degli Studi di Napoli, 5 Via S. Pansini 80131, Naples, Italy. Received for publication 12 April 1991 and accepted in revised form 27 November 1991.

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the genes involved in the pathogenesis of diabetes mellitus. This approach involves the selection of genes whose products may play a role in the disease and the search for mutations of these genes in affected subjects. Time-consuming techniques such as gene sequencing seriously hampered this approach. Most recently, advances in detection of point mutations became widely available, making the screening of a large number of patients more easy (3-5). The insulin receptor gene appears to be a good candidate gene for NIDDM because the receptor plays a critical role in allowing cells to respond to insulin (6), and insulin resistance is a prominent feature of NIDDM (7). In addition, the involvement of insulin receptor defects in the etiology of diabetes mellitus has been suggested by the findings of mutations in the insulin receptor gene in rare patients with genetic syndromes of severe insulin resistance (8-16). Whether the insulin receptor mutations also contribute to the etiology of the common forms of NIDDM is unclear. Because the tyrosine kinase activity of the insulin receptor is required for insulin signal transduction (17,18), we postulate that an abnormal structure of this domain is present in some patients with NIDDM, thus impairing receptor function and contributing to insulin resistance. In this study, we have devised an efficient technique for screening a population of NIDDM patients for mutations in the tyrosine kinase domain of the insulin receptor by combining polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). With this approach, one patient was identified exhibiting a mutated receptor kinase allele. RESEARCH DESIGN AND METHODS The study population consisted of 103 NIDDM patients referred to the Diabetes Clinic of the Medical School (Univ. of Naples). Diagnosis of NIDDM was made according to National Diabetes Data Group criteria (1). All subjects were informed of the aim of the study and gave

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their consent to participate. Forty-three patients were men, and 60 were women. Mean ± SE age was 56.90 ± 1.06 yr, and body mass index was 28.12 ± 0.45 kg/m2. Diabetes duration was 12.97 ± 0.85 yr. A blood sample was drawn from each patient in 50-mM EDTA tubes. Genomic DNAs were prepared according to previously described procedures (19). Amplifications of patient genomic DNAs by PCR were performed separately with five pairs of oligoprimers, one for each exon encoding the tyrosine kinase domain of the insulin receptor (20). The oligoprimer sequences (S, sense; A, antisense) are as follows: exon 17 5'-CATGC TCTGTGTACGTGCCG-3' (S), 5'-CTGGACTCACCACGT GATGG-3' (A); exon 18 5'-TGCTCTGCAGGTGCGCCT CC-3' (S), 5'-GCTTACCTCAGCCTCTGGCC-3' (A); exon 19 5'-GTTTTAGAATAATCCTGGCC-3' (S), 5'-CCAGAC GAACCTCCAATTTT-3' (A); exon 20 5'-GTGTTGTCAGA CTTTGGAAT-3' (S), 5'-CACAACTCACCACATGTCAG3' (A); exon 21 5'-TCATCGGCAGGTCCTTTGGC-3' (S), 5'-CTACACTTACACTCTCTCTG-3' (A); to the S primers GC clamps, 5'-CGCCCGCCGCGCCCCGCGCCCGTC CCGCCGCCCCCGCCCGAATTC-3' were attached (5). Two hundred nanograms of genomic DNA from each patient was amplified in 50 |xl PCR buffer (10 mM Tris-HCI [pH 8.3], 1.5 mM MgCI2, 50 mM KCI, 0.001% gelatin) containing 20 pmol of each oligonucleotide primer and 10 nmol of each DNA triphosphate. One unit of thermus aquaticus DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT) was added, and 100 jxl mineral oil was layered on the samples. Samples were then incubated at 95°C, 55°C, 72°C, respectively, for 45, 50, and 50 s; this cycle was performed 36 times in a DNA-RNA amplifier (Biostar Violet, Rome, Italy). DGGE assays were performed as follows: 10 IJLI of each PCR-amplified DNA fragment was loaded onto a 20 cm x 20-cm, 0.5-mm-thick 10% polyacrylamide gel (acrylamide/bisacrylamide 37.5/L) containing a 30-70% denaturant gradient parallel to the direction of electrophoresis (100% denaturant = 7 M urea and 40% formamide). Electrophoresis was performed at 200 V for 14 h in an aquarium containing TAE (0.04 M Tris-acetate, 0.002 M EDTA) buffer at constant temperature (60°C). After electrophoresis, the gel was soaked for 30 min in 200 mM Tris-HCI, pH 7.5, and then stained with ethidium bromide. Sensitivity of the assay for detection of nucleotide substitution in the region of interest was analyzed before screening with in vitro mutated exon 17-21 DNAs. These were obtained by PCR with antisense primers, one for each exon, carrying the following substitutions: antisense exon 17, A - - > G at position 17; antisense exon 18, G - - > A at position 17; antisense exon 19, T - - > A at position 17; antisense exon 20, T - - > C at position 17; antisense exon 21, T - - > C at position 17. These substitutions have always been detected by DGGE; in vitro mutated DNAs were also used as controls in all gels during the screening. Direct sequencing of amplified genomic DNA. DNA sequencing was performed on double-stranded PCR products by a modified version of a previously described procedure (21). One picomolar of template DNA purified by electroelution was mixed with 25 pmol primer in 14 (xl

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of 40 mM Tris-HCI (pH 7.5), 25 mM MgCI2) 50 mM NaCI, and 10% DMSO. After boiling for 3 min and cooling in ice, 3 JJII labeling mix, 10 ixCi (3.3 pmol) a-[32P]dATP (Amersham, Aylesbury, UK), and 2 |xl of 1.5 U/|xl T7 polymerase were added. The labeling reaction (4.5 (xl) was directly transferred in four prewarmed tubes containing 2.5 |xl each of termination mix (T7 sequencing kit, Pharmacia, Uppsala, Sweden) and incubated at 37°C for 5 min. Termination reactions were stopped by adding 5 |xl stop solution (T7 sequencing kit, Pharmacia). Three microliters of each reaction was loaded onto 8% denaturing polyacrylamide gel. Insulin-receptor autophosphorylation and kinase activity. Autophosphorylation of the insulin receptor was studied in the crude detergent extract of erythrocyte membranes as previously described (22). The crude detergent extracts were normalized for insulin binding and preincubated in the absence or presence of 1 |xM insulin at 4°C for 15 h. Phosphorylation reactions were initiated by addition of MnCI2 (final concn 4 mM), cold ATP (final concn 10 jiM), and 5 |xCi/tube 7-[32P]ATP for 15 min at 20°C. The reaction was stopped by addition of stopping solution (22), and insulin receptors were identified by immunoprecipitation with the B2-anti-insulin receptor antibodies followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (100 mM dithiothreitol) and autoradiography. Partial purification of erythrocyte insulin receptor was performed as previously described (23). Briefly, 1% Triton X-100-solubilized erythrocyte membranes were passed three times over 1 ml agarose conjugated ricinus lectin columns. Glycoproteins were eluted with 0.1 M lactose, and 125l-labeled insulin binding was compared in the eluate and flowthrough from the columns. Typically, 60-70% of the receptors were retained by the columns, 20% were present in the flowthrough, whereas the remaining was lost during column washing. Kinase activity was assayed as described previously (23). Ricin-purified receptor preparations (50 \x\) were adjusted for insulin binding and preincubated at 22°C for 30 min in 50 mM HEPES (pH 7.6), 0.05% albumin in the absence or the presence of 1 ixM insulin (final vol 70 |xl). The substrate poly Naglutamate.tyrosine (4:1) (final concn 2.5 mg/ml) MgCI2 (final concn 20 mM) were added along with ATP (final concn 25 mM) and 1 |xCi (3 Ci/jxmol) 7-[32P]ATP in a total vol of 100 |il. After 20 min, the phosphorylation reaction was terminated by spotting 70-|xl aliquots onto 3 x 3-cm Whatman paper that was soaked four times over 3 h in 10% trichloroacetic acid (vol/vol) with 10 mM sodium pyrophosphate, rinsed with ethanol, dried, and counted in a liquid-scintillation counter. Glucose clamp and forearm glucose uptake. Hypennsulinemic glucose clamp was performed according to DeFronzo et al. (24). Briefly, insulin was infused (24) at a rate of 1.5 mU • kg""1 • min" 1 for 2 h while blood glucose was maintained constant 5.5 mM (100 mg/dl) by a glucose infusion at a variable rate; the amount of glucose infused in the last 30 min in a steady-state condition equals the amount of glucose metabolized and was utilized for the molar concentration (mg glucose

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S. C0C022A AND ASSOCIATES TABLE 1 Clinical and metabolic features of patient

1 Patient

Age (yr) Body mass index (kg/m2) Duration of diabetes (yr) Fasting plasma glucose (mM) 2-h Plasma glucose (mM)* Fasting plasma insulin (mU/L)* 2-h Plasma insulin (mU/L)* Specific 125l-labeled insulin binding to intact erythrocytes (%) Whole-body insulin-mediated glucose disposal (mg • kg~ 1 • min~ 1 )t Forearm glucose uptake during clamp (mg • L~1 • min~ 1 )f

Normal values

31 21 7 8.7 15.8 7.1 13.1

Glu1056) in exon 18 and were Incubated in the absence (lanes 1-3) or presence (lanes 2-4) of a missense mutation (Val971 - - > Met971) and two re1 |iM insulin. D, Controls; • , probands. ported silent polymorphisms (codons 970 and 1046) in exon 17 were found. In our patients, we did not detect measured with that of the proband (Fig. 3A solid bars). any alteration in the DGGE migration pattern of the Next, receptor autophosphorylation was investigated. PCR-amplified fragment corresponding to exon 18, sug1056 in white NIDDM Detergent extracts of erythrocyte membranes were incu- gesting a low frequency of Glu bated in the absence or presence of 1 u.M insulin and subjects. Because all previously described polymor971 autophosphorylated with 7-[32P]ATP. Labeled receptors phisms in this locus, including Met substitution, lie in were then immunoprecipitated with B2 receptor antibod- the primer matching regions, DGGE is unable to detect ies and identified by SDS-PAGE and autoradiography. A them in our conditions. single 95,000-Mr band that exhibited insulin-dependent The proband receptor exhibited no measurable kinase phosphorylation was immunoprecipitated (Fig. 3S). activity toward the synthetic peptide poly Glu.Tyr (4:1). Based on its molecular weight and antibody specificity, This finding could not be ascribed to decreased insulin the band was identified as the insulin receptor p-sub- interaction with the kinase because specific insulin bindunits. With insulin receptors from nondiabetic patients, ing to intact erythrocytes from the patient and binding to autophosphorylation increased threefold and was mea- solubilized receptors were in the normal range. Although sured in the presence of insulin (Fig. 38, lanes 3 and 4). 30-80% decreased kinase activity of the receptor has Autophosphorylation of the proband receptors appeared been described in NIDDM patients (28,29), the defect we to be only slightly reduced compared with control recep- detected with the proband receptor far exceeded that tors (Fig. 3B, lanes 1 and 2). Thus, despite unmeasurable observed in most NIDDM patients. In addition, reduced kinase activity toward the exogenous substrate, insulin receptor kinase in NIDDM appears to be reversible upon receptor preparation from the patient exhibited pre- weight loss and/or achievement of metabolic control, served autophosphorylation. whereas this patient was lean and in good metabolic control since the discovery of diabetes. In fibroblasts from an NIDDM patient, a glycoprotein inhibitor of insulin DISCUSSION The insulin receptor gene appears to be an appealing receptor kinase has been recently identified (30). The candidate for NIDDM, because the receptor plays a inhibitor affected both insulin-dependent autophosphorpivotal role in the cellular responsiveness to insulin and ylation and kinase activity toward exogenous substrates. resistance of the cells to insulin action is a prominent At variance with that case, autophosphorylation apfeature of NIDDM (7). The tyrosine kinase activity of the peared preserved in receptor preparations from the 1152 patient. In addition, differently from the case receptor is required for insulin action (6), leading to the Gin hypothesis that abnormalities in this domain of the mol- reported, antibody-purified receptors from our patient ecule may contribute to insulin resistance in NIDDM. exhibit defective kinase activity. Thus, inhibitors are unHowever, time-consuming techniques have limited these likely to account for defective kinase activity in the 1152 patient. Because no other mutation has been studies to small samples of patients, and thus it is unclear Gin whether receptor mutations contribute to NIDDM etiol- found in the kinase domain of the patient receptor, this ogy. To identify genetic alterations in the tyrosine kinase data suggest a direct relationship between abolished domain of the insulin receptor, we used DGGE, which is kinase activity and the mutation. Undetectable kinase activity of the mutant receptor a gel system that separates DNA fragments according to their melting properties (4). With the GC clamp, DGGE was unexpected because the patient was heterozygous

B

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S. COCOZZA AND ASSOCIATES

for the mutation. It is possible that the Gin1152 substitution impaired the insulin-induced receptor internalization and that the receptors with inactive tyrosine kinase accumulated at the cell surface. However, it is also possible that mutated receptors inhibited the kinase activity of the normal receptors, which was the case for receptors bearing mutations in the ATP binding site (12). Because both patients' alleles of the Ir gene have not been fully sequenced, we do not know whether they are normal or have a second mutation. Despite impaired kinase activity toward the exogenous substrate, autophosphorylation of the proband receptor appeared to be almost unchanged. With in vitro mutagenesis (31), substitution of insulin receptor twin tyrosines immediately before Arg 1152 with phenylalanine abolished insulin-dependent kinase activity toward poly Glu.Tyr (4:1) with almost no effect on receptor autophosphorylation. In this case, a cryptic activity of the receptor has been postulated, which was still capable of undergoing autophosphorylation but unable to perform phosphorylation of lower-affinity substrates such as poly Glu.Tyr (4:1). Alternatively, the region of the molecule including amino acids 1146-1152 may be relevant to receptor interaction with exogenous substrates. The phe1150/1151 receptor did not affect an insulin-dependent increase in glucose uptake and glycogen synthesis, although it could initiate insulin growth responses. We speculate that the Arg 1152 - - > Gin1152 receptor is also unable to signal insulin effects on glucose metabolism because insulin-dependent glucose disposal was impaired in our patient. Because the patient also showed an impaired insulin secretion (Table 1), it is likely that both the impaired insulin secretion and reduced peripheral insulin sensitivity contributed to the development of diabetes. Several studies suggest that insulin receptor mutations are responsible for the altered glucose tolerance of a few patients with rare genetic syndromes of severe insulin resistance (8-16). In these syndromes, insulin resistance is accompanied by various peculiar clinical features (8-16). At variance with those patients, in the one we have described, the clinical phenotype clearly resembles that of an NIDDM patient. Thus, mutations of the insulin receptors also occur in common forms of diabetes mellitus. Further studies are needed to estimate the prevalence of mutations in the insulin receptor gene of NIDDM patients and to evaluate the role of Gin1152 substitution in the pathogenesis of NIDDM. ACKNOWLEDGMENTS This study was supported in part by Progetto Finalizzato Ingegneria Genetica, Progetto Finalizzato "Invecchiamento" of the Consiglio Nazionale delle Ricerche, and the exchange program between Italian scientists and the National Institutes of Health, which is funded by the Italian Dept. of Education (Ministero della Pubblica Istruzione). We thank Drs. S.M. Aloj and G. Salvatore of the Department of Cellular and Molecular Biology and Pathology, University of Naples, for support and advice during this work.

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