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Physiol. Genomics 19: 1-10, 2004. First published July 20, 2004; doi:10.1152/physiolgenomics.00114.2004
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Received 12 May 2004; accepted in final form 19 July 2004.
Physiological Genomics 19:1-10 (2004)
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Call for Papers: Comparative Genomics

Quantitative trait locus dissection in congenic strains of the Goto-Kakizaki rat identifies a region conserved with diabetes loci in human chromosome 1q

Karin J. Wallace*,1, Robert H. Wallis*,1, Stephan C. Collins*,1, Karène Argoud1, Pamela J. Kaisaki1, Alain Ktorza2, Peng Y. Woon1, Marie-Thérèse Bihoreau1 and Dominique Gauguier1

1 The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom
2 Laboratoire de Physiopathologie de la Nutrition, Centre National de la Recherche Scientifique UMR 7059, Université Paris 7, Paris, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Genetic studies in human populations and rodent models have identified regions of human chromosome 1q21–25 and rat chromosome 2 showing evidence of significant and replicated linkage to diabetes-related phenotypes. To investigate the relationship between the human and rat diabetes loci, we fine mapped the rat locus Nidd/gk2 linked to hyperinsulinemia in an F2 cross derived from the diabetic (type 2) Goto-Kakizaki (GK) rat and the Brown Norway (BN) control rat, and carried out its genetic and pathophysiological characterization in BN.GK congenic strains. Evidence of glucose intolerance and enhanced insulin secretion in a congenic strain allowed us to localize the underlying diabetes gene(s) in a rat chromosomal interval of ~3–6 cM conserved with an 11-Mb region of human 1q21–23. Positional diabetes candidate genes were tested for transcriptional changes between congenics and controls and sequence variations in a panel of inbred rat strains. Congenic strains of the GK rats represent powerful novel models for accurately defining the pathophysiological impact of diabetes gene(s) at the locus Nidd/gk2 and improving functional annotations of diabetes candidates in human 1q21–23.

type 2 diabetes mellitus; genetics; comparative genomics


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE INBRED GOTO-KAKIZAKI (GK) rat strain is a well-characterized nonobese model of spontaneous type 2 diabetes mellitus (T2DM), which is widely used for investigating important aspects of the pathogenesis of diabetes (1, 28) and mapping quantitative trait loci (QTL) involved in altered regulation of glucose and insulin levels. Replicated linkage between diabetes phenotypes and rat chromosome (RNO) 2 is suggested by results from QTL mapping studies in crosses derived from GK and the nondiabetic Brown Norway (BN) (13) or F344 rats (10) (loci Nidd/gk2 and Niddm2, respectively) and more recently in a cross involving the spontaneously diabetic Torii (SDT) rat, a new inbred model of nonobese type 2 diabetes (25).

Comparative genome analyses have highlighted the possible conservation of synteny homology between the Nidd/gk2 region and human chromosome 1q21–24 (3), which shows evidence of replicated linkage to T2DM in at least eight populations, including European Americans (7), French whites (34), the UK Warren 2 repository (37), Pima Indians (17), and Chinese (38). This region is therefore the focus of intense interest in T2DM genetics.

Progress in the completion of the rat genome sequence (30) provides a unique opportunity to refine homology relationships between RNO2 and human 1q and take advantage of genome annotations for T2DM candidate gene identification. The integration of comparative genomics and studies in rat congenic strains, which are designed to fine map QTL and test the phenotypic impact of gene variants in well-characterized regions of a QTL (5, 31), allows full utilization of rodent genetic and pathophysiological data in human genetics. The power of this strategy has recently been exemplified with the translation of GK diabetes QTL in T2DM functional and genetic association studies (8, 15, 20, 21, 23).

Following fine genetic mapping of the QTL Nidd/gk2 in the GKxBN F2 cross, we carried out its genetic and pathophysiological characterization in a series of congenic strains designed to contain different GK haplotypes at the locus introgressed onto the genetic background of the BN strain. We were able to localize gene(s) affecting glucose tolerance and insulin secretion in a 3- to 6-cM region of RNO2. Comparative genome analysis provided evidence of strong conservation of homology between this region and an 11-Mb segment of human chromosome 1q21–23, which allowed the selection of strong diabetes candidate genes for transcription studies and sequence variant screening in rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals
A GK colony was initiated in Oxford from rats of the Paris colony used to derive the original GKxBN cross (13). BN rats were obtained from Charles River Laboratories (Margate, Kent, UK) was also maintained. All rats had free access to water and standard laboratory diet pellets (B and K Universal, Hull, UK) and were maintained on a 12-h light/dark cycle. Progeny were weaned at 21 days. Experiments were conducted with Home Office approval and according to the rules of animal use in scientific experiments in the UK.

Microsatellite Marker-Assisted Production of Congenic Rats for the Locus Nidd/gk2
Construction of the congenics was specifically designed to introgress GK alleles from RNO2 regions covering the QTL Nidd/gk2 and Niddm2 onto the genetic background of the BN strain (BN.GK congenics), using a genetic marker-assisted breeding strategy (24), as previously described (35). Although the production of reciprocal congenics (GK.BN) was also initiated, it proved to be problematic, because of a high perinatal mortality rate in (GKxBN)xGK backcross progenies, which remained, however, similar to that observed in GK rats.

At each backcross and inbreeding generation, progeny genotypes were determined across the 20 rat autosomes using markers polymorphic between GK and BN strains (Ref. 3; http://www.well.ox.ac.uk/rat_mapping_resources). A panel of markers was optimized in each successive generation to 1) precisely define the introgressed region; 2) monitor the elimination of GK alleles from the genetic background, in particular in regions containing other QTLs previously identified in the GKxBN (13) and GKxF344 (10) crosses; and 3) ensure the retention of GK homozygous haplotype at Nidd/gk2 in the final congenics. All information regarding breedings and subsequent genetic and physiological analyses of the congenics was stored in our MACS database specifically designed for congenic projects (4).

Genotype Determination
Genomic DNA was prepared from ear clips, and PCRs were performed with 50 ng of DNA as previously described (3). Primer sequences, PCR conditions, and mapping information for all markers used are available at the Wellcome Trust Centre for Human Genetics Rat Mapping Resources web page (http://www.well.ox.ac.uk/rat_mapping_resources).

Phenotype Analysis
Physiological screening was carried out with animals of at least three different litters to minimize possible litter effects on phenotype variability. All phenotypes related to glucose homeostasis and lipid profile were determined in male and female congenic and BN rats at 3 mo. One week later, rats were killed, and liver samples were harvested in overnight fasted rats, immediately frozen in liquid nitrogen, and kept at –80°C for gene expression studies.

Glucose tolerance and glucose-induced insulin secretion tests.
Intravenous glucose tolerance tests (IVGTT) were performed using the protocol previously applied in GKxBN genetic study (13). Rats were anesthetized using ketamine hydrochloride (95 mg/kg body wt, Ketalar; Parke-Davis, Cambridge, UK). A solution of 14% glucose (0.8 g/kg body wt) was injected via the saphenous vein. Blood samples were collected before the injection and 5, 10, 15, 20, and 30 min afterwards. Samples were spun at 8,000 rpm, and plasma was separated. Plasma glucose concentration was measured on a Cobas Mira Plus automatic analyzer (ABX Diagnostics, Shefford, UK). Plasma immunoreactive insulin (IRI) was determined with an ELISA kit (Mercodia, Uppsala, Sweden).

Cumulative glycemia and insulinemia were determined by the total increment of plasma glucose and plasma insulin levels during the IVGTT. The cumulative glycemia reflects the overall glucose tolerance during the test, and the cumulative insulinemia is an indicator of insulin secretory capacity.

Plasma lipids.
Following a 16–18 h fast, blood samples were collected via the tail vein, and plasma concentrations of total cholesterol (TC), HDL-C, LDL-C, triglycerides, and phospholipids were determined on a Cobas Mira Plus analyzer using diagnostic enzymatic/colorimetric kits (ABX Diagnostics). Values for VLDL-C were obtained by subtracting the sum of HDL-C and LDL-C from TC.

RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR
Liver total RNA was isolated following two successive TRIzol (Invitrogen GIBCO, Paisley, UK) extractions followed by chloroform phase separation and ethanol precipitation. For the first-strand cDNA synthesis, total RNA (100 µg) was further purified using the Qiagen RNeasy kit (Qiagen, Crawley, UK) and analyzed on an Agilent 2100 Bioanalyzer (Bracknell, Berks, UK). Total RNA was used to synthesize first-strand cDNA using SuperScript II RNase H – reverse transcriptase (Invitrogen GIBCO) in 1x first-strand buffer (50 mM Tris·HCl, pH 8.3, 75 mM KCl, 3 mM MgCl 2), 0.5 mM dNTP, 10 mM DTT, and 500 µg of oligo-dT primer poly-d(T) 12–18. Residual RNA was removed by Escherichia coli RNase H.

Quantitative real-time PCR (QRT-PCR) was performed using a Rotor-Gene 3000 system (Corbett Research, Milton, UK) using the QuantiTest SYBR Green PCR kit (Qiagen). First-strand cDNA from each individual was used at various concentrations for the detection and quantification of candidate genes or internal housekeeping gene (ß2-microglobulin) transcripts. Gene-specific QRT-PCR primers, which were designed to span an intron/exon boundary, are available through our data repository (http://www.well.ox.ac.uk/rat_mapping_resources) and in the Supplementary Table S1 (available at the Physiological Genomics web site).1 Experiments were performed in triplicate with samples prepared from four animals. Quantitative analysis of the QRT-PCR products was performed using the Rotor-Gene software (version 5.0.47; Corbett Research). Gene dosage was calculated by comparing with the standard curve generated and normalized to the housekeeping gene.

Sequence Analysis of the Genes Encoding Rat Endosulfine-{alpha} (Ensa) and Hydroxyacid Oxidase 3 (Hao3)
Sequence analysis was carried out with genomic DNA of BN and GK rat colonies maintained in our laboratory and rats of three inbred colonies of the Wistar-Kyoto (WKY) strain [Izumo (Izm), Heidelberg (Heid), and Leicester (Leic)]. These strains were chosen because both WKY and GK derive from outbred Wistars, and they may share extensive sequence similarities outside GK-specific diabetes susceptibility alleles. Reference sequences for the rat Ensa (NM_021842) and Hao3 (NM_032082) genes were used to obtain the corresponding genomic sequences (AC121649 and AC123109, respectively). PCR primers were designed to cover all coding regions, about 3 kb of the promoter region and 1 kb of the 3'-end of the two genes (see Supplemental Table S2). PCR products were sequenced using the BigDye Version 3.1 dye terminator kit (ABI, Foster City, CA). The sequencing products were purified on Sephadex G50 Superfine gel (Amersham, Little Chalfont, Bucks, UK) and analyzed on ABI 3700 DNA sequencers (ABI). Sequence Navigator V1.0 (ABI) was used for sequence comparisons.

Statistical Analyses
All phenotypes were regressed for both sex and cross effects as previously described (13) prior to genetic linkage analysis. Linkage between marker genotypes and diabetes-related phenotypes in the GKxBN F2 cross was initially evaluated by an ANOVA test followed by a permutation test as previously used (35). Interval mapping was performed with the MAPMAKER/QTL computer package (22).

SPSS version 11.0 was used for statistical analysis of the physiological data from congenic rats. The univariate general linear model (GLM) was used to analyze all phenotypes. This allows comparisons between the control strain (BN) and congenics as well as between the congenic strains themselves and can account for variance that is not due to the dependent variable. A Bonferroni post hoc test was used to determine strain differences. All results described are analyses of males and females together unless otherwise stated. Threshold for significance was set at P < 0.01.

Comparative Genome Analysis
Existing comparative gene maps (http://www.well.ox.ac.uk/rat_mapping_resources; 36) and rat genome annotations (http://ensembl.ebi.ac.uk) were used to anchor QTL and congenic intervals in the rat genome and subsequently refine homology relationships between the rat and human genomes in the region of Nidd/gk2.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Fine Mapping of the QTL Nidd/gk2 in the GKxBN F2 Cross
Among the 127 RNO2 microsatellite markers that we have now genotyped in the original GKxBN F2 cross (13) since the initial diabetes QTL mapping study was completed, a subset of 21 markers were selected to repeat statistical analyses in the cross. These markers covered the entire chromosomal length (114.4 cM) with an average spacing of ~5 cM (Fig. 1). Marker locus D2Rat41 showed the most significant evidence of linkage to both fasting insulinemia (FI) and the ratio of fasting insulinemia/fasting glycemia (FI/FG) (maximum LOD 4.72; P = 6 x 10 –5). The one-LOD interval around the peak of genetic linkage spans a 15-cM region (between D2Mgh7 and D2Mgh12). The QTL explains up to 17% of the variance of the traits in the cross. As outlined in our previous study (13), marginal linkage to stimulated insulin secretion was detected in the cross. Marker locus D2Mgh12 exhibits the strongest association to this trait (LOD 3.10; P = 9 x 10 –4). Using this refined linkage map of RNO2, no evidence of significant linkage to glucose tolerance, body weight, or adiposity index was found.



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  		Fig. 1. Refined QTL map of the locus Nidd/gk2 in the GKxBN F2 cross. QTL maps for fasting insulin (FI) and the ratio of fasting insulin/fasting glucose (FI/FG) have the same profile. Permutation tests (n = 10,000) were used to determine statistical significance threshold of linkages ( P = 0.001) to FI (LOD = 3.05) and insulin secretion (LOD = 2.97). Niddm2 indicates the approximate position of the QTL identified in the GKxF344 cross (10) around markers D2Mit15 and D2Mit14, which maps 2.5 cM away from D2Wox24 (3). The approximate location of the 1-LOD interval around the locus Gisdt2 (25) identified in the (BNxSDT)xSDT cross is reported. A comprehensive genetic map of RNO2 in the GKxBN cross (36) is available on our public database (http://www.well.ox.ac.uk/rat_mapping_resources).

 
Rats of the F2 cohort carrying the GK homozygous genotype at marker locus D2Rat41 exhibited significant fasting hyperinsulinemia and a higher FI/FG ratio compared with BN homozygous and heterozygous rats (Table 1). GK alleles at marker locus D2Mgh12 were associated with a significant reduction in stimulated insulin secretion in the cross.


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  		Table 1. Summary of the correlations between genotypes at marker loci D2Rat41 and D2Mgh12 and diabetes-related variables quantified in (GKxBN) F2 rats

 
Production of Congenic Strains
Knowledge of BN vs. GK allele variations for over 2,000 rat markers (http://www.well.ox.ac.uk/rat_mapping_resources) and their precise chromosomal location in the GKxBN F2 cross (3) allowed us to select optimal panels of markers for a thorough and accurate genetic screening of congenics. In successive (GKxBN)xBN backcross progeny, the autosomes were screened with up to 238 markers (Table 2), with half of them chosen to monitor the elimination of GK alleles at GK QTL other than Nidd/gk2. Ultimately, the genome of congenics was scanned with an average spacing of <7 cM between loci. Inbreeding was carried out after 7–10 successive backcross breedings after the elimination of GK alleles throughout the genetic background.


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  		Table 2. Congenic strains derived for the QTL Nidd/gk2 and approximate genetic size of regions containing GK homozygous genotypes

 
The genetic characteristics of the congenic strains analyzed in this study are described in Table 2. Two congenic strains (BN.GK2a and BN.GK2c) were initially bred, covering the entire region of Nidd/gk2 (Fig. 2). They contain GK alleles in 33.6 cM (BN.GK2a) and 54.7 cM (BN.GK2c) regions of the QTL. Further congenics were produced, containing GK haplotypes in 23.2 cM (BN.GK2e) and 14.1 cM (BN.GK2k) regions of the QTL.



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  		Fig. 2. Simplified comparative genome maps of RNO2 and detail of the GK haplotype in BN.GK congenics (open bars). Solid bars at the ends of the congenic segments are regions of crossover where genotype is unknown. The linkage map reports only those markers used to characterize the congenics and anchor the genetic map in the most recent assembly of the rat genome sequence (June 2003) (http://www.ensembl.org/Rattus_norvegicus/; annotated version of February 2004). Gene symbols are in parentheses, and approximate positions of rat diabetes QTL are reported. Diabetes candidate genes analyzed in this study for transcriptional changes and sequence polymorphisms are in boldface type in the rat physical map. More comprehensive genetic and comparative genome maps in the region of Nidd/gk2 are publicly available (http://www.well.ox.ac.uk/rat_mapping_resources).

 
Phenotype Analyses
Glucose tolerance.
Results from IVGTT showed that glucose tolerance was similar in BN.GK2a, BN.GK2c, and BN rats, as reflected by identical glycemic profile and cumulative glycemia during the test (Fig. 3A). In contrast, rats of both BN.GK2e and BN.GK2k strains exhibited a marked deterioration of glucose tolerance during the IVGTT, with significantly more elevated glycemia 5 min after glucose injection in both strains compared with BN controls and BN.GK2a rats (F=4.7, P = 0.001). This resulted in a 10% increase in values of cumulative glycemia during the test in BN.GK2e and BN.GK2k congenics compared with BN.GK2a rats and BN controls.



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  		Fig. 3. Glucose tolerance (A) and glucose-induced insulin secretion (B) in 12-wk-old rats of the RNO2 congenic strains BN.GK2a (n = 57), 2c (n = 21), 2e (n = 15), and 2k (n = 25), and BN rats (n = 36). Results are means ± SE. *P < 0.01, statistically significant differences in glycemia between BN.GK2e/2k rats and BN and BN.GK2a rats. {dagger}P < 0.05, statistically significant differences in insulinemia between BN.GK2k rats and BN, BN.GK2a, 2c, and 2e rats.

 
In vivo insulin secretion.
Glucose-induced insulin secretion was similar in BN.GK2a, BN.GK2c, and BN rats (Fig. 3B). Insulin secretion was reduced in BN.GK2e rats compared with BN rats, but differences were not statistically significant. In contrast, rats of the BN.GK2k congenic strain showed a significant enhancement of insulin secretion in response to glucose compared with BN rats and BN.GK2a, 2c, and 2e congenics. As a result, cumulative plasma insulin during the test was higher in BN.GK2k rats than in BN ( P < 0.002), BN.GK2a ( P < 0.005), BN.GK2c ( P < 0.05), and BN.GK2e ( P < 0.003) rats (Fig. 3B).

Body weight and plasma lipids.
There were no differences in body weight between male congenics and BN rats (Table 3). Body weight was only increased in BN.GK2c and BN.GK2e female rats compared with BN controls. No major changes in plasma lipid profile were detected in congenic rats compared with BN controls. Only total cholesterol was significantly lower in BN.GK2c congenic rats than in BN rats. Plasma LDL and VLDL cholesterol concentrations were also lower in BN.GK2c congenic rats than in BN rats, but this effect was significant for LDL in males and for VLDL in females.


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  		Table 3. Body weight and plasma lipid concentrations in 3-mo-old rats of BN.GK congenic strains and age-matched BN controls

 
Comparative Genome Analysis
Detailed in silico comparative genome mapping was specifically carried out for the GK chromosomal interval introgressed in BN.GK2k congenics (Fig. 2). The genomic length covering the entire congenic interval (14 to 17 cM) in this strain is over 60 Mb between markers D2Rat40 and D2Got149. It contains over 140 known genes and 450 EST sequences, which were used to refine its strong homology relationships with regions of human chromosome 1p12-p13 (11 Mb), 1p22, 1q23–23.2 (12 Mb), and 4q36. The 6.6-cM segment most likely to contain the GK variant(s) involved in glucose intolerance in BN.GK2k rats corresponds to a 21.5-Mb genomic length containing more than 78 known genes. This region is conserved with human chromosome 1q21–23 (from 155.9 Mb to 143.3 Mb) and 1p11-p13 (from 116.1 Mb to 119.6 Mb) (Fig. 2). Full results from comparative genome analysis are available on our public database (http://www.well.ox.ac.uk/rat_mapping_resources).

Transcriptional Analysis of Candidate Genes Mapped to the Region of BN.GK2k
Based partially on comparative mapping data from the human genome sequence, we selected nine positional candidate genes localized in the congenic interval of BN.GK2k for transcription studies. These gene encode endosulfine-{alpha} (Ensa, NM_021842), hydroxyacid oxidase 3 (Hao3, NM_032082), ATPase 1a1 (Atp1a1, NM_012504), fatty acid transport protein 3 (Fatp3, XM_215605), HMG-CoA synthase (Hmgcs2, NM_173094), hyperpolarization-activated cyclic nucleotide-gated potassium channel 3 (Hcn3, NM_053685), phosphatidylinositol 4-kinase (Pik4cb, NM_031083), phosphomevalonate kinase (Pmvk, XM_227421), and cellular retinoic acid-binding protein II (Crabp2, NM_017244).

Liver RNA levels of Ensa, Atp1a1, Fatp3, Hmgcs2, Hcn3, Pik4cb, Pmvk, and Crabp2 were not significantly different in BN, GK, and BN.GK2k rats (Fig. 4). The amount of these transcripts was generally higher in the BN.GK2k strain than in the GK rat (up to 129.1 ± 15.1% of BN expression in BN.GK2k vs. 87.1 ± 9.02% of BN expression in GK for Hcn3). In contrast, compared with the BN control, RNA levels of Hao3 were significantly reduced in both GK (67.5 ± 2.5%; P = 0.016) and BN.GK2k congenics (32.9 ± 18.0%; P = 0.014).



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  		Fig. 4. Transcriptional analysis of selected candidate genes mapped to the GK congenic region of the GK.BN2k strain. Pooled BN rat liver first-strand cDNA was used in standard curve determination for both candidate genes and the internal housekeeping gene. The final results are expressed as means ± SE, in percentage of normalized BN values. Statistical analysis was performed using unpaired Student’s t-test. *P < 0.05, statistically significant differences vs. BN controls.

 
Sequence Analysis of the Rat Genes Encoding Ensa and Hao3
Following resequencing of the genes Ensa and Hao3 in five inbred rat strains (BN, GK, WKY-Izm, WKY-Leic, and WKY-Heid), we identified a total of 21 sequence variants, including 16 single nucleotide polymorphisms (Table 4). Five sequence variants were found in Ensa and 16 in Hao3. Sequence variants were identified in the promoter (16 variants), introns (4 variants), and in the 3'-UTR (1 variant) of the genes. No sequence polymorphisms were found in coding regions.


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  		Table 4. Sequence analysis of Ensa and Hao3 in inbred BN, GK, and WKY strains

 
Nineteen of the 21 polymorphisms detected in Ensa and Hao3 were different between GK and BN strains (Table 4). Gene sequences were identical in rats of two WKY colonies (WKY-Izm and WKY-Leic), whereas there were many sequence differences (19/21) between these strains and the WKY-Heid strain. The GK haplotype of Ensa was fully conserved with that of the WKY-Heid strain. In contrast, the GK, WKY-Izm, and WKY-Leic strains share an identical haplotype for Hao3.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study describes the detailed genetic and pathophysiological characterization of the QTL Nidd/gk2 cosegregating with diabetes-related traits in an experimental cross derived from the GK rat model of T2DM (13). Results from the physiological screening in congenic strains derived for the locus suggest that several GK alleles at the QTL induce glucose intolerance and altered insulin secretion. Comparative genome analysis provides confirmation of strong conservation of synteny homology between a segment of the QTL Nidd/gk2 and a T2DM candidate region on human chromosome 1q21–23.

The original QTL mapping studies in the GKxBN and GKxF344 crosses identified significant linkages between diabetes variables quantified in the cohorts and only a few genetic markers on rat chromosome (RNO) 2 (10, 13). We were able to refine the localization of the QTL Nidd/gk2 linked to both hyperinsulinemia and increased ratio of insulin/glucose, which are indicative of insulin resistance, to a region of ~15 cM in the GKxBN F2 cross. We also identified a segment of the QTL that contains GK alleles associated with a sharp reduction in stimulated insulin secretion in the cross, suggesting the involvement of distinct genes at the locus that affect insulin signaling and insulin secretion. Furthermore, this region overlaps with the locus Niddm2, which remains only defined in the GKxF344 cross by linkage between glucose intolerance and two markers mapped 25 cM apart on RNO2 (10).

At this stage of the genetic analysis, statistically defined QTL position and associated subphenotypes provide little information on the number and functional roles of the underlying diabetes susceptibility gene(s). Providing that the GK colonies used in the two QTL mapping studies (10, 13) are genetically identical, the QTL linked to closely related pathophysiological components of diabetes may reflect the action of the same GK variant(s). This implies that different gene variants in the genetic background of the normoglycemic strain (BN or F344) bred to the GK rat modulate the phenotypic expression of GK diabetes susceptibility alleles at the QTL. This hypothesis is supported by the often poor replication of blood pressure QTL in experimental crosses derived from a single hypertensive rat strain bred to different normotensive strains (16). Alternatively, genomic clustering of functionally related QTL may reflect the involvement of several GK variants in the region of Nidd/gk2. Further investigations in BN.GK congenics designed to dissect the locus support this latter hypothesis.

Although investigations in BN.GK2a and 2c congenics primarily aimed at validating the existence of Nidd/gk2 in animals carrying GK alleles on the entire length of the QTL, rats of these strains did not show major impairment of any diabetes phenotypes tested. Rats of the BN.GK2c strain showed a mild deterioration of glucose tolerance, increased body weight, and reduced plasma level of total cholesterol. These results suggest either that the original linkage is a false positive, which by chance colocalizes with Niddm2, or that GK haplotypes in the congenic region contain several gene variants with opposing effects on the overall diabetes phenotypes. Moreover, procedures and assays designed to provide a quantitative evaluation of glucose tolerance and in vivo insulin secretion are relatively complex and may be prone to variability and inaccuracies, especially when carried out in congenics tested over long periods of time. Contrasting phenotypic effects can be detected in an experimental cross due to recombination events, which allow independent mapping of distinct subphenotypes. Diabetes in the GK rat stems from the overall net effect of multiple genetic loci selected over many generations of inbreeding from outbred Wistar rats (14), which together lead to impaired glucose homeostasis through various mechanisms, including insulin resistance and altered insulin secretion (1, 28). In the context of a congenic strain carrying linked GK alleles that independently impair glucose tolerance and raise insulin secretion, their pathological effects may be cancelled out. Similar complex situations have been reported in congenic strains for hypertension QTL in rats (29, 32) and type 1 diabetes QTL in the NOD mouse (33).

Results from the phenotypic screening of congenic strains BN.GK2e and 2k subsequently derived for shorter GK haplotypes at Nidd/gk2 validated the existence of the QTL. The most important observation was a modest but significant deterioration of glucose tolerance in both BN.GK2e and 2k strains compared with BN controls and the other congenics, without major changes in plasma lipid levels, which appear to be specific to the congenic BN.GK2c. This result suggests that the underlying GK variant(s) are localized in a <6.6-cM interval (between D2Rat40 and D2Wox26), corresponding to the congenic interval shared in BN.GK2e and 2k strains, but not with that introgressed in BN.GK2a, which shows normal glucose tolerance. The minimal congenic interval (3 cM) is flanked by D2Wox17 and D2Wox49. The phenotypic effect is consistent with that of Niddm2 in the GKxF344 cross (10). It may also account for the enhanced insulin secretion specifically observed in BN.GK2k congenics, which could originate from the effect of gene(s) at the locus Nidd/gk2 on hyperinsulinemia primarily observed in the GKxBN cross (13).

The absence of alteration in insulin secretion in BN.GK2a, 2c, and 2e congenics suggests that the GK haplotype shared in these strains, possibly telomeric to the congenic region of BN.GK2k (between markers D2Got156 and D2Wox35), may contain gene(s) that can specifically modify insulin secretion. They would only normalize enhanced insulin secretion induced by gene variant(s) in the congenic region of BN.GK2k and may account for the marginally significant QTL for reduced insulin secretion mapped to this region of RNO2 in the GKxBN cross. The relatively modest phenotypic consequences of GK variants at the locus Nidd/gk2 accounts for genetic differences between BN.GK and BN strains in a chromosomal region representing less than 1% of the total rat genome length. The existence of variants in multiple genes contributing to a QTL effect is a hallmark of several attempts to dissect QTL regions, including GK QTL, in congenics (9, 11, 12, 29). These examples, which may be limited to specific strain combinations, particular quantitative phenotypes or QTL, underline the importance of congenics rather than chromosome substitution strains for the dissection of the QTL Nidd/gk2 and the identification of the underlying diabetes genes.

The existence of several diabetes susceptibility loci in human 1q also has been suggested, and two closely linked regions (at ~157 Mb and ~162 Mb) were recently defined in a cohort of American Caucasians (6). They are both conserved with RNO13 where the majority of positional candidate genes already tested for association with T2DM map (APCS, APOA2, CRP, KCNJ9, KCNJ10, LMX1A, MGST3, PBX1, PEA15, RXRG, and SLC19A2). The region of Nidd/gk2 that we can associate with glucose intolerance in BN.GK2k rats corresponds to an 11-Mb interval of human 1q21–23 that is upstream of 157 Mb but which overlaps the 5'-end of a region estimated from several studies with linkage to 1q (18). Further candidate genes can be selected from this region of rat chromosome 2 for expression studies in rat congenics and mutation detection in the rat and human.

Although microarray-based transcription profiling in congenics has been proposed as a tool for facilitating disease gene identification (26, 27), we prioritized our expression studies to positional candidates localized in the critical GK genomic region of the BN.GK2k strain. We focused on genes encoding proteins involved in metabolism (Hao3, Fatp3, Hmgcs2, Pmvk) and cellular physiology (Atp1a1, Hcn3, Pik4cb, Crabp2) including insulin secretion (Ensa). Ensa gene sequencing was also carried out, as liver RNA levels do not necessarily reflect gene expression changes in pancreatic beta cells and their effects on insulin secretion (2). Despite a sharp decrease in Hao3 mRNA levels in both BN.GK2k and GK rats, we did not find GK-specific variants in the gene. Promoter polymorphisms might, however, alter gene transcription. Our results therefore shed light on gene pathways controlled by the GK haplotype in BN.GK2k involving altered peroxisomal fatty acid oxidation (19), which may contribute to impaired glucose regulation in this congenic strain and in GK rats.

In conclusion, the characterization of the locus Nidd/gk2 in both the GKxBN F2 cross and BN.GK congenic strains strongly suggests the implication of GK variant(s) in several diabetes susceptibility genes at the locus. Knowledge of homology conservation between the human and rat T2DM/glucose intolerance loci, combined with the ability to narrow a disease locus by use of congenic strains, emphasizes the importance of comparative genomics in the search for diabetes genes. Pathophysiological and gene expression profiling in existing congenics and new strains designed to further dissect the locus Nidd/gk2, as well as SNP-based haplotype analysis (39), should provide new insights into the functional role of genes and gene pathways underlying these diabetes QTL effects.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work is supported by Wellcome Trust Grant 057733, Wellcome Cardiovascular Functional Genomics Initiative Grant 066780/Z/01/Z, and Diabetes UK Grant RD01/0002160. The production of rat congenic strains was supported by Diabetes UK Grant RD96/0001270 and by EC GIFT QLRT-1999-00546. S. C. Collins is supported by a Wellcome Prize Studentship. D. Gauguier holds a Wellcome Senior Fellowship in Basic Biomedical Science.


   ACKNOWLEDGMENTS
 
We are indebted to Dr. Colin Hetherington for invaluable help in the implementation of congenic colonies.


   FOOTNOTES
 
Article published online before print. See website for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: D. Gauguier, The Wellcome Trust Centre for Human Genetics, Univ. of Oxford, Roosevelt Drive, Headington, Oxford OX3 7BN, UK (E-mail: gdomi{at}well.ox.ac.uk).

* K. J. Wallace, R. H. Wallis, and S. C. Collins contributed equally to this work. Back

1 The Supplementary Material for this article (Supplemental Tables S1 and S2) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00114.2004/DC1. Back


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 MATERIALS AND METHODS
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 DISCUSSION
 GRANTS
 REFERENCES
 

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