Hereditary pancreatitis model WBN/Kob rat strain has a unique haplotype in the Pdwk1 region on chromosome 7.

Abstract

The WBN/Kob rat strain is a hereditary animal model of chronic pancreatitis and diabetes mellitus. The major WBN/Kob loci for pancreatitis (Pdwk1 and Pdwk2) are located on chromosomes 7 and X, respectively. In this study, polymorphisms were sought for candidate genes in the Pdwk1 and Pdwk2 regions. Nucleotide polymorphisms were found in 14 candidate genes examined in the Pdwk1 region. These polymorphisms were not associated with functional changes, and hence were unlikely to be a cause of pancreatitis. Seven nucleotide polymorphisms in three candidate genes, Rac2, Grap2, and Xpnpep3, located within a 3.3-Mb region were not found in 14 other inbred rat strains. These results suggest that WBN/Kob has a unique haplotype block in the chromosomal region contatining Pdwk1.

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Exp. Anim. 58(4), 409–413, 2009
Hereditary Pancreatitis Model WBN/Kob Rat Strain Has a
Unique Haplotype in the Pdwk1 Region on Chromosome 7
Masayuki MORI, Xiaoying FU, Lei CHEN, Guohong ZHANG, and Keiichi HIGUCHI
Department of Aging Biology, Institute on Aging and Adaptation, Shinshu University Graduate School of
Medicine, 3–1–1 Asahi, Matsumoto 390-8621, Japan
Abstract: The WBN/Kob rat strain is a hereditary animal model of chronic pancreatitis and
diabetes mellitus. The major WBN/Kob loci for pancreatitis (Pdwk1 and Pdwk2) are located
on chromosomes 7 and X, respectively. In this study, polymorphisms were sought for candidate
genes in the Pdwk1 and Pdwk2 regions. Nucleotide polymorphisms were found in 14 candidate
genes examined in the Pdwk1 region. These polymorphisms were not associated with
functional changes, and hence were unlikely to be a cause of pancreatitis. Seven nucleotide
polymorphisms in three candidate genes, Rac2, Grap2, and Xpnpep3, located within a 3.3-Mb
region were not found in 14 other inbred rat strains. These results suggest that WBN/Kob
has a unique haplotype block in the chromosomal region contatining Pdwk1.
Key words: gene polymorphism, pancreatitis, WBN/Kob
(Received 27 November 2008 / Accepted 27 January 2009)
Address corresponding: M. Mori, Department of Aging Biology, Institute on Aging and Adaptation, Shinshu University Graduate School of
Medicine, 3–1–1 Asahi, Matsumoto 390-8621, Japan
Chronic pancreatitis (CP) is a continuing or recurring
inflammatory disease of the pancreas that typically
causes pain and leads to irreversible morphological and
functional damage [12]. The etiology of most CP cases
is still unclear. Male WBN/Kob rats represent an animal
model of CP and diabetes mellitus [3, 6, 10]. The ab-
normality initiates at approximately 12 weeks of age
with marked fibrosis around the pancreatic ducts and
blood vessels. Then fibrous tissue gradually and exten-
sively invades the pancreas. The islets are also affected
by fibrotic degeneration, leading to an obvious decrease
in islet number and size. In addition, inflammatory cells
infiltrate and damage tissue around the islets and among
adjacent acinar cells. This endocrine-exocrine dysfunc-
tion eventually leads to diabetes mellitus at 60–90 weeks
of age. The processes leading to pancreatitis in WBN/
Kob have been extensively studied. These studies have
revealed the involvement of sex hormones [4, 7], imbal-
ances of prolyl hydroxylase and collagenase [2], and
autoimmunity [8] in pancreatitis. However, the etiology
of pancreatitis in WBN/Kob rats is still unknown. Ge-
netic factors are strongly suspected. Thus, a genetic
approach should help to elucidate the fundamental cause
of WBN/Kob pancreatitis and diabetes mellitus. Chro-
mosomal mapping of WBN/Kob rats for pancreatitis and
diabetes mellitus genes was performed by breeding F2
hybrid progeny between WBN/Kob and BN [11], and
peaks of likely ratio statistical scores for linkage to pan-
creatitis were observed on two chromosomes. One was
a broad peak between D7Rat97 (70.3 cM) and D7Rat4
(80.5 cM) on chromosome 7, and the other was at
DXRat103 (37.6 cM) on chromosome X. These loci
were designated Pdwk1 and Pdwk2 (pancreatitis and
diabetes mellitus in WBN/Kob locus 1 and 2), respec-
tively. Identification of Pdwk1 and Pdwk2 genes should
shed new light on pancreatitis mechanisms, and to this
—Note—

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M. MOrI, ET AL.
end, we examined polymorphisms in candidate genes
located in the WBN/Kob Pdwk1 and Pdwk2 regions.
Specific pathogen-free 21-week-old male WBN/Kob
and BN/SsN rats were purchased from Japan SLC
(Hamamatsu, Japan). After euthanasia with ether inha-
lation, the pancreas and spleen were removed. WBN/
Kob rats were confirmed to have pancreatitis, while BN
rats had normal pancreata (data not shown). Genomic
DNA and mrNA were isolated by standard procedures.
All experimental procedures were carried out in accor-
dance with the regulations for Animal Experimentation
of Shinshu University.
Candidates with the following criteria were selected
from genes located around D7Rat97, D7Rat4, and
DXRat103 marker loci: (i) genes reported to be associ-
ated with pancreatitis in humans or mice; (ii) genes as-
sociated with pancreatic development or function; (iii)
genes associated with leukocyte development or function.
Fourteen candidate genes on chromosome 7, eukaryotic
transcription initiation factor 3 (Eif3s3), melanoma-de-
rived leucine zipper, extra-nuclear factor (Mlze), soma-
tostatin receptor 3 (Sstr3), rAS-related C3 botulinum
substrate 2 (Rac2), GrB2-related adaptor protein 2
(Grap2), X-prolyl aminopeptidase 3 (Xpnpep3), tumor
necrosis factor receptor superfamily, member 13c (Tnfrs-
f13c), NFAT activating protein with ITAM motif 1
(Nfam1), synaptotagmin X (Syt10), FK506 binding protein
11 (Fkbp11), elastase 1 (Ela1), type II keratin Kb20
(Kb20), keratin complex 2, basic, gene 7 (Krt2-7), and
keratin complex 2, basic, gene 8 (Krt2-8), were examined
(Fig. 1). Seven candidate genes on chromosome X, gas-
trin releasing peptide receptor (Grpr), X-prolyl amino-
peptidase 2 (Xpnpep2), SAM and SH3 domain containing
3 (Sash3), E74-like factor 4 (Elf4), G protein-coupled
receptor 119 (Gpr119), bombesin-like receptor 3 (Brs3),
and CD40 ligand (Cd40lg) were also examined. Primers
for PCr amplification and sequencing were designed
based on the rat genome sequence (rat genome assembly
version 3.4 (rGSCv3.4)), so that approximately 500–1,000
bp of DNA fragments containing exon(s) and partial intron
sequences could be amplified from genomic DNA. PCr
products were purified with an UltraClean PCr Clean-up
DNA Purification Kit (MO BIO Laboratories, Carlsbad,
USA). An aliquot was sequenced by using a BigDye Ter-
minator Cycle Sequencing ready reaction Kit (Applied
Biosystems, Foster City, USA), according to the manu-
facturer’s instructions, and analyzed on an ABI PrISM
Genetic analyzer 3130 (Applied Biosystems).
First, nucleotide sequences of candidate genes were
compared between the WBN/Kob and BN strains. No
polymorphisms were found for seven genes on chromo-
some X. The reason for the absence of polymorphisms
Fig. 1. Maps of candidate genes on chromosomes 7 and X. Arrowheads and circles represent candidate genes and mark-
er loci, respectively. Numbers in parenthesis are exon counts for the genes (up) and for the identified SNPs. A
hatched box indicates the 3.3-Mb region of WBN/Kob-specific haplotype.

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SNP NALYSIS OF WBN/KOB rATS
is unclear. One possibility is that WBN/Kob and BN
share an identical haplotype in this chromosomal region.
In contrast, one hundred and nine nucleotide alterations
were found in fourteen genes within an approximately
53-Mb segment of the Pdwk1 region on chromosome 7.
Sixteen of these were found in Ensembl (http://www.
ensembl.org/). Others were considered to be newly iden-
tified polymorphisms, and registered in the NCBI SNP
database (http://www.ncbi.nlm.nih.gov/SNP/) with a
handle name of DABSUGSM. Other rat strains were
then genotyped for the polymorphisms to examine if the
polymorphisms were unique to WBN/Kob. The WBN/
Kob strain originated from a Wistar rat colony of the
Institute of Experimental Gerontology in Basel and has
been inbred at the Institute of Pathology, University of
Bonn since 1961 [3]. To our knowledge, both the origin
and current state of the Wistar colony are uncertain. Ac-
cording to phylogenetic cluster analysis of rat strains
with single nucleotide polymorphisms (SNPs), WBN/
Kob has a close phylogenetic relationship to the clusters
of BD and ACI, but not to the cluster of WKY (Wistar)
[supplemental information to 9]. Thus, we examined
BDIX (from the BD cluster), ACI, DA, DONrYU, and
SHr/Izm rats. When the polymorphic nucleotides of
the WBN/Kob allele were not found in these strains, IS,
TM, NIG-III, LEW, WM, PVG/c, LOU/M, KHr, and
KMI strains were also examined. Genomic DNA samples
of BDIX, ACI, WM/Ms, PVG/c, LOU/M, IS, DONrYU,
DA, TM, LEW, and NIG-III were kindly donated by Dr.
T Nishikawa of the National Institute of radiological
Sciences, Japan. Genomic DNA samples of KHr and
KMI strains were provided by the Institute of Labora-
tory Animals, Graduate School of Medicine, Kyoto
University, through the National Bioresource Project
(NBrP) of the MEXT, Japan. Most polymorphic nucle-
otides were found in one or more of rat strains. How-
ever, five nucleotide changes in three genes, Rac2,
Grap2, and Xpnpep3 were not found in any of these
strains, suggesting that these changes are specific to the
WBN/Kob strain. G to A nucleotide substitutions in exon
2 of the Rac2 (rGSCv3.4 position 116,530,532) and in
intron 4 of Grap2 (position 118,928,554) were unique
to WBN/Kob rats. Also, WBN/Kob rats had unique
repeat numbers of (TG)n and (AG)n microsatellites in
introns 1 and 2 of Grap2 (positions 118,906,980–
118,907,009 and 118,920,663–118,920,706). Three
polymorphisms in Xpnpep3 on WBN/Kob rats were not
found in the other rat strains examined. One polymor-
phism was a microsatellite in intron 4 (position
119,756,405–119,756,470) in which WBN/Kob unique-
ly had five repeats of GCCTCT, while the other strains
had ten or eleven repeats. The second polymorphism
was a G to A nucleotide substitution at 259 bases up-
stream of the initiating methionine codon (position
119,729,247). The third polymorphism was a G to T
nucleotide substitution in exon 2 (position 119,743,296),
leading to a substitution of a histidine for a glutamine at
codon 28 (Q28H; Fig. 2). In addition to this missense
nucleotide substitution, there was an A to G substitution
in exon 1 between BN and WBN/Kob strains, leading to
a substitution of a threonine for an alanine at codon 15
(A15T). Screening of the other rat strains revealed that
15A is unique to the BN strain. Combining the two
amino acid substitutions, there were three XPNPEP3
isoforms in the rat strains (Fig. 2).
X-prolyl aminopeptidase specifically catalyzes the
removal of any unsubstituted N-terminal amino acid
adjacent to a penultimate proline residue (N-terminal
imido bond). The fibrotic area in the WBN/Kob rat pan-
creas is mainly composed of type-III collagen [2]. N-
terminal imido bonds are common to several collagen
degradation products. Thus, it is possible that a mutation
in the Xpnpep3 gene disturbs collagen metabolism, even-
tually leading to fibrosis. Both of the amino acid sub-
stitutions in WBN/Kob rats are located in the putative
mitochondrial localization signal sequence of XPNPEP3
[1]. The Q28H substitution in particular is located at
three amino acids upstream of the predicted cleavage
site of the signal sequence. The influence of the substi-
tutions on mitochondrial localization signal function was
then examined. DNA fragments containing the coding
sequence of rat Xpnpep3 were PCr-amplified from 1st-
strand pancreas cDNA of SCr, BN, and WBN/Kob rats,
and were inserted into the plasmid vector pEGFP-N3
(Clontech, Palo Alto, USA) so that the XPNPEP3 protein
was expressed as a green fluorescent protein (GFP) fu-
sion protein. Cultured COS cells were transfected with
expression plasmids. Two days after transfection, cells
were fixed with 4% formalin, and observed by fluores-
cence microscopy. Mitochondria were stained with

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M. MOrI, ET AL.
MitoTracker (Molecular Probes, Eugene, USA). All
three isoforms transiently expressed in COS cells were
localized in mitochondria (Fig. 2). Furthermore, all
isoforms had identical molecular weight by western blot
analysis with anti-GFP antibody (rockland, Gilberts-
ville, USA). These results indicate that these amino acid
substitutions do not infl uence mitochondrial localization
of XPNPEP3 and subsequent removal of the signal pep-
tide. Also, rT-PCr analysis of the spleen revealed the
same transcript level with the expected size for Xpnpep3,
as well as for Rac2, and Grap2 for WBN/Kob and BN
rats (Fig. 2). Thus, the alterations in the three genes were
indicated to be functionally neutral.
In general, two conditions must be fulfi lled in order
to establish an etiological link between nucleotide al-
teration in the candidate gene to the disease. One is to
demonstrate that the alteration is found only in the strain
with the disease (strain-specifi city). The other is to dem-
Fig. 2. Mitochondrial localization of three XPNPEP3 isoforms. (A) Comparison of nucle-
otide and deduced amino acid sequences for three Xpnpep3 alleles derived from
ACI, BN, and WBN/Kob rat strains. Nucleotide and amino acid substitutions are
indicated in bold. Position of a cleavage site predicted by the TargetP program
[http://www.cbs.dtu.dk/services/TargetP/] is indicated by an arrow. (B) COS cells
expressing XPNPEP3 isoforms derived from ACI and WBN/Kob rat strains. (C)
Western blot analysis of three XPNPEP3 isoforms expressed in COS cells. (D) rT-
PCr analysis of Rac2, Grap2, and Xpnpep3 transcripts for WBN/Kob and BN.

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SNP NALYSIS OF WBN/KOB rATS
onstrate that the alteration functionally changes the gene.
In this context, the nucleotide changes reported above
are unlikely to be a cause of pancreatitis of WBN/Kob
rats. However, if a threshold exists for the pathogenesis
in WBN/Kob pancreatitis, one gene may not be enough
to cause a functional difference, but two or more genes
may have a coordinated action, and result in an altered
function. Indeed, it was revealed in a previous study
that the two loci on chromosomes 7 (Pdwk1) and X
(Pdwk2) were genetically associated with WBN/Kob
pancreatitis [11]. If complex interactions of the genes
underlie WBN/Kob pancreatitis, a certain type of poly-
morphism which is specific to the WBN/Kob strain
would not have been found by the molecular genetic
strategy employed in this study. One standard way to
confirm an effect of each locus and precisely define the
chromosomal region is to breed congenic strains in which
the Pdwk1 or Pdwk2 regions are separately introduced
into the background of a normal rat strain. Also, there
remains the possibility that a nucleotide alteration for
pancreatitis exists in introns of the candidate genes. In
this study, only exons and portions of introns were ex-
amined. A large part of introns awaits further study. In
fact, recent genome-wide association studies have re-
vealed many intronic SNPs associated with common
diseases in humans.
Further continuation of the candidate gene approach
will hopefully lead to the identification of Pdwk1 and
Pdwk2. Notably, five WBN/Kob-specific nucleotide al-
terations were found in three genes located within a 3.3-
Mb region. The results obtained in this study suggest that
a distinct haplotype is preserved in this relatively small
chromosomal segment of WBN/Kob. It is unlikely that
these nucleotide changes occurred during or after the
establishment of the WBN/Kob strain as a spontaneous
pancreatitis model. rather, it would be reasonable to
assume that the chromosomal segment with a unique
haplotype was introduced to the WBN/Kob strain from
its ancestry. It is premature to conclude, but tempting to
speculate that Pdwk1 might be located within the unique
haplotype block. Perhaps, Pdwk1 itself has a peculiar
nucleotide alteration(s). This may be why the WBN/Kob
strain uniquely develops hereditary pancreatitis. It was
impossible to precisely define the chromosomal region
of the particular haplotype because only portions of se-
lected genes were examined in this study. According to
Ensembl, approximately 100 genes are in the region be-
tween the Rac2 and Xpnpep3 loci. In this study, candidate
genes with a reported association with pancreatitis in
humans or mice, or genes associated with pancreas de-
velopment or function, or genes associated with leukocyte
development or function, were examined. It is possible
that Pdwk1 does not have any of the functional categories
of the candidate genes that were examined. If this is the
case, identification of the gene(s) should shed new light
on the mechanisms of pancreatitis.
Acknowledgment(s)
We are thankful to the National Bioresource Project
for the rat in Japan (http://www.anim.med.kyoto-u.ac.
jp/NBr/) for providing rat strains. We also thank Dr T.
Nishikawa at the National Institute of radiological Sci-
ences, for providing genomic DNA samples from labora-
tory rat strains. This work was supported in part by a
Grant-in-Aid for Scientific research (No. 19300145)
from the Ministry of Education, Culture, Sports, Science
and Technology, Japan.
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