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From: TSS ()
Subject: Association of a Bovine Prion Gene Haplotype with Atypical BSE
Date: March 21, 2008 at 10:54 am PST

PLoS ONE. 2008; 3(3): e1830.
Published online 2008 March 19. doi: 10.1371/journal.pone.0001830. PMCID:

Copyright This is an open-access article distributed under the terms of the
Creative Commons Public Domain declaration which stipulates that, once
placed in the public domain, this work may be freely reproduced,
distributed, transmitted, modified, built upon, or otherwise used by anyone
for any lawful purpose.

Association of a Bovine Prion Gene Haplotype with Atypical BSE

Michael L. Clawson,1* Juergen A. Richt,2 Thierry Baron,3 Anne-Gaëlle
Biacabe,3 Stefanie Czub,4 Michael P. Heaton,1 Timothy P. L. Smith,1 and
William W. Laegreid1¤
1United States Department of Agriculture (USDA), Agricultural Research
Service (ARS), U.S. Meat Animal Research Center (USMARC), Clay Center,
Nebraska, United States of America
2USDA, ARS, National Animal Disease Center, Ames, Iowa, United States of
3Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Unité ATNC,
Lyon, France
4National & OIE BSE Reference Laboratories, Pathology/Virology/Wildlife
Diseases, Animal Diseases Research Institute/Canada, Food Inspection Agency,
Lethbridge/Alberta, Canada
Matthew Baylis, Academic Editor
University of Liverpool, United Kingdom
* E-mail:
Conceived and designed the experiments: MC WL. Performed the experiments: MC
JR TS. Analyzed the data: MC JR WL. Contributed reagents/materials/analysis
tools: TB AB SC JR MH. Wrote the paper: MC. Other: Reviewed the paper: WL TS
¤Current address: Department of Pathobiology, University of Illinois at
Urbana-Champaign, Urbana, Illinois, United States of America

Received December 20, 2007; Accepted February 14, 2008.


Atypical bovine spongiform encephalopathies (BSEs) are recently recognized
prion diseases of cattle. Atypical BSEs are rare; approximately 30 cases
have been identified worldwide. We tested prion gene (PRNP) haplotypes for
an association with atypical BSE.

Methodology/Principle Findings

Haplotype tagging polymorphisms that characterize PRNP haplotypes from the
promoter region through the three prime untranslated region of exon 3 (25.2
kb) were used to determine PRNP haplotypes of six available atypical BSE
cases from Canada, France and the United States. One or two copies of a
distinct PRNP haplotype were identified in five of the six cases
(p=1.3×10-4, two-tailed Fisher's exact test; CI95% 0.263–0.901, difference
between proportions). The haplotype spans a portion of PRNP that includes
part of intron 2, the entire coding region of exon 3 and part of the three
prime untranslated region of exon 3 (13 kb).


This result suggests that a genetic determinant in or near PRNP may
influence susceptibility of cattle to atypical BSE.


Transmissible spongiform encephalopathies (TSEs), or prion diseases, are
infectious, invariably fatal neurodegenerative disorders that occur in
humans, ruminants, cats, and mink [1]. TSEs are unique in their ability to
manifest through acquired, inherited, and sporadic routes [1]. Classical
bovine spongiform encephalopathy (BSE) is an acquired cattle TSE of unknown
origin that spreads through the consumption of meat and bone meal
contaminated with the infectious prion agent [2]. Classical BSE is accepted
as the probable cause of the human TSE variant Creutzfeldt-Jakob Disease
(CJD) [3], [4]. Two BSEs distinct from classical BSE, so called “atypical
BSEs” (H-type and L-type) have recently been identified in Asian, North
American and European cattle [2]. Approximately, 30 atypical BSEs have been
identified worldwide and their etiology is unclear.

Variation in the prion gene (PRNP) correlates with TSE susceptibility in
some mammals including cattle [1], [5]–[7]. The deletion alleles of two
bovine PRNP insertion/deletion polymorphisms, one within the promoter region
and the other in intron 1, associate with classical BSE susceptibility
[5]–[7]. These same alleles do not correlate with atypical BSE
susceptibility [8]. In 2006, a United States atypical BSE case was
identified and subsequently found to have a PRNP nonsynonymous polymorphism
(E211K) that is homologous to the human PRNP E200K polymorphism (observation
by J.A.R). The human K200 allele is a highly-penetrant risk factor for
genetic CJD [9]. To date, the K211 allele has not been observed in other
atypical BSE cases or reported in healthy cattle [10], [11]. Thus, while the
K211 allele may have been a genetic cause for one case of atypical BSE, it
has not accounted for the majority of atypical cases. Consequently, any
association of PRNP alleles with atypical BSE was largely unknown prior to
this study.

PRNP variation in cattle is complex. Bovine PRNP polymorphism alleles
reflect a region of high linkage disequilibrium (LD) from the promoter
through a portion of intron two, and a region of low LD from intron two past
the three prime untranslated region. This genetic architecture is present
across populations of Bos taurus breeds and a similar trend has been
observed in a small sampling of Bos indicus influenced breeds [11]. A set of
19 haplotype tagging polymorphisms (htSNPS) was previously developed that
accounts for the genetic architecture of PRNP and characterizes haplotype
diversity within and across PRNP [11]. In this study, we used the htSNPs to
test PRNP haplotypes for an association with atypical BSE and report the
association of a relatively uncommon PRNP haplotype with atypical BSE.


The 19 PRNP htSNPs were used to determine PRNP haplotypes of six available
atypical BSE cases that originated from Canada, France and the United
States. The haplotypes were phased in previously defined PRNP regions of
high and low LD (Fig. 1A; network 1 spans the high LD region, network 2
spans the low LD region). Additionally, the entire prion protein (PrP)
coding region was sequenced for each of the six atypical BSE cases. None of
the cases contained previously unknown SNP alleles in the PrP coding region
or the K211 allele. However, one or two copies of a distinct haplotype were
identified by haplotype reconstructions in five of the six cases. The
haplotype spans a portion of intron 2, the entire coding region, and a
portion of the 3' UTR of PRNP (13 kb), (haplotype “o”, Fig. 1B and 1C, Table

The frequency of the implicated haplotype in atypical BSE cases was compared
to its frequency in a control group of 114 diverse DNA samples representing
21 breeds of U.S. beef and dairy cattle, since unaffected controls from the
farms where the atypical BSE cases originated are not available, nor are
diversity panels of beef and dairy cattle in Canada and France. However, the
control group of U.S. cattle represents germplasm that is collectively found
in Canada, France, and the United States, and current evidence from the
international bovine HapMap project indicates that diversity within Bos
taurus breeds is similar between countries (personal communication from
T.P.L.S.). Therefore, we used the group of U.S. cattle as a surrogate
control in this study which involves natural occurrences of atypical BSE
cases from three countries on two different continents. The implicated
haplotype was observed in both Bos taurus and Bos indicus individuals in the
control group and had a frequency of 0.050, ten-fold less than the atypical
BSE-cases (frequency=0.50). A Fisher's exact two-tailed test showed a
significant association of the haplotype with atypical BSE (p=1.3×10-4), as
did the difference between proportions (CI95% 0.263–0.901).

This result suggests that a genetic determinant in or near PRNP may
influence susceptibility of cattle to atypical BSE. The causative allele(s)
remains to be identified and probably occurs on the background of the
implicated PRNP haplotype. Complete sequencing of PRNP from atypical BSE
cases and BSE negative controls that both have the implicated haplotype may
reveal PRNP alleles with predictive power for atypical BSE. The implicated
haplotype itself does not effectively predict atypical BSE because of its
frequency in healthy cattle. However, our results combined with the
discovery of the PRNP K211 allele suggest that atypical BSE may be managed
through the identification of cattle with known genetic risk factors for the
disease and their removal from livestock populations.


Composition of atypical BSE group
Atypical BSE cases were selected for this study solely on the basis of
available DNA for PRNP sequencing and genotyping. DNA samples were obtained
from six unrelated BSE cases confirmed as atypical H or L type BSE by
Western blot profile (high or low molecular mass of unglycosylated
protease-resistant prion protein (PrPres) [12]–[14]. Two atypical L-type and
two atypical H-type BSE cases originated in France. Two additional atypical
H-type BSE cases originated from Canada and the United States.

Composition of cattle control group

Samples from two cattle DNA diversity panels were used to construct the
cattle control group; the U.S. Meat Animal Research Center (USMARC) Beef
Cattle Discovery Panel 2.1 (MBCDP2.1) [15] and the USMARC Dairy Cattle Panel
(MDCP1.5) [11], [16]. Breeds in this group include Angus (n=8) Hereford
(n=8), Limousin (n=8), Simmental (n=7), Charolais (n=6), Beefmaster (n=5),
Red Angus (n=6), Gelbvieh (n=6), Brangus (n=5), Salers (n=5), Brahman (n=6),
Shorthorn ( n=5), Maine-Anjou (n=5), Longhorn (n=4), St. Gertrudis (n=4),
Chianina (n=4), Holstein (n=8), Jersey (n=7), Guernsey (n=3), Aryshire
(n=2), and Brown Swiss (n=2). A total of 21 breeds and 114 individuals are
represented in the group.

PRNP amplification and sequence-based genotyping of htSNPs
Twelve segments of PRNP were amplified for sequence-based genotyping of 19
htSNPs (Table S1). In addition, the complete prion protein coding region was
sequenced. All but two PRNP segments were amplified with the following
reagents (per 55 uL reaction), 1.25 units of Thermo-Start DNA Polymerase,
2.3 mM MgCl2, 0.181 mM dNTPs, 0.4 uM forward and reverse amplification
primer, and 50 ng genomic DNA. Two segments were amplified with identical
concentrations of Taq, dNTPs, primers, and genomic DNA as described above.
However, one segment, BTAPRNPDS13a2, was amplified with 1.36 mM MgCl2 and 3%
DMSO and the other, segment BTAPRNPDS13b, was amplified with 1.36 mM MgCl2
and 2% DMSO. PCR conditions for the 12 segments were the following: 94°C for
15 min, 40 cycles of 94°C for 20 sec, 58°C for 30 sec (excluding
BTAPRNPDS13a2), 72°C for 60 sec, and a final incubation at 72°C for 3
minutes. The primer extension temperature for segment BTAPRNPDS13a2 was
conducted at 53°C for 30 sec. Following an Exonuclease I digestion [17], the
amplicons were sequenced with BigDye terminator chemistry on an ABI 3730
capillary sequencer (PE Applied Biosystems, Foster City, California). All
sequencing primers listed in Table S1 were used in duplicate or
quadruplicate for each atypical BSE sample to obtain multiple genotypes of
each htSNP.

SNP genotyping, haplotype phasing and statistical testing
PRNP sequences were processed for polymorphism detection and genotyping with
Phred, Phrap, Polyphred, and Consed software [18]. Haplotype phase was
determined with Phase (version 2.1) [19], [20]. The frequencies of PRNP
haplotype “o” in the atypical BSE case group and the control group were
tested for significance with a Fisher's exact two-tailed test in WinPepi
(version 4.5) [21]. The 95% confidence interval for the difference between
the frequency proportions with continuity correction was also calculated in

SUPPORTING INFORMATION ......snip........end

see full text ;

Sunday, March 16, 2008

MAD COW DISEASE terminology UK c-BSE (typical), atypical BSE H or L, and or
Italian L-BASE


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