Vol.1, No.1, 39-43 (2010)s
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/as/
Agricultural Science
Somatic cell mutations in cerebral tissue of cattle
affected by bovine spongiform encephalopathy
Matteo Busconi, Corrado Fogher*
Istituto di Agronomia, Genetica e Coltivazioni erbacee, Sezione di Botanica e Genetica vegetale, Università Cattolica S. Cuore,
Piacenza, Italy; *Corresponding Author: corrado.fogher@unicatt.it
Received 16 April 2010; revised 28 April 2010; accepted 6 May 2010.
In animals the prion disease includes sheep and
goat scrapie and the bovine spongiform en-
cephalopathy (BSE). While several polymor-
phisms of the prion (PRNP) gene have been
identified in sheep and some of them have been
associated with susceptibility to scrapie, few
mutations are reported in cattle and no correla-
tion with BSE have been demonstrated. Genetic
screening for mutants in the PRNP gene of 21
BSE positive animals by direct sequencing of
the amplified gene, using DNA extracted from
brain as template, confirmed that only few
polymorphisms are present. However DNA
molecules cloned and sequenced from the
population of fragments considering a total of
90 clones from 9 BSE positive and 70 clones
from 7 BSE negative animals, gave a highly
significant differences in the frequency of mu-
tations (p = 0.01). The high frequency and type
of variants found cannot be explained only w ith
misincorporation error of the Ta q polymerase.
Interestingly one of the mutations found in the
BSE positive animals (F209S) corresponds to a
mutant that causes a familiar form of prion dis-
ease in humans (F198S). These data can be ex-
plained with the presence of somatic mutations
modifying the PRNP gene in single brain cells.
Keywords: BSE; Prion; Somatic Mutations
The transmissible spongiform encephalopathies (TSE) in
humans and animals are caused by anomalous aggrega-
tion of a cell-surface protein known as prion protein
(PrP). This aggregation is a consequence of a tem-
plate-induced conformational change of the normally
expressed protein (PrPC) into the pathogenic missfolded
form (PrPSc) [1]. In humans the TSE known as Creutzfeldt-
Jakob disease (CJD) has been categorised into three
main forms: 1) sporadic cases with no known environ-
mental cause (85%) [2]; 2) familial cases with a domi-
nant inherited mutation of the PRNP gene (10-15%) [3];
3) cases transmitted from a known or presumed envi-
ronmental source (e.s. iatrogenic, rare) [4]. In animals
the prion disease includes sheep and goat scrapie and the
bovine spongiform encephalopathy (BSE). While several
polymorphisms of the PRNP gene have been identified
in sheep and some of them have been associated with
susceptibility to scrapie [5], few mutations have been
found in cattle and no correlations with BSE have been
postulates [6].
Molecular genetic studies of the inherited form of
CJD indicate that this disease is caused by mutations in
the prion protein gene (PRNP) and the most frequent
mutations described are P102L, D178N, F198S, E200K,
and V210I [7]. No mutations genetically linked to the
disease have been found in animals, but amino acid
changes at codons 136, 154 and 171 have been shown to
be associated with susceptibility to scrapie in sheep [8].
In contrast to the many PrP polymorphisms found in
sheep, only few PrP polymorphisms have been found in
cattle regarding the number of octapeptide repeat units
(five-seven), present in the first half of the gene [9], or
changes in the amino acids S46I and S146N found in
healthy animals [6,10]. Genetic studies have not shown
an association between numbers of repeats and BSE
susceptibility [9,11].
A new variant of CJD (nvCJD) has been demonstrated
to be caused by the same prion strain that causes BSE
rising concern for a possible human epidemic following
meat from affected animals entering the human food
chain, in late 1980s and early 1990s [12]. In terms of
control of BSE it is important to know whether this dis-
ease, apart from the origin from infected meat and bone
meals, is conditioned by genetic factors. The aim of this
work is to show that the brain tissue of BSE affected
cattle contains somatic mutations, one of which is ho-
mologous to a PRNP mutation responsible for a familial
form of prion disease in humans.
M. Busconi et al. / Agricultural Sciences 1 (2010) 39-43
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2.1. DNA Extraction
Twenty-one BSE positive samples diagnosed with rapid
methods and confirmed with histopathology, immuno-
histochemistry and Western blot as described by Casa-
lone et al. [13] and seven BSE negative samples, were
used for PCR amplification of the entire PRNP gene
(Acc. n. X55882). DNA extraction from obex region of
the medulla oblongata in the brain stem has been done
with manual Qiagen kits.
2.2. PCR Amplification
All the PCR amplifications were performed with a Ge-
neAmp PCR system 9700 thermal cycler (Applied Bio-
systems). PCR reactions were carried out in a final vol-
ume of 20 l (1X PCR buffer; 2 mM Mg++, 150 M
dNTPs, 5 pmol of each primer (Forward IGP553
GAAAAATGAGG), 1.5 U AmpliTaq Gold (Applied
Biosystems) and 5 ng of genomic DNA). The following
cycle was applied: 10 min at 95°C, 35 cycles of 30 s
denaturation at 95°C, 1 min at 56°C, 1.5 min extension
at 72°C. The amplified products were resolved by elec-
trophoresis on agarose gel and visualised by ethidium
bromide staining. The expected size of PCR products
was 807 bp (795 bp from PRNP gene and 12 bp more
from primers). Fragments were recovered from the gel
and directly sequenced or ligated in a T-vector (pGEM-T
easy, Promega), then transformed by electroporation into
E. coli strain DH5α. Ten isolated clones were sequenced
for each PCR sample reactions of 9 positive and 7 nega-
tive animals after plasmid purification. Cycle-sequen-
cing was performed in a final volume of 20 μl using the
big dye v3 chemistry (Applied Biosystems) and, after
purifications, the sequences were loaded and ran on the
ABI Prism3100 Genetic Analyzer (Applied Biosystems)
following the manufacturer’s protocols.
Using the sequence data of the clone with the mutation
F209S, presenting a change from T to C in position 626,
a reverse internal primer (IGP717 5’-CATCTTGATGT-
CAGTTTCGGTGG-3’) was designed in order to per-
form a mutation screening.
All the original twenty one positive BSE samples,
plus three BSE negative samples, were screened for the
presence of the mutation F209S. Reactions were carried
out in a final volume of 20 l (1X PCR buffer; 2 mM
Mg++, 150 M dNTPs, 5 pmol of each primer (IGP553
and IGP717), 1.5 U AmpliTaq Gold. The product of the
first reaction (IGP553/554) from different independent
amplifications at different dilutions (ranging from 10-3 to
10-9) was used as template for PCR. The following PCR
touch down program was applied: 10 min at 95°C, 12
cycles of 30 s denaturation at 95°C, 30 s at 62°C (–0.5°C
for each cycle), 1 min 30 s extension at 72°C, followed
by 35 cycles of 30 s denaturation at 95°C, 30 s at 56°C,
1 min 30 s extension at 72°C. Amplified products were
resolved by electrophoresis on agarose gel.
Genetic screening for mutants in the PRNP gene of 21
positive BSE animals diagnosed in Italy in the past years,
by direct sequencing of the amplified gene fragment
using DNA extracted from brain as template, confirmed
the previous knowledge that only few polymorphisms
are present in cattle. However, some amplified PCR
products (9 out of 21) were not directly sequenced since
the signal was not clear due to the heterozygosity of the
number of octapeptide repeats. Therefore we decided to
clone the PCR product of those 9 samples and to con-
sider single clones for sequencing. The amplified frag-
ments obtained using the DNA recovered from the brain
tissue of 7 negative BSE animals of the same age as the
positive ones were cloned too and used as control. It is
noteworthy to stress that all the amplifications for both
positive and negative samples were carried out in the
same conditions: using the same reagents, the same am-
plification cycle and thermal cycler.
By sequencing a total of 90 clones of BSE positive
animals and 70 clones of BSE negative animals, we
found a highly significant difference in the frequency of
mutations (p = 0.001, Tab le 1 ). 26 out of 90 clones ana-
lysed from the BSE positive samples showed nucleotide
changes while only 4 out of 70 clones analysed from the
BSE negative samples were characterised by sequence
variations. Moreover all the mutations from the positive
BSE animals, with no amino acid changes, were ran-
domly distributed all over the full sequence, while the
mutations with amino acid changes were concentrated
mainly in the carboxy-terminal domain of the protein
(Figure 1).
So we found that DNA molecules cloned and se-
quenced from the population of the fragments amplified
from a tissue sample of 25 mg (corresponding to about 4
× 106 cells if we consider the size of the bovine brain cells
similar to the human brain cells [14]) from the positive
BSE animals contain several single nucleotide polymor-
phisms (SNP) and interestingly one of these mutations
(F209S) corresponds to a mutant which causes a familiar
form of prion disease in humans (F198S, Figure 1).
The high frequency and type of variants cannot be due
only to misincorporation and frame shift errors of the
Taq polymerase since 1) for the BSE negative samples
we reported a significantly lower variation rate (2 =
17.92, p = 0.001); 2) the distribution of the mutations
M. Busconi et al. / Agricultural Sciences 1 (2010) 39-43
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Figure 1. Variations and mutations of the prion protein gene in humans (CJD) and variations and polymorphisms found in
cattle positive and negative to BSE. The mutations causing prion disease are above the line of the human sequence. Below
the lines are the polymorphisms, some but not all of which are known to influence the phenotype of disease (adapted from
17). For the bovine sequence, the variations determining amino acids changes are above the line and the polymorphisms
are below the line. Those found in BSE positive animals by direct sequencing are in green. All the bovine variations and
polymorphisms, with the exception of those in position 46 and 146, are from this work. Parentheses in the bovine se-
quences indicate corresponding human codons.
Table 1. Frequency of mutations and polymorphisms found in amplified and cloned sequences of the PRNP gene from cerebral tissue
of positive and negative BSE cattle.
Samples N° of clones sequenced N° of variants
Percentage of variation
9 BSE positive 90 26 28.9
7 BSE negative 70 4 5.71
with amino acid changes shows hotspot areas, the major
of which corresponding to the human gene hotspot mu-
tation area (codon 174 to 264, Figure 1); 3) all the ob-
served variants are base substitutions, with no frameshift
errors [15]; 4) if misincorporation was the only mutants
cause, there would be an even distribution of changes
in the three codon positions (1:1:1) instead of the
1.6:1.2:0.2 found in the hotspot area with a 2 of 5.2 (p =
0.1); 5) the KA/KS ratio [16], where KA is the number of
non-synonymous substitutions per non-synonymous site
(20/508.5 = 0.0393) and KS is the number of synony-
mous substitutions per synonymous site (6/283.5 =
0.0211), is greater than 1.0 (1.86).
Furthermore based on the sequence of the mutant
F209S, we designed a reverse primer and we used it in a
mutation screening on all the 21 BSE positive plus 3
negative samples. Using the genomic DNA as template
for the reaction, we replicated the PCR three times in-
dependently and obtained always the amplification of
the expected 627 bp fragment only for the sample in
which the mutation was originally found (animal n. 7)
and never on the other positives and negatives (Figure
2). The mutation screening was performed also using the
product of the first reaction (IGP553/554) at different
M. Busconi et al. / Agricultural Sciences 1 (2010) 39-43
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Figure 2. Results of the PCR mutation screening for the variant F209S using the primer IGP553-IGP717. Lines
1-4: negative control with the PCR product of a BSE negative animal at serial dilutions (1: 10-6; 2: 10-7; 3: 10-8; 4:
10-9). Lines 5-8: BSE positive animal n. 15 at serial dilutions (5: 10-6; 6: 10-7; 7: 10-8; 8: 10-9). Lines 9-12: BSE
positive animal n. 7 at serial dilutions (9: 10-6; 10: 10-7; 11: 10-8; 12: 10-9). Line M: 100 bp ladder.
dilutions (ranging from 10-3 to 10-9) as template for PCR,
again we obtained the amplification only for the ex-
pected sample and never in the remaining with the ex-
ception of positive n. 15 were at some sample dilutions
the band appears (Figure 2).
Considering that different amino acid variations could
alter more or less heavily both the structure and the
functionality of the protein, we analysed the mutated
sequences with the software CODDLE (Codon Opti-
mized to Discover Deleterious Lesions, www.proweb.
org/coddle) in order to have some information about the
most putative dangerous mutations. Analysing the se-
quence of the bovine prion gene against the prion block
(IPB000817, from BLOCKS Database Version 14.3) the
software identifies the presence of five highly conserved
amino acid blocks and shows all the possible deleterious
changes according to the mutation method selected. The
analysis was repeated considering several mutation
methods and, finally, 9 out of 20 amino acid changes we
have found were considered important for the function
of the protein: N50S, M145V, Y174C, V200A, F209S,
T212A, M216T, I252F, L253P.
A possible explanation of the sequence variations found
in the analysed clones could be a polymerase error dur-
ing amplification or the presence of somatic cell muta-
tions in the cerebral tissue.
The first hypothesis do not explains 1) the great dif-
ference in the variation rate between positive and nega-
tive samples amplified in the same conditions and con-
sidering a similar number of clones analysed, 2) the dis-
tribution of the putative amino acid variation is very
correspondent to the distribution of human pathogenic
mutations (Figure 1) defining a main hotspot area in the
carboxy-terminal region of the protein, and 3) the use of
a selective primer designed on a mutation which corre-
spond to a pathogenic human mutation gives amplifica-
tion always in the expected sample and never in the oth-
ers (Figure 2).
Furthermore 8 out of 9 of the most dangerous putative
mutations predicted by using the software CODDLE
were grouped in the second part of the gene and this
agree with the situation found in human prion gene
where all the pathogenic mutations up to now are in the
second part of the gene (Figure 1).
To our opinion the reported data on sequence variants
can be explained with the presence of somatic mutations
modifying the PRNP gene in single cells. These somatic
mutations will usually be unnoticed because they are
surrounded by millions of normal cells, producing the
PrPSc isoform that causes disease by amplification of the
somatic genetic effect with seeding of the PrPC protein in
the surrounding cells. Somatic mutations have been as-
sumed also due to the origin of CJD [17]. Somatically
generated variations, determining a seeding point from
which the pathological form of the protein spreads
around, can produce enough protease-resistant PrPSc to
allow diagnosis of the pathology with hysto- and im-
muno-techniques. In order to investigate the role of these
somatic mutations, a higher number of animals, includ-
ing BSE negative animals both exposed and unexposed
to the risk factor will be analysed. Depending on being
an early or late event in the cerebral development, the
somatic area of the mutated cells can be limited or ex-
panse. This will be further studied coupling sequencing
and Western analysis of fine topographical sections of
BSE positive brains.
We wish to thank P.L. Acutis and M. Caramelli of CEA, Centro di
Referenza Encefalopatie Animali, for providing the positive samples.
This work was supported by a MIUR grant to Chelab Srl.
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