Download Quantitative trait locus dissection in congenic strains of the Goto

Physiol Genomics 19: 1–10, 2004.
First published July 20, 2004; doi:10.1152/physiolgenomics.00114.2004.
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 Argoud,1 Pamela J. Kaisaki,1
Alain Ktorza,2 Peng Y. Woon,1 Marie-Thérèse Bihoreau,1 and Dominique Gauguier1
1
The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom; 2Laboratoire de
Physiopathologie de la Nutrition, Centre National de la Recherche Scientifique UMR 7059, Université Paris 7, Paris, France
Wallace, Karin J., Robert H. Wallis, Stephan C. Collins,
Karène Argoud, Pamela J. Kaisaki, Alain Ktorza, Peng Y. Woon,
Marie-Thérèse Bihoreau, and Dominique Gauguier. Quantitative
trait locus dissection in congenic strains of the Goto-Kakizaki rat
identifies a region conserved with diabetes loci in human chromosome
1q. Physiol Genomics 19: 1–10, 2004. First published July 20, 2004;
doi:10.1152/physiolgenomics.00114.2004.—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
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).
* K. J. Wallace, R. H. Wallis, and S. C. Collins contributed equally to this
work.
Article published online before print. See web site 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: [email protected]).
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
GK⫻BN 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
Animals
A GK colony was initiated in Oxford from rats of the Paris
colony used to derive the original GK⫻BN 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.
1094-8341/04 $5.00 Copyright © 2004 the American Physiological Society
1
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Submitted 12 May 2004; accepted in final form 19 July 2004
2
COMPARATIVE MAPPING OF A RAT DIABETES LOCUS IN CONGENIC STRAINS
Microsatellite Marker-Assisted Production of Congenic Rats for the
Locus Nidd/gk2
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 GK⫻BN 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.
Physiol Genomics • VOL
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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 ␮g) 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 1⫻ first-strand buffer (50 mM
Tris 䡠 HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2), 0.5 mM dNTP, 10 mM
DTT, and 500 ␮g 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
(␤2-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 RotorGene 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-␣ (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
GK⫻BN 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
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.
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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 (GK⫻BN)⫻GK 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 GK⫻BN (13) and GK⫻F344 (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).
RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR
COMPARATIVE MAPPING OF A RAT DIABETES LOCUS IN CONGENIC STRAINS
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.
3
the cross. Marker locus D2Mgh12 exhibits the strongest association to this trait (LOD 3.10; P ⫽ 9 ⫻ 10⫺4). Using this
refined linkage map of RNO2, no evidence of significant
linkage to glucose tolerance, body weight, or adiposity index
was found.
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.
RESULTS
Among the 127 RNO2 microsatellite markers that we have
now genotyped in the original GK⫻BN 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 ⫻ 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
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 GK⫻BN F2
cross (3) allowed us to select optimal panels of markers for a
thorough and accurate genetic screening of congenics. In
successive (GK⫻BN)⫻BN 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.
The genetic characteristics of the congenic strains analyzed
in this study are described in Table 2. Two congenic strains
Fig. 1. Refined QTL map of the locus Nidd/gk2 in the GK⫻BN 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 GK⫻F344 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 (BN⫻SDT)⫻SDT
cross is reported. A comprehensive genetic map of RNO2 in the GK⫻BN cross (36) is available on our public database
(http://www.well.ox.ac.uk/rat_mapping_resources).
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Production of Congenic Strains
Fine Mapping of the QTL Nidd/gk2 in the
GK⫻BN F2 Cross
4
COMPARATIVE MAPPING OF A RAT DIABETES LOCUS IN CONGENIC STRAINS
Table 1. Summary of the correlations between genotypes at marker loci D2Rat41 and D2Mgh12 and
diabetes-related variables quantified in (GK⫻BN) F2 rats
GK/BN
GK/GK
F
P Value
37
97⫾6
6.56⫾0.18
15.13⫾0.98
322⫾6
494⫾36
11.96⫾0.94
66
88⫾4
6.50⫾0.10
13.40⫾0.76
325⫾5
524⫾26
11.98⫾0.62
36
120⫾10
6.51⫾0.15
18.92⫾1.73
322⫾6
422⫾31
9.47⫾0.75
6.3
0.05
6.7
0.3
1.8
2.0
0.0005
NS
0.0003
NS
NS
NS
31
94⫾6
6.51⫾0.13
14.70⫾0.97
319⫾6
519⫾39
12.40⫾1.01
75
94⫾5
6.62⫾0.08
14.48⫾0.76
327⫾4
527⫾25
12.08⫾0.60
33
113⫾10
6.38⫾0.10
17.62⫾1.62
319⫾7
377⫾25
8.36⫾0.54
3.6
0.8
3.7
0.7
4.2
4.8
0.02
NS
0.01
NS
0.007
0.003
The sum of blood glucose during an intravenous glucose tolerance test (IVGTT) is used as an index of glucose tolerance (GT). Plasma insulin 5 min after
stimulation was used as an index of acute insulin secretion (AIS), and the sum of plasma insulin (SPI) during the test evaluates the overall insulin secretory
capacity. For all phenotypes, means ⫾ SE were calculated for each genotype at the locus. FI, fasting insulin; FG, fasting glucose; BN/BN, homozygous for the
BN allele; GK/BN, heterozygous; GK/GK, homozygous for the GK allele; n ⫽ number of observations. ANOVA was applied to test for linkage. NS, not
significant.
(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.
BN.GK2k congenics compared with BN.GK2a rats and BN
controls.
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
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
Table 2. Congenic strains derived for the QTL Nidd/gk2 and approximate genetic size of regions
containing GK homozygous genotypes
Congenic
Strain
BN.GK2a
BN.GK2c
BN.GK2e
BN.GK2k
Minimum Interval
Maximum Interval
Marker
Typed
Resolution,
cM
Generation at
Inbreeding
D2Wox26–D2Mit16
(33.6 cM)
D2Mit6–D2Got147
(54.7 cM)
D2Wox17–D2Rat63
(23.2 cM)
D2Wox17–D2Got156
(14.1 cM)
D2Wox49–D2Rat70
(38.1 cM)
D2Wox30–D2Wox68
(69.2 cM)
D2Rat40–D2Wox35
(29.5 cM)
D2Rat40–D2Got149
(17.4 cM)
227
(119)
226
(119)
233
(121)
238
(124)
6.8
(4.6)
6.8
(4.1)
6.6
(4.8)
6.5
(4.8)
7
7
8
10
Homozygous genotypes for the BN or the GK allele at chromosome 2 marker loci were used to define the maximum and minimum congenic interval,
respectively, containing GK haplotypes; the length of the congenic region in parentheses was obtained in the linkage map of the GK⫻BN intercross (10). “Marker
Typed” indicates total number of markers used for the genetic screening of the QTL Nidd/gk2 and the genetic background in progenies of backcross breedings;
the number of markers used to screen chromosomal segments containing known GK QTL, including Nidd/gk2, is shown in parentheses. “Resolution” indicates
average spacing between markers used for the genetic characterization of the congenics; results from the screening of chromosomes containing known GK QTL
are shown in parentheses. The last column (“Generation at Inbreeding”) shows the backcross generation at which GK alleles were eliminated from the genetic
background and inbreeding was initiated.
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D2Rat41
n
F1, pmol/l
FG, mM
FI/FG
GT, mM
AIS, pmol/l
SPI, nmol/l
D2Mgh12
n
FI, pmol/l
FG, mM
FI/FG
GT, mM
AIS, pmol/l
SPI, nmol/l
BN/BN
COMPARATIVE MAPPING OF A RAT DIABETES LOCUS IN CONGENIC STRAINS
5
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.
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.
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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).
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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).
6
COMPARATIVE MAPPING OF A RAT DIABETES LOCUS IN CONGENIC STRAINS
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 candi-
date genes localized in the congenic interval of BN.GK2k for
transcription studies. These gene encode endosulfine-␣ (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,
Table 3. Body weight and plasma lipid concentrations in 3-mo-old rats of BN.GK congenic strains
and age-matched BN controls
BN
Body wt, g
Males
Females
TC, mmol/l
Males
Females
Triglycerides, mmol/l
Males
Females
Phospholipids, mmol/l
Males
Females
HDL-C, mmol/l
Males
Females
LDL-C, mmol/l
Males
Females
VLDL-C, mmol/l
Males
Females
BN.GK2a
BN.GK2c
BN.GK2e
BN.GK2k
F
P
236⫾5 (28)
159⫾2 (36)
247⫾4 (27)
170⫾2 (46)
250⫾6 (16)
174⫾4* (17)
238⫾10 (11)
178⫾8* (6)
248⫾4 (15)
158⫾5 (8)
1.2
6.6
NS
0.00009
1.60⫾0.03 (29)
1.72⫾0.05 (18)
1.58⫾0.05 (16)
1.67⫾0.05 (14)
1.48⫾0.05* (15)
1.48⫾0.04* (11)
1.63⫾0.04 (10)
1.82⫾0.07 (6)
1.73⫾0.04 (10)
1.55⫾0.09 (6)
3.1
3.8
0.02
0.009
0.79⫾0.03 (22)
0.88⫾0.04 (17)
0.75⫾0.04 (16)
1.00⫾0.04 (14)
0.70⫾0.05 (15)
0.78⫾0.06 (11)
0.72⫾0.04 (10)
0.81⫾0.06 (6)
0.85⫾0.05 (10)
0.71⫾0.10 (6)
2.1
3.6
NS
0.01
1.14⫾0.04 (20)
1.44⫾0.05 (15)
1.14⫾0.04 (15)
1.32⫾0.04 (14)
1.17⫾0.04 (15)
1.26⫾0.08 (8)
1.16⫾0.04 (10)
1.25⫾0.19 (6)
1.25⫾0.05 (10)
1.22⫾0.14 (6)
0.9
1.6
NS
NS
0.70⫾0.02 (26)
0.84⫾0.02 (22)
0.76⫾0.03 (16)
0.82⫾0.02 (14)
0.69⫾0.03 (15)
0.77⫾0.02 (11)
0.72⫾0.03 (10)
0.88⫾0.03 (6)
0.84⫾0.01 (10)
0.84⫾0.02 (6)
3.1
1.3
0.02
NS
0.40⫾0.03 (26)
0.22⫾0.01 (20)
0.41⫾0.01 (15)
0.21⫾0.02 (14)
0.30⫾0.02* (13)
0.23⫾0.02 (11)
0.31⫾0.02* (10)
0.27⫾0.01 (6)
0.33⫾0.01 (10)
0.20⫾0.02 (6)
4.6
1.0
0.003
NS
0.53⫾0.03 (23)
0.64⫾0.03 (20)
0.41⫾0.07 (15)
0.63⫾0.04 (14)
0.50⫾0.06 (13)
0.48⫾0.04* (11)
0.59⫾0.05 (10)
0.67⫾0.06 (6)
0.56⫾0.03 (10)
0.51⫾0.06 (6)
1.7
4.0
NS
0.007
Values are means ⫾ SE; number of rats is in parentheses. Analysis of variance using the general linear model (GLM) was applied to test statistical differences
across the five rat groups. A Bonferroni post hoc test was used to determine differences between lines (*P ⬍ 0.01 significantly different to BN rats). TC, total
cholesterol; HDL-C, HDL cholesterol; LDL-C, LDL cholesterol; VLDL-C, VLDL cholesterol.
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Fig. 3. Glucose tolerance (A) and glucoseinduced 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. †P ⬍ 0.05, statistically significant differences in insulinemia between BN.GK2k
rats and BN, BN.GK2a, 2c, and 2e rats.
7
COMPARATIVE MAPPING OF A RAT DIABETES LOCUS IN CONGENIC STRAINS
NM_173094), hyperpolarization-activated cyclic nucleotidegated 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).
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 WKYHeid), 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 vari-
Table 4. Sequence analysis of Ensa and Hao3 in inbred BN, GK, and WKY strains
Rat Strains
Polymorphisms
Ensa
86485-TCTT-Ins
86760-TTC-Ins
87852-(AAAC)n
89931-G/A
93167-G/A
Hao3
102541-A/G
102021-7A/8A
101956-GAG-Del
101810-C/T
101779-C/T
101030-T/C
100390-G/A
99912-C/A
99843-7G-9G
99733-C/T
99607-G/A
99462-7A/8A
99372-C/T
99136-T/C
90477-C/T
88282-C/A
Description
BN
GK
WKY-Izm-Leic
WKY-Heid
Promoter
Promoter
Promoter
Intron 1
3⬘-UTR
DD
DD
n⫽6
GG
GG
II
II
n⫽5
AA
AA
DD
DD
n⫽6
GG
GG
II
II
n⫽5
AA
AA
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Intron 1
Intron 3
Intron 4
AA
7A
II
CC
CC
TT
GG
CC
7G
CC
GG
7A
CC
TT
CC
CC
GG
8A
DD
TT
TT
CC
AA
CC
9G
TT
AA
8A
TT
CC
TT
CC
GG
8A
DD
TT
TT
CC
AA
CC
9G
TT
AA
8A
TT
CC
TT
CC
AA
7A
II
CC
TT
TT
AA
AA
7G
CC
GG
7A
CC
TT
CC
AA
Polymorphism locations refer to the GenBank sequences NM_021842 (Ensa) and NM_032082 (Hao3). I, insertion (Ins); D, deletion (Del).
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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 ttest. *P ⬍ 0.05, statistically significant differences vs.
BN controls.
8
COMPARATIVE MAPPING OF A RAT DIABETES LOCUS IN CONGENIC STRAINS
ants), and in the 3⬘-UTR (1 variant) of the genes. No sequence
polymorphisms were found in coding regions.
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.
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 GK⫻BN and
GK⫻F344 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 GK⫻BN 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
GK⫻F344 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
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DISCUSSION
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 GK⫻F344 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 GK⫻BN 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 GK⫻BN 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
COMPARATIVE MAPPING OF A RAT DIABETES LOCUS IN CONGENIC STRAINS
ACKNOWLEDGMENTS
We are indebted to Dr. Colin Hetherington for invaluable help in the
implementation of congenic colonies.
GRANTS
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 QLRTPhysiol Genomics • VOL
19 •
1999-00546. S. C. Collins is supported by a Wellcome Prize Studentship. D.
Gauguier holds a Wellcome Senior Fellowship in Basic Biomedical Science.
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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
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proteins involved in metabolism (Hao3, Fatp3, Hmgcs2,
Pmvk) and cellular physiology (Atp1a1, Hcn3, Pik4cb,
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