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Research Division, Joslin Diabetes Center, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Alessandro Doria, M.D., Ph.D., Section on Genetics and Epidemiology, Joslin Diabetes Center, One Joslin Place, Boston, Massachusetts 02215. E-mail: . alessandro.doria{at}joslin.harvard.edu
Abstract
Recent evidence indicates that I
B kinase ß (Ikkß) may be a mediator of acquired forms of insulin-resistance. In this study, we examined whether genetic variability at the Ikkß locus (IKBKB) contributes to the development of genetic forms of early-onset type 2 diabetes transmitted with an autosomal dominant mode of inheritance. Linkage with four markers flanking the IKBKB gene was evaluated in 32 multigenerational families. Included in the study were 233 diabetic (mean age at Dx = 37 ± 18) and 152 nondiabetic subjects. The overall LOD scores were negative (-54.9 and -46.2 on the centromeric and telomeric sides, respectively) indicating that variability in IKBKB was not a major determinant of diabetes in these families. Positive values, however, were observed for selected pedigrees. All 17 families for which linkage with the IKBKB locus could not be excluded were screened for sequence differences in the 22 exons and 1.6 kb of the 5' flanking region by dideoxyfingerprinting or direct sequencing. Polymorphisms were identified in the 5' flanking region (-1775del/insC and -1547T > A), exon 11 (c.1083A > G, L361L) and in intron 12 (IVS12+14t > a). However, no mutations segregating with diabetes could be found in these families. Furthermore, all four polymorphisms had similar allele frequencies in the 32 family probands, 171 individuals with common, later-onset type 2 diabetes, and 182 nondiabetic controls. We conclude that sequence differences in the IKBKB gene do not play a major role in either early-onset, autosomal dominant type 2 diabetes, or common forms with a later-onset.
GENETIC FACTORS ARE important determinants of insulin resistance and type 2 diabetes, but the genes that are involved remain mostly unknown (1). Until recently, IB kinase ß (Ikkß) was known primarily for its role in phosphorylating IB and activating NF-B in response to stimuli related to inflammation and cell survival (2). New evidence, however, points to Ikkß as an important mediator of insulin resistance. Inhibition of Ikkß with salicylates or through targeted gene disruption causes a dramatic improvement of insulin sensitivity in animal models of insulin resistance such as ob/ob mice and obese Zucker fatty rats (3, 4). Reports in the literature of blood glucose lowering after high dose aspirin in type 2 diabetic subjects strongly suggest that Ikkß acts as a crucial regulator of insulin-sensitivity also in humans (5). The specific mechanisms underlying this Ikkß action are unclear at this time, but an increased serine/threonine phosphorylation of IRS1 appears to be involved. Because of this inhibitory effect on insulin action, Ikkß mutations/polymorphisms determining gain of function may contribute to genetic forms of insulin-resistance and type 2 diabetes. In the present study, we examined whether genetic variability at the Ikkß locus (IKBKB) is involved in the etiology of early-onset, autosomal dominant type 2 diabetes—a subtype of diabetes that is more strongly determined by genetic factors than common type 2 diabetes but is also characterized by the presence of insulin resistance (6).
Subjects and Methods
Families
Thirty-two families with early-onset, autosomal dominant type 2 diabetes were included in this study. The ascertainment of these families has been previously described (6). Diabetes was diagnosed: 1) if an individual was treated with insulin or oral agents, 2) if results of an oral glucose tolerance test met World Health Organization criteria, or 3) if the level of HbA1c was >7.0% in individuals who declined an oral glucose tolerance test or were not fasting when examined. Families were of Caucasian origin with the exception of four Hispanic and two African-American pedigrees. Included were 233 family members with diabetes, impaired glucose tolerance or previous gestational diabetes mellitus and 152 nonaffected members. All families were negative for mutations in the HNF-4
, HNF-1, IPF1, and NEUROD1 genes. Analysis with markers flanking the glucokinase locus also excluded linkage with diabetes (Doria, A., unpublished results). The clinical characteristics of diabetes in 29 of these families have been previously reported in detail (6). This form of diabetes is on average diagnosed at an older age than maturity-onset of diabetes of the young, and is frequently associated with obesity and insulin resistance (6). The study protocol and informed consent procedures were approved by the Human Subjects Committee of the Joslin Diabetes Center.
Radiation hybrid mapping
The IKBKB gene was mapped by screening the Stanford G3 Radiation Hybrid panel (Research Genetics, Inc., Huntsville, AL) with an STS derived from BAC RPCI-11–384-C-8 in the GenBank GSS division (entry no. AQ533730), one end of which was found to contain bp 1522–1722 of the IKBKB cDNA. Screening was performed by standard PCR using primers 5'-TTTGGGATCAGTG AGTGTGC-3' and 5'-CACCCCCTTGGTAAGTTCC3-3'. Scores were submitted to the Stanford on line RH Server for a two-point statistical analysis with markers in the version 2.0 SHGC G3 map.
Linkage studies
Marker genotypes were determined by 32P-labeled PCR followed by denaturing PAGE and autoradiography. Multipoint parametric and nonparametric analyses were performed using GENEHUNTER (version 1.2.1) (7). The parametric analysis assumed an autosomal dominant mode of inheritance with a disease allele frequency of 0.001 and four age-related liability classes, as previously described (8).
Exon-intron boundaries
A BAC clone (RPCI-11-384-C-8) containing the portion of the IKBKB gene 3' to bp 1522 was identified through a BLAST search of the GSS division of GenBank. Another BAC clone overlapping with RPCI-11-384-C-8 and covering the 5' half of the gene was isolated from the CITB human genomic BAC library (Research Genetics, Inc., Huntsville, AL) by PCR-based screening using the same primers that were used for RH mapping. Exon-intron boundaries were defined by directly sequencing these two BAC clones using the Sequiterm EXCEL II DNA sequencing kit (Epicentre, Madison, WI) with 32P-deoxy-ATP.
Mutation screening
The coding sequence of IKBKB was screened for sequence differences by dideoxyfingerprinting (ddF) (9), the promoter region by direct sequencing. DNA fragments (170–600 bp) covering the exons, exon-intron boundaries, and 1,600 bp of 5' flanking region were amplified by PCR from the DNA of one affected member from each family for which linkage could not be excluded (LOD score > -2.0). The primers and annealing temperatures are available from the authors. PCR products were purified and subjected to Sanger’s dideoxy chain termination reaction using dideoxy-GTP in a 10-µl reaction as described by Sarkar et al. (9). To increase the sensitivity, reactions were performed twice, with the forward and the reverse primer. After adding 20 µl of stop/denaturing solution (7 M urea, 50% formamide, bromophenol blue, and xylene cyanol) and heating the samples at 95 C for 5 min, 4 µl were electrophoresed overnight in a nondenaturing 0.75x mutation detection enhancement gel in 0.5x Tris-borate EDTA on a sequencing apparatus at a constant power of 6 W at room temperature. Dried gels were autoradiographed overnight. Samples with changes in band patterns were sequenced by the Big-Dye Terminator Cycle Sequencing Kit version 2.0 (PE Applied Biosystems, Foster City, CA) using an ABI 310 Genetic Analyzer. DNA fragments from the promoter region were directly sequenced without prior ddF screening. Allele frequencies were determined in the 32 original family probands, 171 patients with type 2 diabetes, and 182 nondiabetic controls by PCR, dot-blotting and allele specific hybridization. The -1775del/insC polymorphism was genotyped by fragment length analysis on an ABI 310 Genetic Analyzer. Subjects with type 2 diabetes were randomly selected from a sample of Joslin Clinic patients aged 40–64 yr who met the following criteria: 1) having diabetes that was diagnosed after age 35; and 2) being treated with diet or oral agents for at least two years after diagnosis of diabetes. Nondiabetic controls consisted of nondiabetic spouses of family members. All these subjects were of Caucasian origin.
Results
To screen the IKBKB locus for major gene effects, we examined its linkage with diabetes in 32 families with a transmission of early-onset type 2 diabetes consistent with an autosomal dominant mode of inheritance. Consistent with its previous mapping by FISH (10), the IKBKB gene was mapped by radiation hybrid screening to chromosome 8p at 7 centirays (about 200 kb) from marker SHGC-9715. The LOD score for this location was 17.1. The closest polymorphic marker on the version 2.0 SHGC G3 map was identified as Genethon marker AFM156xa3 (D8S268) located 18 centirays (about 500 kb) centromeric to SHGC-9715. Based on this mapping information, four markers (D8S1821, D8S532, D8S1460, and D8S538) spanning about 5 centimorgans in this region were chosen for linkage analysis. Strong evidence against linkage was observed at each marker position. Results are reported in Table 1
for the two markers flanking the gene on the centromeric and telomeric sides (D8S532 and D8S1460). In a parametric analysis using GENEHUNTER, both markers produced strongly negative total LOD scores (-54.9 and -46.2 for D8S532 and D8S1460, respectively). Results were similarly negative when the six non-Caucasian families where considered separately. A multipoint LOD score smaller than -2.0, excluding linkage, was observed at either marker position in 15 families. Of the remaining pedigrees, four had a positive LOD score, reaching in one case (family 29) a value of 1.6. All seven affected members of this family shared the same D8S532-D8S1460 haplotype, which was present in only one of the five nondiabetic members who were examined. However, the evidence of linkage heterogeneity was not significant, with a maximum heterogeneity LOD score of +0.05. Nonparametric analysis, which considers only affected individuals, also failed to detect significant evidence of linkage at either marker position. The smallest P value (0.02) was observed in family 29.
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). The gene organization and exon-intron boundaries were subsequently confirmed by the draft genome sequence published by the Human Genome Project (contig no. NT_017505). The sequence information was used to amplify all coding exons, exon-intron boundaries, and about 1,600 bp of the 5' flanking region from one proband for each family. After screening these fragments by ddF and/or direct sequencing, we found a germline variant in the 5' flanking region (-1775del/insC) in one family, but this did not segregate with diabetes. Polymorphisms were identified in the 5' flanking region (-1547T > A), exon 11 (c.1083A > G, L361L), and intron 12 (IVS12+14t > a) (Fig. 1). Two candidate single nucleotide polymorphisms (SNPs) were reported in exon 19 in the public SNP database (SNP nos. 1057763 and 1057764), but these could not be confirmed when the exon was directly sequenced in the family probands. All polymorphisms had similar allele frequencies in the 32 family probands, 171 individuals with later-onset type 2 diabetes, and 182 nondiabetic controls, indicating that these sequence differences did not contribute to the common form of type 2 diabetes in this population (Table 2). Results were similarly negative when study subjects were stratified by body mass index or age at diagnosis, or when all polymorphisms were considered together as haplotypes (data not shown).
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Several studies have emphasized the role of naturally occurring inhibitors of insulin action such as TNF-, -HS glycoprotein, membrane glycoprotein PC-1, ras associated with diabetes, and resistin in the etiology of insulin-resistance and type 2 diabetes (11, 12, 13, 14, 15). An amino acid polymorphism in one of these molecules (PC-1 K121Q) has been recently found to be associated with insulin resistance, raising the hypothesis that sequence variants in natural inhibitors of insulin action may influence susceptibility to type 2 diabetes (16). Our results indicate that genetic variability in Ikkß—another important mediator of insulin resistance—is unlikely to contribute to the development of early-onset, autosomal dominant type 2 diabetes. The similarity of allele frequencies in unrelated cases with later-onset diabetes and nondiabetic controls also argues against a major role in common type 2 diabetes. In fact, the IKBKB gene sequence appears to be remarkably well conserved among individuals. Based on a genome-wide average of one common (>5%) SNP every 450 bp (16), one would expect at least four or five sequence differences in the IKBKB cDNA. In contrast, we identified only one SNP, which did not affect the amino acid sequence. The reasons for this low variability are unclear, but an important factor may be the negative selection of polymorphisms because of Ikkß importance in regulating inflammation and apoptosis (2). It is possible that sequence differences were missed by our mutation screening, but this seems unlikely, especially for frequent polymorphisms. First, ddF is a robust mutation screening technique having a sensitivity of almost 100% if both forward and reverse primers are used for the screening as was done in our study (9). Second, the number of individuals who were screened (17 subjects, corresponding to 34 chromosomes) provides more than 90% power to detect alleles having frequencies as low as 0.05 (17). Third, no suggestive evidence of undetected polymorphisms could be found in silico by aligning cDNA sequences or expressed sequence tags from different sources.
Negative results for Ikkß are not completely unexpected because efforts to link type 2 diabetes with genetic variation in other important mediators of insulin action (e.g. IRS-2) have similarly failed (1, 18, 19). At the same time, it must be emphasized that our efforts do not completely exclude the IKBKB locus as a gene for type 2 diabetes. Our screening was limited to the coding exons and to the 1.6 kb immediately 5' of the transcription start site. It is still possible that sequence differences in other regulatory regions that are not in linkage disequilibrium with the polymorphisms found in this study may affect susceptibility to diabetes. Moreover, the recent findings with the NIDDM2/calpain 10 gene suggest that intronic SNPs might be important in regulating susceptibility to common disorders (20). One must also consider that Ikkß is part of a complex signaling network (2), and therefore the site of variability, if any, may be in upstream or downstream proteins rather than Ikkß itself. Efforts are in progress to identify proteins that activate or are activated by Ikkß, so that the impact of genetic variability in this new insulin-resistance pathway can be fully investigated.
Acknowledgments
Footnotes
This study was supported by NIH Grants DK-55523 (to A.D.), DK-51729 (to S.E.S.), DK-47475 (to J.H.W.), and Joslin’s Diabetes Endocrinology Research Center Grant DK-36836 (Genetics Core). C.M. was a recipient of a Postdoctoral Fellowship from the Juvenile Diabetes Foundation International. N.P. was supported by a Fellowship Grant from Siriraj Hospital (Mahidol University, Thailand).
Abbreviations: ddF, Dideoxyfingerprinting; Ikkß, IB kinase ß; SNP, single nucleotide polymorphism.
Received September 5, 2001.
Accepted January 3, 2002.
References
B kinase-ß: NF-B activation and complex formation with IB kinase-. Science 278:866–869: direct role in obesity-linked insulin resistance. Science 259:87–91 2-HS-glycoprotein is an inhibitor of the human insulin receptor at the tyrosine kinase level. Mol Endocrinol 7:1445–1455This article has been cited by other articles:
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  J. M. Fernandez-Real and W. Ricart Insulin Resistance and Chronic Cardiovascular Inflammatory Syndrome Endocr. Rev., June 1, 2003; 24(3): 278 - 301. [Abstract] [Full Text] [PDF]
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