Download Distinct genetic regulation of progression of diabetes and renal

Physiol Genomics 39: 38–46, 2009.
First published July 7, 2009; doi:10.1152/physiolgenomics.90389.2008.
Distinct genetic regulation of progression of diabetes and renal disease
in the Goto-Kakizaki rat
Marcelo A. Nobrega,1,2* Leah C. Solberg Woods,2,3* Stewart Fleming,4 and Howard J. Jacob1,2
1
Department of Physiology, 2Human and Molecular Genetics Center, and 3Department of Pediatrics, Medical College
of Wisconsin, Milwaukee, Wisconsin; and 4Department of Pathology, University of Dundee, Dundee, United Kingdom
Submitted 5 December 2008; accepted in final form 2 July 2009
animal models; genetic mapping
DIABETIC NEPHROPATHY (DN) is the most common cause for
end-stage renal disease (ESRD) in the United States (18, 43),
accounting for ⬎44% of the new cases of kidney failure
(http://www.diabetes.org/diabetes-statistics/complications.jsp).
While multiple factors, including duration of diabetes, levels of
microalbuminuria, degree of glycemic control, and hypertension, contribute to the progression of DN (4, 8), no single
factor can predict those diabetic patients who will develop
renal disease and those who will not. Type 2 diabetes (T2D)
itself has a strong genetic component, with monozygotic twins
exhibiting from 50% to 60% concordance rates (2, 28). Strong
familial aggregation is also seen for DN (40). Because only a
subset of diabetic patients develop overt proteinuria and even
fewer progress to ESRD, it is likely that DN is regulated, at
least partially, by genetic pathways independent from those
involved in T2D onset and progression.
* M. A. Nobrega and L. C. Solberg Woods contributed equally to this work.
Addresses for reprint requests and other correspondence: M. A. Nobrega,
Dept. of Human Genetics, Univ. of Chicago, Chicago, IL 60637 (e-mail:
[email protected]); H. J. Jacob, Dept. of Physiology, Medical Coll. of
Wisconsin, Milwaukee, WI 53266 (e-mail: [email protected]).
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T2D and DN are complex disorders that progress over time.
Evidence exists to suggest that the genetic pathways involved
in the onset of these disorders may differ from those involved
in disease progression, as elegantly shown several years ago
for rheumatoid arthritis (44). For T2D, loci have been identified specifically for age of onset (48) or early onset (45), and
one study found that loci for onset of diabetes differ from those
involved in progression of diabetes (7). Furthermore, when age
of onset of T2D is taken into account, significance for loci
associated with T2D can increase (25). Additional studies
looking at the genetics of DN have found genes involved in
overt proteinuria separate from those involved in decreased
kidney function (review in Ref. 34). Despite these examples,
however, there is a general lack of information on the genetic
differences involved in disease onset versus disease progression. The notion of complex diseases having developmental
components, potentially determined by different genetic factors, is in sharp contrast to most experimental designs in human
linkage and association studies, because most studies include
individuals from a wide range of ages or disease stages in a
common group. Under the hypothesis of independent genetic
components determining a trait at different ages or developmental stages, this pooling of individuals of different age
groups may blunt the ability to detect linkage (24).
The use of an animal model can facilitate the search for
genetic components involved in the onset and progression of
diabetes and renal disease. The Goto-Kakizaki (GK) rat and
related substrains are models of nonobese T2D and some
aspects of renal disease. The strain exhibits glucose intolerance
as early as 2 wk of age (high basal plasma insulin levels) and
elevated plasma glucose levels after the administration of a
glucose load by 4 wk of age (15, 31, 36). Aging GK rats show
zones of scarring in the pancreatic islets that eventually outnumber preserved islets (35). The GK histological changes in
the kidney include thickening of the glomerular basement
membranes, mild mesangial matrix expansion, and glomerular
hypertrophy (33, 37, 38). Some substrains exhibit increased
urinary protein and albumin either naturally (37, 38) or as a
result of induced hypertension (17).
Recently we developed a new rat model of DN, using GK
and fawn-hooded (FHH) rats (29). T2DN rats are ⬎97%
genetically identical to GK rats and spontaneously develop
diabetes with timing and intensity similar to those in GK rats.
Moreover, these rats also display significant proteinuria as
early as 3 mo of age and progress to ESRD by 18 mo of age.
Together with overt proteinuria, T2DN rats also develop focal
glomerulosclerosis, mesangial matrix expansion, and thickening of basement membranes at 3 mo of age. With time, these
renal lesions progress to diffuse global glomerulosclerosis with
nodular formation and arteriolar hyalinosis by 18 mo of age,
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Nobrega MA, Solberg Woods LC, Fleming S, Jacob HJ. Distinct genetic regulation of progression of diabetes and renal disease in
the Goto-Kakizaki rat. Physiol Genomics 39: 38 –46, 2009. First
published July 7, 2009; doi:10.1152/physiolgenomics.90389.2008.—
Goto-Kakizaki (GK) rats develop early-onset type 2 diabetes (T2D)
symptoms, with signs of diabetic nephropathy becoming apparent
with aging. To determine whether T2D and renal disease share similar
genetic architecture, we ran a quantitative trait locus (QTL) analysis
in the F2 progeny of a GK ⫻ Brown Norway (BN) rat cross. Further,
to determine whether genetic components change over time, we ran
the QTL analysis on phenotypes collected longitudinally, at 3, 6, 9 and
12 mo, from the same animals. We confirmed three chromosomal
regions that are linked to early diabetes phenotypes (chromosomes 1,
5, and 10) and a single region involved in the late progression of the
disorder (chromosome 4). A single region was identified for the onset
of the renal phenotype proteinuria (chromosome 5). This region
overlaps the diabetic QTL, although it is not certain whether similar
genes are involved in both phenotypes. A second QTL linked to the
progression of the renal phenotype was found on chromosome 7.
Linkage for triglyceride and cholesterol levels were also identified
(chromosomes 7 and 8, respectively). These results demonstrate that,
in general, different genetic components control diabetic and renal
phenotypes in a diabetic nephropathy model. Furthermore, these
results demonstrate that, over time, different genetic components are
involved in progression of disease from those that were involved in
disease onset. This observation would suggest that clinical studies
collecting participants over a wide age distribution may be diluting
genetic effects and reducing power to detect true effects.
DISTINCT LOCI CONTROL SAME DISEASE AT DIFFERENT STAGES
METHODS
Animals
A single male GK rat obtained from the Karolinska Institutet
(Stockholm, Sweden), a kind gift of Dr. Holger Luthman, was initially
mated with two BN female rats from the Medical College of Wisconsin. This single GK rat is the same animal used to develop the T2DN
rat described above (29).
Animals from the F1 generation were brother-sister mated to obtain
204 GK ⫻ BN F2 intercross male rats. The F2 generation animals
obtained from this cross were subjected to a renal disease and diabetes
characterization protocol as described below. Animals were maintained under a 12:12-h light-dark cycle and fed a standard Purina rat
diet containing 1.0% NaCl by weight ad libitum. All protocols were
approved by the Institutional Animal Care and Use Committee at the
Medical College of Wisconsin.
Phenotypic Characterization
GK ⫻ BN F2 intercross rats were characterized for renal and
diabetic phenotypes at 3, 6, 9, and 12 mo of age. At each time point
animals were initially placed in metabolic cages for 24-h determination of proteinuria. Intraperitoneal glucose tolerance test (IPGTT) was
also administered as described below. In addition, a plasma lipid
profile consisting of plasma total cholesterol and triglycerides was
collected at 6 and 12 mo of age in these rats.
Proteinuria. Urine samples were collected while the animals were
housed in Nalgene metabolic cages containing a conical separation
device for feces and urine. The animals were allowed to adapt to the
metabolic cage for 24 h. Urine was then collected during the consecutive 24-h period. Total protein concentration in the urine was
determined colorimetrically by the Bradford method (Bio-Rad, Hercules, CA) (3).
IPGTT. Rats were initially subjected to two or three training
periods. Each training period consisted of being placed in a restrainer
for a 4- to 5-h period. Before the IPGTT, rats were fasted for 12–18
h. During the IPGTT, rats were restrained and basal fasting glucose
was assayed from a tail vein blood sample. Animals were then
injected intraperitoneally with 1 g/kg body wt of a 2.8 M glucose
solution. Tail blood samples (⬃10 ␮l) were collected at 30, 60, 90,
and 120 min after the glucose challenge. Glycemia was measured with
reagent strips read in a glucose meter (Bayer, Elkhart, IN). The area
under the curve (AUC) for glycemia was calculated by the summation
of the four individual areas in the glycemic profile, each representing
a 30-min segment of the IPGTT.
Determination of lipid profiles. Rats were fasted for 12–18 h. While
rats were under light anesthesia (Methoxyfluorane), we collected
500 –700 ␮l of blood from the tail. Total cholesterol and triglycerides
were determined with kits from Sigma Diagnostics (St. Louis, MO).
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Kidney histology. At the time of euthanasia, the right kidney was
removed and placed in 10% formalin, followed by paraffin blocking.
Four-micrometer sections were stained with periodic acid-Schiff and
studied by routine light microscopy for analysis of patterns of injury
(i.e., vascular sclerosis, interstitial fibrosis) and degree of glomerular
sclerosis and mesangial expansion. Sclerosis was defined as collapse
and/or obliteration of the glomerular capillary tuft, accompanied by
hyaline material, increase of matrix, and/or adhesion of the tuft to
Bowman’s capsule. Lesions in individual glomeruli were scored from
0 to 4⫹, with 0 being normal, 1⫹ up to 25% sclerosis of the tuft, 2⫹
up to 50% sclerosis, 3⫹ up to 75% sclerosis, and 4⫹ more than 75%
of the tuft sclerosis. A total of 30 –35 glomeruli per kidney were
analyzed, and an average score (sclerosis index) was calculated. A
detailed description of the procedure is reported elsewhere (29).
Genetic Analysis
A genomewide scan was carried out in 204 GK ⫻ BN F2 animals.
A total of 183 microsatellite markers, polymorphic between GK and
BN rats, were selected from the SHR ⫻ BN V.7 rat genetic map
(http://rgd.mcw.edu/GENOMESCANNER). Genotypes were assayed
by PCR as previously described (27). The genotypes obtained were
used to construct a genetic map with the MAPMAKER/ExP computer
package.
Before analysis, phenotypes were transformed (logarithm, natural
logarithm, or square root) to fit a normal distribution. Nonparametric
statistics were used for phenotypes that failed to follow a normal
distribution pattern after three transformation attempts. Correlation
between traits was determined by simple regression analysis.
The MAPMAKER/QTL computer package was used to carry out
linkage analysis in this data set. The threshold for suggestive linkage
was set at ␣ ⫽ 0.016, representing a logarithm of odds (LOD) score
of 2.8, and the threshold for significant linkage was set at ␣ ⫽ 0.0005,
representing a LOD score of 4.3 (23). The particular genetic mode of
inheritance fitting each quantitative trait locus (QTL) was also determined with MAPMAKER/QTL. Further analysis to detect possible
allelic interactions between QTLs controlling the same trait was
performed by plotting genotype effect plots that incorporated the
effects of two QTLs simultaneously.
RESULTS
Longitudinal Linkage to Diabetes
Phenotypical values on the parental and F1 rats from 3 to 12
mo of age are illustrated in the supplemental material for this
article and in Ref. 29.1
We identified separate loci for the early stages and late
progression of diabetes (see Fig. 1). At 3 mo of age, three
QTLs were identified for post-glucose injection glycemia at all
time points after the glucose challenge as well as AUC. These
QTLs were altered over time by becoming stronger, disappearing, or changing shape. At 12 mo of age a separate locus on
chromosome 4 (LOD ⫽ 10.0 at D4Rat16), absent at the other
ages (see Fig. 1), with a recessive mode of inheritance was
identified for post-glucose injection glycemia.
We identified a single locus with a recessive mode of
inheritance on chromosome 5 for fasting glycemia at 3 mo of
age with a peak LOD of 4.2 at marker D5Mit10. We also
identified linkage on chromosomes 1, 5, and 10 for postglucose injection glycemia at 30, 60, 90, and 120 min after the
glucose challenge, as well as AUC. Because the 60 min time
point was representative of the phenomena observed at other
1
The online version of this article contains supplemental material.
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closely resembling the renal functional and structural alterations seen in DN (29). This study helped to establish the GK
rat and the T2DN strain derived from it as attractive models of
spontaneous diabetes-associated renal disease.
The goal of this study was to dissect the nature of the
relationship between diabetes and late-onset renal disease and
to determine whether genetic factors determining early stages
of these disorders are independent from those controlling their
progression. To this end, a linkage analysis for glucose intolerance and urinary protein excretion (proteinuria) in the segregating F2 generation of a GK ⫻ Brown Norway (BN) cross
at four different time points (3, 6, 9, and 12 mo of age) was
carried out. The BN rat is a well-characterized strain that has
been shown to be nondiabetic (12) and not to develop any of
the structural lesions or functional renal abnormalities seen in
diabetic rats such as the T2DN model (29).
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40
DISTINCT LOCI CONTROL SAME DISEASE AT DIFFERENT STAGES
post-glucose injection times, for simplicity blood glucose at 60
min after challenge will be used to discuss longitudinal dynamics observed in the QTLs across different ages (3, 6, 9, and
12 mo). Linkage at the chromosome 1 locus (D1Rat75 to
D1Mgh13) decreased from 7.2 at 3 mo of age to 3.1 at 12 mo.
Longitudinal linkage changes were also evident on chromosome 5, although the pattern was more complex. A peak with
a LOD of 4.7 at D5Mgh11 was identified at 3 mo of age. While
this peak disappeared at 6 mo, a biphasic peak mapping to a
similar region emerged at 9 mo of age (LOD of 4.6 at D5Mit4
and LOD of 3.5 at D5Mit10) and remained suggestive at 12 mo
of age. A third QTL mapping on chromosome 10 displayed
suggestive linkage at 3 mo of age (LOD of 3.7 at D10Rat20).
Interestingly, this linkage increased to significant levels at 6
mo of age (LOD of 6.1), then decreased at 9 mo of age (LOD
of 4.3), and was not detected by 12 mo of age. The change in
shapes and the appearance of the locus on chromosome 4
suggests that different QTLs contribute to initiation, maintenance, and progression of this trait.
Genetics of diabetes progression. To evaluate whether susceptibility to early-onset diabetes and future progression were
related, the animals were separated according to their genotypes in the QTLs linked to early stages (chromosome 1, 5, and
10) and progression (chromosome 4) and their resultant phenotypes were studied. Four groups emerged: 1) G/G early-B/B
progression, 2) G/G early-G/G progression, 3) B/B early-G/G
progression, and 4) B/B early-B/B-progression, where G represents the GK allele, B represents the BN allele, “early” refers
to genotypes at D1Rat75, D5Mgh11, or D10Rat20, and “progression” refers to genotype at D4Rat16. It was found that
susceptibility to early development and progression were inPhysiol Genomics • VOL
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dependent in this animal model (see Fig. 2). In other words,
rats homozygous for the GK allele on chromosomes 1, 5, and
10 exhibited increased glycemia relative to rats homozygous
for the BN allele on these chromosomes, and this effect was
independent of the genotype on chromosome 4. The progression of glycemia from 3 to 12 mo showed that the genotype at
D4rat16 is the strongest predictor of the degree of glycemia at
12 mo. Thus G/G early-B/B progression rats, which were
diabetic at 3 mo, did not progress with age, indicating not only
that chromosome 4 is required for progression of diabetes but
also that the progression of the disease in this model is all but
an inexorable event, once the disease is established. G/G
early-G/G progression rats (also diabetic since 3 mo of age)
become significantly more hyperglycemic at 12 mo of age,
indicating that the superimposition of late effects due to the
locus on chromosome 4 worsens the level of diabetes in this
group. Finally, independent of the genotype on chromosomes
1, 5, and 10, at 12 mo of age BN homozygosity on chromosome 4 confers protection against hyperglycemia.
Longitudinal Linkage to Renal Disease
Phenotypical values in parental and F1 rats from 3 to 12 mo
of age are illustrated in the Supplemental Material and in Ref.
29. We identified a QTL on chromosome 5 (D5Rat13) that was
significantly linked to proteinuria at all time points studied (3,
6, 9, and 12 mo). The LOD score of this QTL varied from 6.2
at D5Rat13 at 3 mo to 4.7 at 12 mo (see Fig. 3). At 12 mo of
age, a second QTL, on chromosome 7 (LOD of 5.2 at
D7Mgh6) was linked to proteinuria (see Fig. 3). This locus
appears to be critical for the progression of proteinuria, bewww.physiolgenomics.org
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Fig. 1. Longitudinal linkage for post-glucose injection glycemia in Goto Kakizaki (GK) ⫻ Brown Norway (BN) F2 rats. Logarithm of odds (LOD) plots are
shown for chromosomes 1, 4, 5, and 10.
DISTINCT LOCI CONTROL SAME DISEASE AT DIFFERENT STAGES
41
cause the LOD score at this locus increases to 7.8 when linked
to the change in proteinuria levels from 6 to 12 mo, an index
of disease progression.
Suggestive linkage for structural damage of the kidney
(glomerular and tubular sclerosis) was also identified on chromosomes 5 and 7, in similar locations as the loci for proteinuria
(see Fig. 4).
Plotting the proteinuria levels according to the genotypes at
D5Mgh11 and D7Mgh6 revealed strong epistasis between the
two loci, as shown in Fig. 5A. Rats harboring GK alleles at
D5Mgh11 had moderately or considerably more proteinuria
than those that had BN alleles at the same locus, depending on
the genotype at D7Mgh6. Thus progression of proteinuria only
occurs if the genotype at D7Mgh6 is GK/GK, but for progression to occur at least one GK allele is also required at
D5Mgh11. In addition, the effect plot between these two loci
demonstrates an interaction similar to that found for proteinuria
(Fig. 5B), providing corroborative evidence that these loci are
involved in kidney disease in this rat model. This pattern
contrasts with that seen in diabetes, where the progression
locus on chromosome 4 influenced glycemia independently
from the early-stage diabetes QTLs.
Correlational Analysis of Chromosome 5 Locus
The QTL on chromosome 5 for proteinuria, which displays
an additive mode of inheritance, overlaps with the QTLs for
both fasting and post-glucose injection glycemia described
above, immediately raising the possibility that this might
reflect an interaction between renal disease and T2D in the GK
rat model. Nevertheless, no significant correlation was found
between glycemia and proteinuria in the F2 rats (data not
shown). The analysis of correlation was carried out in subgroups of animals, breaking down the 204 animals into groups
according to genotype at D5Mgh11 (maximum likelihood of
linkage for glycemia) and D5Rat13 (maximum likelihood of
linkage for proteinuria). No evidence of a possible interaction
between these two traits at these loci was observed (data not
shown).
Linkage to dislipidemia. Plasma cholesterol and triglyceride
levels were assayed at 6 and 12 mo of age. Figure 6 shows the
linkage for these blood parameters. The only QTL identified
for triglycerides mapped, at both ages, to chromosome 7 at
D7Mgh6, overlapping with the QTL for proteinuria. At 6 mo of
Physiol Genomics • VOL
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age, the maximum LOD score at this locus was 7.1, and at 12
mo of age it was 6.4.
Plasma cholesterol levels mapped to chromosomes 1 and 8.
At 6 mo, the QTL on chromosome 1 had a suggestive LOD
score of 2.9 at D1Pas1. On chromosome 8, the LOD score was
2.4 at D8Rat47. At 12 mo of age the QTL on chromosome 1
disappeared, while the LOD score on chromosome 8 increased
to 4.1.
DISCUSSION
Through genomewide analysis of a GK ⫻ BN F2 intercross,
we have identified several loci involved in glucose tolerance
and urinary proteinuria. Over time, loci for both phenotypes
change, demonstrating that separate genetic mechanisms are
involved in disease onset versus disease progression. These
results highlight the importance of taking into account the stage
of disease in genetic studies of complex traits. To our knowledge, this is the first study to look at the influence of genetics
on the progression of diabetes and renal disease in a rat model
of DN.
We confirmed three loci involved in the presence of glucose
tolerance (chromosomes 1, 5, and 10) and one locus for the
onset of proteinuria (chromosome 5). In addition, we identified
one locus involved in the progression of T2D (chromosome 4)
and a locus involved in the progression of urinary proteinuria
(chromosome 7). While loci for both glycemia and proteinuria
were found on chromosome 5, no correlation was found
between these traits, suggesting that separate genetic mechanisms are involved in these phenotypes in this rat model.
The early-stage diabetes QTLs (chromosomes 1, 5, and 10)
decreased in significance over time, such that they were no
longer present when the animals were 12 mo old. Each of these
three QTLs has previously been identified in other F2 intercrosses. The chromosome 1 locus was the most significant and
has been identified previously by multiple investigators using
the GK rat (10, 12) and other rat models of T2D (19, 47).
Recently, congenic and expression studies have shown that
multiple loci within this region play a role in glycemic control
(14, 46). Another study in GK substitution congenic rats found
separate loci within the chromosome 1 region at 3 versus 6 mo
(6). This QTL also coincides with age-of-onset QTL identified
in the human population (7), as well as several other loci
identified in human linkage and association studies for T2D
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Fig. 2. Longitudinal genotype ⫻ phenotype relationships determining progression of glycemic levels in GK ⫻ BN F2 rats. Groups are based on pairwise
comparisons contrasting the impact of genotypes on loci mapping to early detection of hyperglycemia at D1Rat75 on chromosome 1 (A), D5Mgh11 on
chromosome 5 (B), and D10Rat20 on chromosome 10 (C) and genotype on the locus mapping to hyperglycemia progression (D4Rat16 on chr. 4). *Different
from group matched at 3 mo; #different from groups with BN genotypes at D4Rat16 at 12 mo; ␾different from groups with GK genotypes at D1Rat75 at 3 mo
of age (different ⫽ P ⬍ 0.05). Number of rats within each group ranged from 10 to 19.
42
DISTINCT LOCI CONTROL SAME DISEASE AT DIFFERENT STAGES
Fig. 3. Longitudinal linkage for proteinuria in GK ⫻ BN F2 rats. A: linkage
to chromosome 5. B: linkage to chromosome 7. 6 –12 mo, Ratio of proteinuria
at 6 mo to proteinuria at 12 mo.
(13, 26, 49). In addition, several genes identified in recent
genomewide association studies reside in this region (see Ref.
9). Specifically, TCF7L2, whose introns 3 and 4 have repeatedly shown the strongest association with the risk of developing T2D in humans (9, 41), maps within this QTL. Nevertheless, a recent study in congenic strains from GK rats excluded
Tcf7l2 from the minimal critical region linked to diabetes (14).
However, SORCS1, a gene recently identified for T2D in the
mouse (5), resides in one of the minimal congenics (14) and
therefore may be a candidate gene in the GK rat. Both the
chromosome 5 and 10 loci have also previously been identified
for glucose tolerance in the GK rat (10, 12), while the chromosome 5 locus has also been found in a cross using the
diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rat
(47). Interestingly, CDKN2A/B, recently identified by human
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Fig. 4. Linkage for structural damage in GK ⫻ BN F2 at 12 mo of age.
Glomerular sclerosis and tubular sclerosis represent scores obtained by multiplying the average injury index per kidney by the number of glomeruli or
tubules injured. A: linkage at chromosome 5. B: linkage at chromosome 7.
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genomewide association studies (41), resides within this chromosome 5 locus.
Only one locus (chromosome 4) was identified for glucose
intolerance when the animals were 12 mo old, suggesting that
this locus is involved in the progression of a decline in
glycemic control. Another diabetes-related QTL was identified
previously in a GK ⫻ BN cross, linked to plasma insulin
levels, but not glucose levels, in 4-mo old rats (12). This QTL
does not overlap with the one that we found linked to glycemic
levels at 12 mo. This diabetes progression QTL that we
uncovered has not previously been identified, likely because a
longitudinal study design was not employed, and appears to act
independently from the three early-stage diabetes QTLs. A GK
allele at this locus results in a worsening of glycemic control
even if the alleles at the early-stage QTLs (on chromosome 1,
5, or 10) are from the BN strain. In fact, animals with a GK
DISTINCT LOCI CONTROL SAME DISEASE AT DIFFERENT STAGES
43
Fig. 5. Longitudinal genotype ⫻ phenotype relationships determining progression of renal disease in GK ⫻ BN F2 rats. A: proteinuria. BB, BN/BN; BG,
BN/GK; GG, GK/GK. B: glomerular sclerosis. Genotypes at D5Mgh11 and
D7Mgh6 were used as references to calculate average phenotypic values.
*Different from groups with same genotype at D7Mgh6.
allele at chromosome 4 and a BN allele at one of the earlystage loci achieve the same degree of hyperglycemia at 12 mo
of age as those animals that carry a GK allele at both chromosome 4 and any one of the early-stage QTLs. Interestingly, a
decline in glycemic control does not progress over time in
animals that have a BN allele at the chromosome 4 locus, even
in animals that carry a GK allele at one of the early-stage QTLs
and therefore initially exhibit hyperglycemia. This important
locus, which is involved in determining disease prognosis,
would not have been identified if we had not studied the
animals at 12 mo of age, emphasizing the importance of timing
in genetic studies of complex traits.
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Fig. 6. Linkage for plasma lipid parameters. A: plasma cholesterol levels
mapping to chromosome 8. Maximum LOD score was calculated at D8Rat47.
B: plasma triglyceride levels at 6 and 12 mo of age mapping to chromosome
7. Linkage to proteinuria is shown to denote the overlapping of the quantitative
trait loci.
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We identified a QTL for proteinuria on chromosome 5 at a
location similar to the locus identified on this chromosome for
glucose tolerance. However, no correlation was found between
glycemia and proteinuria in the F2 generation, suggesting that
similar genetic mechanisms are not involved in these traits in
the GK rat. Interestingly, genes identified in recent genomewide association studies for T2D (e.g., TCF7L2, HHEX) differ
from those recently identified for DN (e.g., ELMO1 and
CNDP1) (16, 50), supporting a separate genetic basis for these
two traits.
In addition to the chromosome 5 locus for proteinuria, a
second locus was identified for this trait on chromosome 7.
The chromosome 7 locus was only found when the animals
were 12 mo old, suggesting that it is involved in the
44
DISTINCT LOCI CONTROL SAME DISEASE AT DIFFERENT STAGES
ACKNOWLEDGMENTS
The authors are thankful to Masahide Shiozawa and other Jacob lab
members for technical assistance and to Nancy Schick for help in writing the
manuscript.
H. J. Jacob and M. A. Nobrega designed the study. M. A. Nobrega carried
out the experiments. M. A. Nobrega and H. J. Jacob analyzed the data. S.
Fleming scored the renal ultrastructural abnormalities. L. C. Solberg Woods
wrote the manuscript with the assistance of M. A. Nobrega and H. J. Jacob.
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progression of proteinuria. In contrast to the progression
QTL for glucose tolerance, the proteinuria QTL on chromosome 7 acts in conjunction with the onset QTL on chromosome 5 such that high levels of proteinuria are only found if
both the chromosome 5 and 7 alleles are from the GK strain.
While the chromosome 5 locus has not previously been
identified in other F2 intercrosses, the chromosome 7 locus
has previously been identified for progression of albuminuria in the Munich Wister Fromter (MWF) rat (39), while it
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A QTL on chromosome 7 for high triglyceride levels overlaps with the progression QTL for proteinuria, suggesting a
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Because high cholesterol and triglyceride plasma levels have
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We have identified independent loci involved in onset and
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the early-stage loci for glucose intolerance were identified
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and animals. Clustering of participants across a wide range of
ages in studies using humans is likely to reduce power to detect
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