Diss. ETH No. 14853
Comparative
approach
with
to
and molecular
identify
genetic
genes associated
"Congenital progressive
in
spastic paresis"
ataxia and
pigs
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY
ZÜRICH
for the
degree
of
Doctor of Natural Sciences
presented by
ANTKE KRATZSCH
med. vet.
born December
citizen of
accepted
1970
Germany
the recommendation of
on
Prof. Dr. G.
Stranzinger, examiner
Vögeli and
Bertschinger, co-examiners
Prof. Dr. P.
Prof. Dr. H.U.
29,
2002
We shall not
cease
And the end of all
from
our
Will be to arrive where
And know the
exploration
exploring
we
started
place for the first
time.
T.S. ELLIOT
Contents
Table of contents
i
vii
Summary
Zusammenfassung
ix
List of
xi
figures
List of tables
xiii
Abbreviations
1
xv
Introduction
1.1
1.2
1.3
1
Congenital progressive
ataxia and
1.1.1
Congenital splayleg
1.1.2
Myasthenia gravis
spastic paresis
disease
2
2
How to get from the marker to the gene ?
positional candidate
1.2.1
The
1.2.2
The pure
gene
approach
positional cloning approach
Candidate genes
1.3.1
1.3.2
1
2
3
5
5
Ion channels
6
calcium channels
1.3.1.1
Voltage-gated
1.3.1.2
Calcium
1.3.1.3
Voltage-gated Na+ channelopathies
10
1.3.1.4
Inwardly rectifying potassium channelopathies
11
1.3.1.5
Cholinergic receptor
channelopathies
Spastin
diseases
7
8
12
13
i
Contents
ii
1.4
2
Objectives
of this
study
14
Methods
2.1
2.2
15
Animals and
samples
2.1.1
Breeding
2.1.2
Tissue
2.1.3
Blood
15
studies
15
samples
15
samples
16
Clinical examinations
16
2.2.1
Clinical, neurophysiological, biochemical, and pharmaco¬
observations
logical
2.2.2
16
Neuropathology
17
2.3
Primers
17
2.4
DNA methods
20
2.5
2.4.1
Isolation of
genomic DNA from tail biopsies
20
2.4.2
Isolation of
genomic DNA from blood samples
21
2.4.3
Isolation of PAC DNA
2.4.4
Isolation of
2.4.5
Quantification of DNA
2.4.6
Polymerase
2.4.7
Restriction enzyme
2.4.8
Gel
2.4.9
DNA extraction from agarose
2.4.10
Genescan
2.4.11
Sequencing
2.4.12
Sequence analysis
2.4.13
Gene
2.4.14
Fluorescence
2.4.15
Library screening
25
2.4.16
Ligation
26
2.4.17
Transformation
A-phage
DNA
21
22
chain reaction
electrophoresis
22
digestion
22
of DNA
23
gel
23
analysis
23
23
24
mapping by using
2.4.18
Organization
2.4.19
Screening
2.4.20
Single
in
situ
somatic cell
of the PAC
24
25
27
library
27
library
stranded conformation
RNA extraction
hybrids
hybridization
of the PAC
RNA methods
2.5.1
21
27
polymorphism analysis
.
.
28
29
29
Contents
2.6
3
iii
2.5.2
Quantification of RNA
2.5.3
Gel
2.5.4
Reverse
2.5.5
Rapid amplification of cDNA ends
30
2.5.6
Northern blot
30
2.5.7
Slot blot
29
of RNA
electrophoresis
29
29
transcription
analysis
analysis
30
Statistics
32
Results
3.1
3.2
3.3
33
Phenotype approach
33
3.1.1
Clinical examination
33
3.1.2
Consanguinity
34
3.1.3
Neurophysiological
3.1.4
Hematology
and chemical parameters
35
3.1.5
Pathological
and
36
3.1.6
Clinical
Assignment
studies
34
histological
examination
of CPA
diagnosis
36
of CPA
37
3.2.1
Mode of inheritance
3.2.2
Genetic
3.2.3
CPA
mapping
37
of the CPA
phenotype
37
diagnostics
42
Candidate genes
3.3.1
44
Calcium channel
3.3.1.1
3.3.1.2
ß± subunit
Regional
3.3.3
45
pigs
3.3.1.3
Expression study
3.3.1.4
Drug
Chromosomal
3.3.2.1
assignment
50
of other ion channel genes
Chromosomal assignment of
alpha subunit cluster
Chromosomal
3.3.2.3
Chromosomal
Spastic paraplegia
3.3.3.2
47
treatment
3.3.2.2
3.3.3.1
44
Characterization of the cDNA of affected and
unaffected
3.3.2
44
localization
4
a
.
.
sodium channel
51
assignment of KCNJ3
assignment
52
of CHRNA1
53
SPG4
Mapping of SPG4 by
53
somatic cell
Characterization of the
and unaffected
pigs
51
hybrids
...
53
SPG4 cDNA of affected
54
Contents
iv
Histological
3.3.3.3
3.4
PAC
58
3.4.1
Contig
3.4.2
Single
around microsatellite SW902
nucleotide
58
polymorphism
59
3.4.2.1
SNP in A340D12-SP6
59
3.4.2.2
SNPs in A78G1-SP6
61
Linkage analysis
and fine
61
mapping
Discussion
4.1
4.2
4.3
65
Diagnosis of CPA
65
picture of CPA
4.1.1
The clinical
4.1.2
Microsatellite SW902 and CPA
Chromosomal
cluster
of
assignment
a
65
67
sodium channel
(SCN1A-SCN3A), KCNJ3,
and CHRNAl
4.5
68
69
4.3.1
Chromosomal
4.3.2
Mutation
4.3.3
CACNB4 expression
70
Comparison of CPA with the clinical picture of lethargic
epilepsy and ataxia in humans
70
69
assignment
screening
of the cDNA
mice and
4.4
alpha
subunit
CACNB4
The candidate gene
4.3.4
5
58
contig
3.4.3
4
examination
The candidate gene
SPG4
71
4.4.1
Chromosomal
4.4.2
Mutation
4.4.3
Comparison of CPA with
The PAC
69
71
assignment
screening of the cDNA
pure HSP
71
72
72
contig
4.5.1
General aspects of
4.5.2
Discrepancies
generating
in the
Conclusions and further
contig
perspectives
a
contig
72
73
75
Bibliography
76
Appendix
90
A
Materials
91
A.l
Equipment
91
A.2
Substances/chemicals
93
Contents
A.3
Media/Solutions
v
96
Curriculum Vitae
101
Acknowledgment
103
Summary
In
1996, the Congenital progressive ataxia and spastic paresis (CPA)
was
observed for the first time in Switzerland.
in
pigs
The disease manifests itself
shortly after birth as a severe neuropathy. Affected animals show spastic gait,
incoordination, and rapidly progressive ataxia in the hind limbs. Clinical and
neurophysiological observations, as well as biochemical and pharmacological
studies did not reveal any
an
significant
abnormalities.
The CPA is inherited
as
autosomal recessive trait.
In the first part of this
project a genome scan revealed that in our family
size in bp), located on
pigs the microsatellite SW902189 allele (189
Sus scrofa chromosome 3 (SSC3) co-segregated 100% with the recessive allele
of 206
=
involved in the disease
,
while the
SW902197, SW902'20i,
or
SW90221i alleles
co-segregated 100% with the normal allele. SW902 is genetically mapped in
close proximity to the IL1 locus on SSC3ql3-q21. Comparative maps predict
correspondence of this region to HSA2ql-q2, where a sodium channel a subunit cluster (SCNA), a calcium channel ß subunit gene (CACNB4), a potassium
(KCNJ3) and a cholinergic receptor a subunit
mapped. Epilepsy, ataxia and paralysis seem to be caused
by mutations in these genes. Physically, SW902 was mapped to SSC3q21-q27
which is equivalent to HSA2pl3-p24, where the spastin (SPG4) gene is local¬
ized. Mutations in SPG4 may be responsible for Hereditary Spastic Paraplegia
(HSP). As ataxia, paralysis and Spastic Paraplegia resemble the phenotypical
inwardly-rectifying
gene
(CHRNAl)
channel gene
are
appearance of CPA in the
In
a
pig, these
second part, the genes
of
chosen
were
SCNA, KCNJ3,
to SSC15 and therefore excluded
to
genes
as
as
candidate genes.
and CHRNAl
candidate genes.
CACNB4
were
was
mapped
mapped
SSC3ql4-q21 and SPG4 to SSC3q21-q27. Sequence analysis of the cDNA
porcine CACNB4 did not reveal any mutation in the amino acid sequence
in affected
CACNB4
pigs. Northern blotting revealed
with ethosuximide did not
analysis
no
in brain and muscle of affected and
of the cDNA of
difference in the
healthy pigs.
expression of
Drug
treatment
improve the condition of affected animals. Sequence
porcine SPG4
mutation in the amino acid sequence. No
VII
in affected
degeneration
pigs did
not
reveal any
of motor axons, which is
Summary
vm
suffering from HSP, was observed in affected piglets.
findings, the hypothesis that CACNB4 or SPG4 is identical
CPA gene was rejected.
found in humans
of these
In
the
a
third part,
a
region harboring
around SW902
was
out that the
pure
positional cloning approach
responsible for CPA the
the gene
started.
Altogether,
five clones
was
initiated. To isolate
creation of
were
Because
with the
a
PAC
arranged.
contig
It turned
starting clone containing SW902 was a chimeric clone with two
co-ligated
deriving from SSC3 and SSC4. Additionally, it was confirmed
by linkage analysis, and genetically and physically mapping of STSs and SNPs
found in the contig that a gap between the starting and the subsequent clones
inserts
existed.
Zusammenfassung
Die
erblich
bedingte progressive Ataxie
und
spastische
Schweines wurde 1996 erstmals in der Schweiz beobachtet.
tome dieser
Krankheit, die
Parese
(CPA)
Die ersten
des
Symp¬
lassen, zeigen sich kurz
spastischem Gang, Inkoordina-
ein Nervenleiden vermuten
nach der Geburt. Betroffene Tiere leiden unter
tion und Ataxie der
Hintergliedmassen, die sich innert weniger Tage verschlim¬
neurophysiologische, sowie biochemische und pharmakolo¬
gische Untersuchungen weisen keine signifikanten Veränderungen auf. Die CPA
mert.
Klinische und
wird autosomal rezessiv vererbt.
ersten Teil dieses Projekts wurde eine Genomanalyse durchgeführt.
zeigte, dass bei allen 206 Tieren unserer Familie das Allel 189 (189
Basenpaargrösse) des Mikrosatelliten SW902, welcher auf dem Schweinechro¬
mosom 3 (SSC3) lokalisiert ist, zu 100% mit dem rezessiven Allel, welches
mit der Krankheit assoziiert ist, segregiert.
Die anderen Allele, SW902197,
Im
Diese
SW90220i oder SW90221i segregieren
=
zu
100% mit dem normalen Allel. SW902
genetisch in der Nähe des IL1 Locus kartiert, der auf SSC3ql3-q21 lokalisiert
wurde. Vergleichende Genkarten zeigen eine Übereinstimmung dieser Region
mit dem Abschnitt ql-q2 des menschlichen Chromosomes 2 (HSA), wo Gene für
eine Natriumkanal-ct-Untereinheit (SCNA), eine Kalziumkanal-/3-Untereinheit
ist
(CACNB4), einen einwärtsgleichrichtenden Kaliumkanal (KCNJ3) und eine
Cholinrezeptor-a-Untereinheit (CHRNAl) kartiert sind. Mutationen in diesen
Genen scheinen für einige Formen von Epilepsie, Ataxie und Paralyse verant¬
wortlich zu sein. SW902 wurde physikalisch auf SSC3q21-q27 lokalisiert. Diese
Region entspricht dem HSA2pl3-p24, wo das Gen Spastm (SPG4) lokalisiert
ist. Mutationen in diesem Gen scheinen für die Krankheit Hereditäre Spastische
Paraplégie (HSP) verantwortlich zu sein. Da Ataxie, Paralyse und Spastische
Paraplégie dem Krankheitsbild der CPA ähneln, wurden diese Gene als Kan¬
didatengene ausgewählt.
In einem zweiten Teil der Studie wurden die Gene
CHRNAl dem Schweinechromosom 15
SCN2A,
KCNJ3 und
zugeordnet und daher als Kandidaten¬
gene ausgeschlossen. CACNB4 wurde dem SSC3ql4-q21 zugeordnet, während
SPG4 auf SSC3q21-q27 kartiert wurde. Die Sequenzierung des CACNB4 Gens
ix
Zusammenfassung
X
gesunder Schweine zeigte keine Mutation auf, die zu einer
Aminosäuresequenzänderung führte. Es wurde kein Unterschied in der Ex¬
pression des CACNB4 Gens im Gehirn und Muskel von erkrankten und gesun¬
den Tieren mit Hilfe der Northen Blot Technik festgestellt. Eine Behandlung
mit dem Medikament Ethosuximid zeigte keine Verbesserung des Krankheits¬
bildes. Die Sequenzierung des SPG4 Gens erkrankter und gesunder Schweine
zeigte keine Mutation, die zu einer Aminosäuresequenzänderung führte. Eine
Degeneration der motorischen Axone, wie bei Menschen zu beobachten ist, die
an HSP leiden, wurde nicht gefunden.
Aufgrund dieser Ergebnisse wurde die
Hypothese, dass die Gene CACNB4 oder SPG4 identisch mit dem CPA Gen
erkrankter und
sind, verworfen.
In einem dritten Teil versuchten
Klonieren
zu
charakterisieren.
wir, das CPA Gen mittels positionellem
Um die
Region
zu
isolieren, die das CPA Gen
PAC-Kontigs um den Marker SW902
begonnen. Insgesamt
angeordnet. Es zeigte sich
dass
sich
bei
dem
der
den
Marker SW902 enthält,
es
Ausgangsklon,
jedoch,
umfasst,
wurde mit dem Aufbau eines
herum
wurden fünf Klone
entait, die sowohl von SSC3,
Kopplungsanalysen und
und
der
STSs
und
SNPs, die im Kontig
genetischer
physikalischer Zuweisung
Lücke
zwischen
dem
eine
Ausgangsklon und den nachfolgen¬
gefunden wurden,
den Klonen nachgewiesen.
um
einen chimären Klon
handelt, der
als auch SSC4 stammen.
zwei Inserts
Zusätzlich wurde mittels
List of
1.1
Figures
Comparative
(HSA2)
porcine (SSC3 and SSC15) and human
of
map
chromosomes
4
Ca2+ channel
1.2
Subunit structure of
1.3
Locations of mutations in
1.4
Subunit structure of
1.5
Putative membrane
1.6
Putative
2.1
topology
Alignment
Needle
an
3.2
3.3
3.4
a
8
CACNB4
9
Na+ channel
10
topology
of
KCNJ channel
a
11
of the nAChR channel
13
of deduced amino acid sequences of four neuronal
calcium channel
3.1
a
ß subunits
31
electromyography (EMG)
pig
infraspinatus muscle of
of the
affected
and
Physical
mapping
genetic linkage
map
35
(sex-averaged)
of SSC3 and
of CPA gene
40
Electropherogram of microsatellite
family affected by CPA
Fluorescence
situ
in
SW902 alleles
on
SSC3 of
a
41
hybridization (FISH) analysis
of
porcine
CACNB4
3.5
45
Nucleotide
(cDNA)
porcine CACNB4
3.6
A
3.8
47
gene
comparison of the porcine, human, rat, bovine and
calcium channel
3.7
and deduced amino acid sequence of the
Northern blot
Slot blot
ß\ subunit (CACNB4)
analysis
analysis
of
for
Regional
porcine CACNB4 mRNA expression
localization of SCN2A
matic cell
.
porcine CACNB4 mRNA expression.
bellum and muscle of affected and
3.9
mouse
amino acid sequence.
hybrids
by
in
analysis
49
cere¬
healthy pigs
PCR
48
50
on
porcine
so¬
51
xi
xii
List
3.10 Chromosomal
cell
3.11
assignment
hybrid panel
Chromosomal
somatic cell
assignment
hybrid panel
of
porcine KCNJ3 using the somatic
of
porcine CHRNAl
52
using
the INRA
53
assignment of porcine SPG4 using the INRA
hybrid panel
3.12 Chromosomal
matic cell
3.13 Nucleotide
(cDNA)
porcine SPG4
3.14 A
of Figures
so¬
and deduced amino acid sequence of the
56
gene
comparison of the porcine, human and
mouse
spastin amino
acid sequence
57
3.15 The sequence
tagged
region deriving from
site
(STS) contig
map for the chromosomal
microsatellite SW902
58
3.16 PCR-SSCP pattern of STS A340D12-SP6 of four
tropherogram
of STS A340D12-SP6
flanking
pigs and elec¬
the identified
mu¬
tation
3.17
60
Polymorphism M143 of STS A78G1-SP6 resulting
fragment polymorphism
in
a
restric¬
tion
3.18 Genetic
some
3
54
mapping
of SNP A78G1_SP6_M143
on
porcine
62
chromo¬
64
List of Tables
2.1
Sequence information of the primers used
2.2
Primer
matic cell
3.1
study
17
pairs, annealing temperature Ta(°C), fragment size and
GenBank Accession of the genes used for
2.3
in this
mapping with the
so¬
hybrid panel
Components
25
of the SSCP
gel
28
Quantitative Electromyography (EMG) and Electroencephalog¬
of two affected and one unaffected pig
raphy (EEG)
3.2
Motor
nerve
3.3
nerve
conduction
Clinical
chemistry
3.5
one
pigs
of the
venous
35
plasma of
and marker
located
different
SSC3 in
phenotype
offspring, produced by
of CPA and
Two-point linkage analysis
bination fraction
Assignment
(9)
of the
and lod
binary
6 unaf¬
36
Transmission patterns of CPA
on
seven
estimation of
scores
sex
SW902,
mating pairs.
averaged
recom¬
(Z)
code for the
38
chromosome 3-
43
polymorphism
in STS
A340D12-SP6
3.7
34
peroneal
piglet
at rest
specific marker loci used for
3.6
amplitude
unaffected
data determined in
fected and 13 affected
3.4
latency
of two affected and
and
61
Two-point linkage analysis of A78G1-SP6JVI143,
seven
chromo¬
3-specific marker loci and the CPA locus used for estima¬
of sex-averaged recombination fractions (0) and lod scores
some
tion
(Z)
63
xm
Abbreviations
AAA
ATPase associated with diverse cellular
AD-HSP
autosomal dominant HSP
AMP
adenosine
AMV
avian
monophosphate
APS
myeloblastosis virus
ammonium peroxodisulfate
AR-HSP
autosomal recessive HSP
AS
Andersen
ASAT
aspartate aminotransferase
BAC
bacterial artificial chromosome
bp
base
BSA
bovine
serum
°C
degree
Celsius
Ca2+
calcium ion
CACNB4
calcium channel
cDNA
activity
syndrome
pair
albumin
ß± subunit
DNA derived from mRNA
CHRNA1/4
complementary
cholinergic receptor, nicotinic,
CK
creatine kinase
a
polypeptide 1/4
Cl¬
chloride ion
em
centimeter
cM
CTAB
centimorgan
Congenital Progressive Ataxia and Spastic
cetyltrimethylammonium bromide
DA
Dalton
DEAE
diethylaminoethyl
diethyl pyrocarbonate
deoxyribonucleic acid
deoxyribonucleotide triphosphates
2'-deoxyuridine 5'-triphosphate
CPA
DEPC
DNA
dNTPs
dUTP
EDTA
EEG
EMG
ethylenediaminetetraacetic
electroencephalography
electromyography
XV
acid
Paresis
Abbreviations
XVI
EtBr
ethidium bromide
FISH
fluorescence
FITC
fluorescein
FSHR
in situ hybridization
isothiocyanate
follicle-stimulating hormone receptor
g
gram
GULOP
acceleration of gravity
gamma-aminobutyric acid
Genetics Computer Group
generalized epilepsy with febrile seizures plus type
guanidine isothiocyanate
L-gulono-7-lactone oxidase
L-gulono-7-lactone oxidase pseudogene
h
hour
HCl
HE
hydrochloric acid
haematoxylin and
H20
water
x
g
GABA
GCG
GEFS+2
GITC
GULO
eosin
HSA
Homo sapiens chromosome
HSP
hereditary spastic paraplegia
Hz
Hertz
IL1(A/B)
interleukin 1
INRA
Institut National de la Recherche
K+
potassium ion
kbp
kilo base
kDA
kilo Dalton
KCNJ3
potassium channel, inwardly-rectifying,
Kir
subfamily J, member 3
inwardly rectifying potassium
1
liter
LB
Luria Bertani
LCT
Ih
lactase-phlorizin hydrolase
lactate dehydrogenase
lethargic
LMN
lower motor
LDH
LOD
(alpha/beta)
Agronomique
pair
channel
neuron
of the odds
MgS04
logarithm
molarity (= mol/liter)
magnesium sulfate
min
minute
MOPS
3-(N-morpholino)propanesulfonic
mRNA
messenger RNA
mi
milli-x
jJLX
micro-x
Na+
sodium ion
(n)ACh(R)
(nicotinic) acetylcholine (receptor)
M
acid
(= 10"3)
(= 10-6)
2
NaCl
sodium chloride
NaOH
sodium
NCBI
National Center for
NCS
OD
OD
o/n
ORF
hydroxide
Biotechnology
study
osteogenic disorder locus in pig
optical density
over night
open reading frame
Information
conduction
nerve
PAC
Pl-derived artificial chromosome
PCR
RN
polymerase chain reaction
plaque forming units
phase lock gel
plate pool
protein kinase, AMP-activated, noncatalytic, 7-3
pico-x (= 10-9)
Q-bands by fluorescence
using quinacrine or quinacrine mustard
rapid amplification of cDNA ends
restriction fragment length polymorphism
Rendement Napole
pfu
PLG
PP
PRKAG3
px
QFQ-banding
RACE
RFLP
RNA
ribonucleic acid
rpm
rounds per minute
r.t.
room
RT
reverse
SCN1A
sodium
SCN2A
sodium
temperature
transcription
SCN3A
channel, neuronal type I, a subunit
channel, voltage-gated, type II, a subunit
sodium channel, neuronal type III, a subunit
SDS
sodium
sec
second
SNP
single
nucleotide
SP
super
pool
SPG4
spastic paraplegia
SSC
saline-sodium citrate buffer
SSC
Sus
dodecyl
sulfate
polymorphism
gene 4
(Spastin)
SSP
scrofa chromosome
single strand conformation polymorphism
super/super pool
STS
sequence
SUISAG
public
SSCP
tagged
site
company for
pig breeding
services in Switzerland
TBE
Tris-borate-EDTA buffer
TE
Tris-EDTA buffer
TEMED
N,N,N',N'-tetramethylethylenediamine
0
recombination fraction
Abbreviations
xvm
TNE
Tris-natrium-EDTA
U
unit
5'/3'
UTR
5'/3'
untranslated
UV
ultraviolet
V
Volt
VDCC
voltage-dependent
v/v
w/v
volume per volume
region
calcium channel
X-Gal
weight per volume
xylene cyanol FF
5-bromo-4-chloro-3-indolyl /3-D-galactopyranoside
X-HSP
X-linked HSP
YAC
yeast artificial chromosome
Z
lod
XCFF
score
Chapter
1
Introduction
Congenital progressive
paresis
1.1
In
1996,
a
previously
ataxia and
undescribed neuromuscular disease
was
spastic
observed for the
first time in two litters of
pigs. The animals were derived from two dams and
one sire, all of Large White origin. The dams were cousins and not related to
the sire, referring to the last two generations. One third of the offspring were
affected while the other two third appeared normal. The severity of the symp¬
toms varied within the litters and occurred in both
showed
sexes.
Affected animals
spastic gait, incoordination, progressive distal weakness and atrophy
of the limbs
leading to an inability to walk. Due to this disability the severely
piglets died due to starvation or they were crushed by the sow. The
observed ratio of approximately 3:1 (healthy:affected) pigs suggested that the
disease may be controlled by a recessive allele. The disorder was named "Con¬
genital progressive ataxia and spastic paresis (CPA)".
affected
A whole genome
scan
relatively high although
not
between CPA and SW1066
was
mapped
to
performed in our institute revealed
statistically significant linkage (Z=1.81, 0=0.00)
of these 26 animals
(Gmür, 1997).
The microsateUite locus SW1066
porcine chromosome 3 by Rohrer
A disease similar to CPA
et al.
(1996).
Finland, called "Progres¬
pigs" (Rimaila-Pärnänen,
1982). The newborn pigs showed the first clinical signs at the age of 10 days
to 2 weeks. The etiology of the disease is unknown. Any specific chromosomal
changes could not be found (Veijalainen and Rimaila-Pärnänen, 1978). The
possible hereditary nature of this disease was investigated on one specific farm,
where the suspected boar produced 37 litters totally. In one third of the litwas
sive ataxia and incoordination
described in 1982 in
syndrome
1
in Yorkshire
Chapter
2
ters, ataxia and incoordination
an
was
noticed. Rimaila-Pärnänen
autosomal recessive mode of inheritance.
were
no
further
(1982)
showed
investigations
made.
Congenital splay leg
1.1.1
The disease
"Congenital splayleg
to CPA. Affected animals
are
in
disease
piglets"
severe cases
the fore
legs
exhibits
are
also affected
similar clinical
picture
properly due
hind legs are splayed sideways and
(Curvers et al, 1989). If the hind
not able to stand and
to muscular weakness of the hind limbs. The
in
However,
Introduction
1.
a
can
not walk
distance, the animals recover from the disease and
legs
bandaged
Landraces
are more frequently affected than other breeds and
develop normally.
the incidence in male piglets is reported to be up to twice as high as in females
(Van der Heyde et al., 1989). Several investigations indicate an immaturity
of the skeletal muscle of the hind legs at birth with subsequent compensatory
hyperplasia (Ducatelle et al., 1986). Defects of the cholinergic system and/or
to 5
are
cm
delayed maturation of motor nerves may furthermore be involved (Le Hong
et al., 1990).
Nevertheless, the pathogenesis of this disease is still not fully
understood. Analyses of the mode of inheritance favor a two locus model but
a polygenic inheritance can not be excluded (Stigler et al., 1991).
Myasthenia gravis
1.1.2
Specific muscle weakness, which
increases with exercise is the main clinical
symptom in myasthenia gravis in humans and dogs.
This weakness is due
global disturbance of the function of the neuromuscular end plate: the
density of the subsynaptic acetylcholine receptors is reduced and therefore,
the transmitter acetylcholine, released in normal quantity can bind only to a
few receptors. Additionally, the patients develop antibodies against their own
acetylcholine receptors which leads to a complement-mediated lysis and an in¬
creased rate of receptor degradation. Consequently, the end plate potentials
are substantially reduced in amplitude, which in turn leads to muscle weak¬
ness (Ito et al., 1978; Vincent, 1980).
Myasthenia gravis can be treated with
anticholinesterase drugs (such as Konstigmin®), which inhibit the breakdown
of acetylcholine.
to
a
1.2
How to
get from the marker
Once the chromosomal
known,
a
strategy
to
position of
identify
to the gene ?
a
certain disease is
the disease gene would be the
positional cloning
a
marker linked with
1.2.
How to
get from the marker
to the gene ?
3
approach. Positional cloning implies identifying a gene on the basis of its chro¬
(Collins, 1992, 1995). The positional cloning approach can
be divided into the positional candidate gene approach and the pure positional
cloning approach.
mosomal location
The
1.2.1
The
positional
positional candidate
candidate gene
gene
approach relies
approach
on
a
three-step
process:
(1)
localizing a disease gene to a chromosomal subregion, generally by using tradi¬
tional linkage analysis; (2) searching databases for an attractive candidate gene
within that subregion; if no suitable gene can be found comparative gene maps
of other species can be used, and (3) testing the candidate gene for diseasemutations.
causing
For the identification of
L-gulono-gamma-lactone oxidase
deficiency in pigs, the positional can¬
didate gene approach was used. These pigs manifest deformity of the legs,
multiple fractures, osteoporosis, growth retardation and haemorrhagic tenden¬
cies. The trait is controlled by a single autosomal recessive allele designated as
od (osteogenic disorder). Hasan et al. (1999) showed by linkage analysis that
the OD locus is located in the subcentromeric region of SSC14 and mapped the
GULO gene to Sus scrofa chromosome (SSC) 14ql4 by FISH. This region cor¬
responds to an evolutionary conserved segment on human chromosome 8 where
the non-functional human pseudogene GULOP has been mapped (Nishikimi et
al., 1994). Sequencing analysis of the genomic DNA in deficient od/od pigs
identified a frameshift deletion leading to a truncated protein where the partial
C-terminal was altered. In addition, the expression of the deficient od allele
is less compared to the normal OD allele (Hasan et al., 2002). These find¬
ings may explain the absence of GULO activity in the liver microsomes where
GULO usually is produced.
a
deletion in the
GULO gene, which leads to vitamin C
on
Initial mapping of the CPA locus showed possible linkage to marker SW1066
porcine chromosome 3. On this chromosome SW1066 is mapped in close
to the interleukin 1 (ILl) locus. ILIA is mapped to SSC3ql2-ql3
al, 1992; Mellink et al, 1994; Johansson et al, 1994), while IL1B is
located on SSC3qll-ql4 (Krull et al, 1992; Mellink et al, 1994; Rohrer et al,
1996). The human ILIA and IL1B loci were mapped to HSA2ql3-q21 by Webb
proximity
(Krull
et al
et
(1985)
and
Lafage
conserved segment
on
(CACNB4), the
3Ä), the potassium
subunit gene
(SCN1A
-
the nicotinic
which
were
result in
a
et al
(1989)
as
shown in
sodium channel
channel
cholinergic receptor
chosen
as
Fig
1.1.
The
evolutionary
/?4
human chromosome 2 contains the calcium channel
ct\
phenotype (Werderlin, 1986;
subunit type I
subfamily J,
(CHRNAl),
candidate genes.
a
-
member 3 gene
and the
spastin
III genes
(KCNJ3),
(SPG4)
gene
Mutations in these genes in humans
Meisler et
al, 2001) which shows high
Chapter
4
1
Introduction
IL1B
I
IL1A
SSC3
Figure
HSA2
Comparative
1.1:
of
porcine
(SSC3
and
SSC15)
and
hu¬
of
(under¬
ßn subunit, CHRNAl, cholin¬
FSHR, follicle-stimulating hor¬
ergic receptor, nicotinic, a polypeptide 1,
ILIA
1
&
interleukin
mone receptor,
a & ß,
KCNJ3, potassium chan¬
B,
member
nel, inwardly-rectifying, subfamily J,
3, LCT, lactase-phlorizin hy¬
drolase, SCN1A & SCN3A, sodium channel, neuronal type I & III, a subunit, SCN2A, sodium channel, voltage-gated, type II, a subunit, SPG4,
Picture adapted from INRA-Toulouse
spastic paraplegia gene 4 (Spastin)
toulouse
mra
http //www
fr/Ige/pig/compare/HSA htm
man
lined)
(HSA2)
map
SSC15
is
chromosomes
shown
CACNB4,
The localization
calcium channel
the candidate genes
Candidate genes
1.3.
to the CPA
similarity
1.2.2
5
phenotype
in
pigs.
positional cloning approach
The pure
If the
positional candidate gene approach fails, another possibility to charac¬
responsible for CPA is the positional cloning process. After a
is localized to a chromosomal region by linkage studies, multipoint map¬
terize the gene
gene
ping with additional polymorphic loci can be used to pinpoint the site of the
disease gene and identify the closest flanking marker site. At best, the level of
resolution between marker sites is 1 cM, which spans about 1 x 106 bp. On
the average, this amount of DNA contains 20
1999).
50 different genes
(Pasternak,
cloning approach is to discover
which of
genes between two marker sites is actually the disease
gene. A physical map of the region containing the disease gene is generated
by retrieving clones with overlapping segments from a genomic DNA library.
On the basis of the overlaps and other positional information, a continuum of
The
of the
objective
the possible
positional
-
gene
Sets
can be established for this chromosome region (Pasternak, 1999).
contiguously ordered clones (contigs) have been generated from YAC, BAC,
PAC, PI, and cosmid libraries. A contig can be assembled from as few as two
clones, as long as both clones share an overlapping DNA region within their
inserts (Coulson et al, 1986; Olson et al, 1986).
clones
of
The identification of PRKA G3
was
as the causative gene for the RN phenotype
positional cloning approach in a farm animal species
The dominant Rendement Napole (RN~) allele occurs
the first successful
(Milan
al, 2000).
Hampshire pigs and increases the glycogen content in white skeletal mus¬
cle by about 70% (Estrade et al, 1993; Enfält et al, 1997). Törnsten et al.
(1998) mapped the RN locus to SSC15q25. Comparative mapping showed
that the RN region shares homology with parts of human chromosome 2q and
et
in
mouse
chromosome 1. Since
construction of
with RN~
a
were
no
obvious candidate genes
were
identified in these
positional cloning project
including the
markers
that
showed
Two
complete association
contig.
clones.
Of
clone a shotgun
two
on
one
present
overlapping
chromosome segments,
a
was
initiated
BAC
constructed and more than l'OOO individual sequences were deter¬
library
mined, including the PRKAG3 gene. Milan et al (2000) demonstrated that
the RN phenotype is due to a missense mutation in this gene encoding a novel
isoform of the regulatory 7 subunit of AMP-activated protein kinase.
was
1.3
Candidate genes
As described in
genes
chromosomal
in diseases which exhibit simi-
chapter 1.2.1, the candidate
position and their involvement
were
chosen due to their
Chapter
6
lanties to CPA
different
ion
In the
1
following chapters an overview is given
coding for spastin
Introduction
over
both the
channel genes and the gene
Ion channels
1.3.1
Inherited movement and
the molecular
However,
etiology
seizure
remains
disorders
are a
major clinical burden of which
undefined for the most part of the diseases
studies have indicated that mutations
recent
channels
in
neuronal
major contributors to these conditions
are
gated ion
Jan, 1999, McNamara, 1999)
Ion channels
are
voltage-
(Cooper
and
membranous structures formed
contain aqueous central pores
(Celesia, 2001)
by aggregated proteins and
They either produce action po¬
graded potentials, the basis for communication among neurons The
channel specific properties are summarized by Siegelbaum and Koester (2000)
as follows
(1) they conduct ions, (2) they recognize and select among specific
and
(3) they open and close in response to specific electrical, mechanical
ions,
These changes generate either 'all or none' action poten¬
or chemical signals
tials or graded potentials causing an increase or decrease in cell membrane
polarization The resting membrane potential as well as the activation of the
membrane potential depends on a variety of ion channels and other membrane
tentials
or
transporters
The expression of the channel proteins
can
be observed
in
the
system (Koester,
2000) as well as in the neuromuscular junction and in the muscle membrane
(Aidley and Stanfield, 1996), and also in external and internal membranes of
almost all cells (Lehmann-Horn and Jurkat-Rott, 1999)
cell soma,
dendrites,
axons
and at the synapses of the
nervous
In cell membranes, three types of channels can be classified non-gated, di¬
rectly gated and second messenger gated channels In comparison to non-gated
channels, which open or close in relation to simple ion concentration gradients,
the gated channels require a 'key' to open the gate of the channel
Among
the important directly gated channels are voltage-gated (Na+, K+, Ca2+, Cl~)
and hgand-gated (ACh, glutamate, GABA, glycine) channels (Celesia, 2001)
Ligand-gated
channels mediate local
increases in ion
conductance at chemical
thereby depolarize or hyperpolanze the subsynaptic area of the
In contrast, voltage-sensitive ion channels mediate rapid, voltage-gated
cell
changes in ion permeability during action potentials in excitable cells and also
modulate membrane potentials and ion permeability in many mexcitable cells
synapses and
(Catterall, 1988)
The channels
brane and
has
a
1999)
units
are
are
macromolecular protein
complexes within the lipid
divided into distinct protein units called subunits
by different genes (Hille and Catterall,
principal subunit and 3-4 auxiliary subal, 1997, Hille and Catterall, 1999) The principal subunit
specific function and
is
encoded
Most of the channels contain
(Fontaine
et
mem¬
Each subunit
a
Candidate genes
1.3.
7
a subunit which is capable of carrying out
channels, while the auxiliary subunits, according to Hille
and Catterall (1999) 'improve expression and modulate physiological proper¬
ties'. Furthermore, there are several isoforms of each protein subunit and each
in the
voltage-gated
channels is the
the functions of the
isoform is encoded
channel subunits
In neuronal
by
are
another gene. Until
(Hille
known
signaling,
and
channels have
today, more than
Catterall, 1999).
a
50 genes
encoding
fundamental role. Gene mutation
can
produce aberration in channel
given
function. Thus, channel dysfunction may result in a variety of neurological
disorders that span from myopathy to epilepsy (Celesia, 2001). Disorders of
channel function are called channelopathies (Ashcroft, 2000; Lehmann-Horn
and Jurkat-Rott, 1999).
easily
alter the structure of
Here,
each ion
1.3.1.1
we
will
channel,
They
ter from
a
short introduction about the function and structure of
its genes and the known
Voltage-gated
Ca2+ ions play
tions.
give
a
channel and
channelopathies.
calcium channels
regulation of a variety of cellular func¬
contraction, trigger the release of neurotransmit¬
terminals and of hormones from secretory cells, regulate gene
the cell cycle, and mediate cell death (Ashcroft, 2000). The
an
important role
in the
initiate muscle
nerve
expression and
intracellular Ca2+ concentration
([COg+])
is much lower than that of outside
the cell. A transient rise in internal Ca2+ acts
as a
second messenger
receptor activation to many cellular processes. This increase in
diated
coupling
[Ca2+]
by voltage-gated Ca2+ channels that regulate Ca2+ influx
is
across
me¬
the
plasma membrane.
Voltage-dependent Ca2+ channels (VDCCs) are heteromeric complexes
plasma membrane of almost all cell types (Walker and De Waard,
1998). Distinguished by their sensitivity to pharmacological blockers, singlechannel conductance, kinetics and voltage dependence they have been classified
as L-, N-, T-, P/Q- and R- types (Perez-Reyes and Schneider, 1994; Ashcroft,
2000; Catterall, 2000a).
found in the
Associated
a VDCC is illustrated in Figure 1.2.
pore-forming ct\ subunit are the membrane anchored, largely extra¬
cellular (X2-Ö subunit, the cytoplasmatic ß subunit and sometimes a transmem¬
brane 7 subunit (Isom et al, 1994). The a\ subunit is functionally the most
important as it acts as the channel pore, the voltage sensor, and the receptor
for many drugs. The other subunits have auxiliary roles: when co-expressed
with the «i subunit they enhance the current magnitude and alter its kinetic
properties (Ashcroft, 2000). Channel kinetics are modulated by the several
functional domains of the N- and C-terminal portions of the a.\ and ß subunits
(Walker et al, 1999). Walker et al (1998) proposed that the C-terminus of the
The subunit structure of
with the
Chapter
Ca
w
1
1
1
w
rssn^
!
8
J
!
^ i
oc-i
Introduction
2+
w
Y
1.
oc2
-r-T
1111 m
membra ne
/
/
é)
ß
Subunit structure
of
Ca2+ channel, modified from
Figure
1.2:
(1994).
The arrangement and biochemical properties
a
of probable N-linked glycosylation; P,
protein phosphorylation; -S-S-, mter-subunit disulfide
trated.
^,
site
of
Isom et al.
the subunits
site
are
illus¬
of cAMI'-dependent
bond.
ßi subunit interacts directly with the C-terminus of the cï\a subunit (Fig 1.3)
and is required for a proper regulation of channel inactivation.
eight genes are known, four genes
ß subunits ß\-ß\ (Lehmann-Horn and JurkatRott, 1999). The amino acid sequences of the four mammalian ß subunits differ
by approximately 20%. Expression of ß\ is predominant in muscle, ßi is the
major subunit of the heart, while ß$ and ß± are mainly expressed in brain, with
high concentration of ß± in the cerebellum (Castellano and Perez-Reyes, 1994).
While for the
major
a.\
subunit at least
have been identified that encode
1.3.1.2
Calcium
channelopathies
Mutations in the a,
ß and
7 calcium channel subunits have been associated
example, mutations in the «1^4 subunit of the
voltage-gated Ca2+ channel cause three human diseases: spinocerebellar ataxia
type-6 (Zhuchenko et al, 1997), episodic ataxia type-2 and familial hémiplégie
migraine (Ophoff et al, 1996).
with different diseases.
In the
mouse
and
For
human,
two diseases
were
associated with mutations in
ßi subunit (CACNB4): an autosomal recessive neurological disorder in
the mouse mutant lethargic (Burgess et al, 1997) and idiopathic generalized
epilepsy and episodic ataxia in human (Escayg et al, 2000a). CACNB4 is
mapped to human and mouse chromosome 2 (Chin et al, 1995; Taviaux et al,
the
Candidate genes
1.3.
9
COOH
of mutations in CACNB4- In the mouse Ih mutant, the
of the a.\ binding site. In human, the mutation
R482X causes a protein truncated in the middle of a domain that interacts with
the C-termmus of the a.\ subunit.
Figure
1.3: Locations
protein
is
truncated upstream
1997; Escayg
et
al, 1998).
lethargic (Ih) exhibits ataxia, episodic dyskinesia, and
generalized epilepsy. Burgess et al (1997) identified a four base pair insertion
into a splice donor site within the CACNB4 gene. The mutation results in
aberrant pre-mRNA splicing and translational frameshift and is predicted to
encode a severely truncated ß\ protein missing 60% of the C-terminus rela¬
tive to wild type, including the essential ct\-ß interaction domain (Fig. 1.3).
ß subunits expressed with deletions of this domain are unable to modulate
Thus, the absence of
«i subunit function in vitro (De Waard et al, 1994).
detectable wild-type transcripts by RT-PCR of lethargic brain RNA suggests
that the mutation represents a null allele of the CACNB4 gene. McEnery et
al (1998) demonstrated that neither full-length nor truncated ß± protein is
expressed in Ih/lh mice using /^-specific antibodies.
The
mutant
mouse
Escayg et al (2000a) identified a premature-termination mutation R482X
CACNB4 gene in a patient with juvenile myoclonic epilepsy. The R482X
protein lacks the 38 C-terminal amino acids containing part of an interaction
in the
domain for the a\ subunit
(Fig. 1.3).
truncated
Xenopus laevis oocytes demonstrated
crease
protein R482X
in
The results of functional tests of the
a
small de¬
in the fast time constant for inactivation of the cotransfected a.\ subunit.
Additionally,
with generalized epilepsy and praxis-induced
family with episodic ataxia, a missense mu¬
tation C104F was found (Escayg et al, 2000a). This mutation does not alter
channel kinetics, but the replacement of cysteine with a large hydrophobic
seizures and
in
a
a
German
family
French Canadian
Chapter
10
1.
Introduction
Na+
w
w
\|/\|/
rs sn w
i
i
i
a
ß2
ßl
Figure
1.4:
mpmhranp
Subunit structure
of
a
Na+ channel.
The arrangement and bio¬
site of probable N-linked
glycosylation; P, site of c AMP-dependent protein phosphorylation; -S-S-, mter-subunit disulfide bond. The drawing is modified from Isom et al. (1994).
chemical property
phenylalanine
of
the subunit
illustrated.
is
^,
residue in the C104F mutation could
of this domain.
Walker and De Waard
disrupt the conformation
(1998) reported
that the evolutionary
phenylalanine residue is thought to be involved in interaction with
proteins. Such interactions could be important to channel clustering or
conserved
other
targeting without affecting the channel gating.
1.3.1.3
Voltage-gated Na+ channelopathies
Voltage-gated Na+ channels are responsible for the Na+ current that underlies
the rapid upstroke of the action potential in nerve and muscle fibers. Membrane
depolarization of excitable cells causes sodium channel activation in a positivefeedback mechanism
field.
The
resulting
ciated with further
along
both the concentration
gradient
and the electric
increase in sodium conductance of the membrane is
depolarization and
The channels' intrinsic inactivation
repolarization of the membrane
asso¬
activation of further sodium channels.
occurs
within
a
few milliseconds and leads
in the absence of any
voltage-gated
potential, the cell membrane is inexcitable
for a short period of time. The duration of this period of time is regulated by
the kinetics of recovery of the channels from inactivation and is the limiting
factor for the firing rate of the cells (Lehmann-Horn and Jurkat-Rott, 1999).
to
potassium channel. After
an
even
action
Brain Na+ channels have two unrelated
and
auxiliary subunits designated ß\
ßi, while the skeletal muscle Na+ has only a single ß subunit (Catterall,
Candidate genes
1.3.
11
outside
COOH
Figure 1.5: Putative membrane topology of a KCNJ channel. The locations
of identified Andersen syndrome mutations in Kir2.1 are identified by dots.
2000b). Morgan
et al
(2000)
identified
an
additional
subunit, /53, which
is most
ß\.
closely
pore-forming
ß subunits (Fig. 1.4) is sufficient for functional expression, but the kinetics and
voltage dependence of channel gating are modified by the ß subunits (Goldin
et al, 2000).
related to
Until
The
a
eleven different genes
today,
in the humane genome that
subunit which is associated with the
(SCN1A-SCN11A)
known to encode
have been identified
subunits of
voltage-gated
al, 2000). A cluster of genes on human chromosome
2q21-q24 encodes the following three neuronal voltage-gated sodium channel a
subunits: SCN1A, SCN2A and SCN3A (Plummer and Meisler, 1999).
sodium channels
Generalized
(Goldin
epilepsy
are
a
et
with febrile seizures
tosomal dominant disorder characterized
plus type
2
(GEFS+2)
is
an
au¬
variable
phenotype combining
febrile seizures, afebrile generalized seizures (tonic-clonic, absence, myoclonic
or atonic) and partial seizures (Scheffer and Berkovic, 1997). GEFS+2 has been
mapped to human chromosome 2q21-q33 (Baulac et al, 1999; Moulard et al,
1999).
order
Mutations in the SCN1A gene
by Escayg
et al
(2000b), Escayg
The mutations
are
located in
channel, which
are
known to have
It
was
suggested
highly
a
by
were
a
described in families with this dis¬
at al
(2001),
and Wallace et al
(2001).
conserved transmembrane segments of the
role in channel
gating (Wallace
et
al, 2001).
that these mutations may reduce the rate of inactivation of
SCN1A and therefore result in
a
more
depolarized membrane potential and
hyperexcitability.
1.3.1.4
Inwardly rectifying potassium channelopathies
Inwardly rectifying potassium channels (Kir) are important in maintaining the
resting potential and in controlling the excitability of a cell by allowing K+
influx with little K+ outflux through a nonvoltage-gated mechanism (Kubo et
Chapter
12
al, 1993;
As
Ho et
shown
1.
Introduction
al, 1993).
in
Fig.
1.5
membrane domains linked
Kir
by
channels
a
possess only two putative
loop which dips back down into the
trans¬
mem¬
(Ashcroft, 2000). Until today, 15
(http://bioinfo.weizmann.ac.il/cards-
brane to line the outer part of the pore
(KCNJ) are known
bin/cardsearch.pl?search=-S_*kcnj*). The KCNJ3 gene, encoding Kir3.1, is
located on human chromosome 2q24 (Stoffel et al, 1994). It is expressed in
cardiac atrial myocytes as well as various neuronal cell types (Schoots et al,
1997).
Kir
channel genes
(2001)
Plaster et al.
identified nine different mutations in the KCNJ2 gene in
syndrome. Andersen syndrome (AS)
by periodic paralysis, cardiac arrhythmias, and
dysmorphic features. Expression of two of these mutations in Xenopus oocytes
revealed loss of function and a dominant-negative effect in Kir2.1 current as
assayed by voltage-clamp. Therefore, they concluded, that AS is caused by
mutations in Kir2.1. Moreover, the findings suggest that Kir2.1 plays an im¬
portant role in developmental signaling in addition to its function in controlling
different families
is
a rare
cell
suffering
from Andersen
disorder characterized
excitability
in skeletal muscle and heart.
Cholinergic receptor
1.3.1.5
diseases
acetylcholine receptors (nAChRs) are ligand-gated ion channels
change in conductance is regulated by its binding to the neurotransmitter
acetylcholine (ACh). They are expressed in both muscle and nerve and play
a key role in fast synaptic transmission both at neuronal-neuronal synapses
within the nervous system and at the neuromuscular junction (Ashcroft, 2000).
In contrast to voltage-gated channels that are allowing either Na+ or K+ influx,
the AChR as it opens becomes permeable to Na+, K+ and Ca2+ (Hille and
Catterall, 1999).
The nicotinic
whose
The nAChR is
ring around a central
comprises 2ct\, ß\, e, ö
(Fig. 1.6).
subunits, while that of embryonic and denervated muscle is composed of 2cti,
ßi, 7, ö (Mishina et al, 1986). The ACh-binding pocket is found in the extra¬
a
pentamer of subunits arranged in
cellular domains at the interface between
2000).
a
(Ashcroft,
(cholinergic re¬
and the other subunits
a
(1989, 1990) assigned
polypeptide 1) to human
Beeson et al
ceptor, nicotinic,
a
The adult muscle nAChR channel
ion pore
the CHRNAl gene
chromosome
2q24-q32.
Myasthenia gravis is an autoimmune disorder in which the body creates
antibodies against its own nicotinic AChRs (Vincent, 1980). The disease is
characterized by muscular weakness and fatigability. The symptoms tend to
fluctuate throughout the day and under different environmental and physiolog¬
ical conditions (see also chapter 1.1.2).
Autosomal dominant nocturnal frontal lobe
epilepsy
is
a
syndrome
where
1.3.
Candidate genes
B
outside
inside
Figure
1.6:
(2000). (A)
topology of the nAChR channel. Modified from Ashcroft
topology of a single nAChR. (B) Subunit pentamer: m
the 7 subunit is replaced by an e subunit.
Putative
Membrane
adult skeletal muscle
motor seizures with tonic
or
hyperkinetic
(CHRNA4)
was
occur exclusively at night. A
acetylcholine receptor 0:4 subunit
(Steinlein et al, 1995).
features
missense mutation in the neuronal nicotinic
associated with this disease
Spastin
1.3.2
Spastin
is
a
member of the AAA
activities) protein family and is
gene (Hazan et al, 1999). The
the presence of
homology
one or more
(ATPases
encoded
by
members of this
highly
as
molecular
spastic paraplegia
family
are
4
(SPG4)
characterized
by
conserved AAA motifs that contain Walker-
domains and harbor the ATPase
considered to act
associated with diverse cellular
the
chaperones
activity. The AAA ATPases are
assembly, function, and disas¬
in the
sembly of protein complexes and play essential roles in a wide variety of cellular
activities, including protein degradation, vesicle-mediated protein transport,
cell-cycle regulation, organelle biogenesis, and gene expression (Patel and Latterich, 1998).
The SPG4 gene, which maps to human chromosome 2p21-p22 (Hazan et al,
1994; Hentati et al, 1994), has been shown to account for ~40% of all autosomal
hereditary spastic paraplegia (AD-HSP) (Casari and Rugarli, 2001).
group of neurodegenerative disorders characterized by progressive
and bilateral spasticity of the lower limbs. Age of onset is generally between
10 and 40 years. HSPs are classified according to their symptoms and mode of
inheritance into 'pure' and 'complicated' forms (Werderlin, 1986). In pure HSP
spasticity occurs in isolation, while for the complicated form additional features
dominant
HSPs
are a
Chapter
14
Introduction
1.
such as mental retardation, dementia, epilepsy, ataxia, ichthyosis, deafness, and
optic atrophy are known. The pure AD-HSP is the most common form of the
disease, however autosomal recessive (AR-HSP) and X-linked (X-HSP) forms
of transmission
are
also known.
Neuropathological analyses indicate that HSP is characterized by axonal de¬
generation involving the more distal portions of the longest motor and sensory
axon of the central nervous system (McDermott et al, 2000).
published reporting the spectrum of SPG4 muta¬
et al, 2000; Lindsey et al, 2000; Santorelh
et al, 2000; White et al, 2000; Hentati et al, 2000; Burger et al, 2000). Missense, nonsense, and splice-site point mutations as well as deletions or insertions
have been observed in the spastin gene, and almost all seem to affect the AAA
Several papers have been
tions in HSP
patients (Fonknechten
motif-encoding region
Until
recently,
of the gene.
the cellular
pathways
in which
spastin operates and its role
causing degeneration of motor axons were unknown. Now, Errico et al
(2002) suggested that spastin interacts dynamically with microtubules and that
this association is regulated through the ATPase activity of the AAA domain.
Microtubules are highly dynamic polymers of a- and /3-tubulin subunits. Errico
in
et al
(2002)
the AAA
further showed that all the
domain, previously identified
to microtubules and lead to
a
spastin missense mutations located
in HSP
redistribution of the microtubule
Therefore, they suggested that HSP due to SPG4 mutations
an impairment of microtubule dynamics in long axons.
1.4
Objectives
Besides
providing
study
aims of this
a
of this
the
to describe the CPA disease
•
to
more
analysis
of the
by
clinical and
genome, the
pathological examinations,
linkage between CPA and
matings of the type SW1066194
confirm
informative
porcine
following:
•
statistically
cytoskeleton.
depend on
may
study
contribution to the
were
in
patients, bind constitutively
SW1066
x
by producing
SW1066194,
analyze all available microsatellites located close to SW1066 to
strengthen the location of CPA on SSC3ql-q2 and to narrow the region
harboring the disease locus,
•
to
•
to find and
analyze
candidate genes for CPA
by comparative mapping,
and
•
if the gene
a
pure
causing the disease has not been characterized
positional cloning approach will be initiated.
in any
species
Chapter
2
Methods
2.1
Animals and
Breeding
2.1.1
The disease
samples
studies
pigs, derived from two dams and
sire,
Large
origin (family 1, matings 1 and 2, Table 3.4). The
dams were cousins and not related to the sire, referring to the last two gener¬
ations. From these two litters two males and five females, all phenotypically
normal animals were mated to each other and produced 167 progeny. Of the
167 descendants, 35 animals showed ataxia and paresis syndromes. The ani¬
mals were bred and kept at the Faculty of Veterinary Medicine of the University
of Zürich under supervision of Dr. E. Bürgi.
was
first observed in two litters of
all of
one
White
Additionally,
ilies which
Altogether,
while 156
2.1.2
the
of the disease
occurrence
was
confirmed in five other fam¬
originate from different farms (families 2-6, Table 3.4).
pigs
206 animals
were
Tissue
were
examined: 50 showed the
typical signs
of
CPA,
healthy.
samples
(Table 3.4),
cm was taken from every piglet
days after birth. Affected piglets were
euthanized, when their condition progressively worsened. Portions of the brain,
specifically of cerebrum and cerebellum were collected, as well as portions of
M. quadriceps femoris, M. triceps brachii and M. biceps brachii. Samples were
taken also from healthy control animals.
The tissues were frozen in liquid
nitrogen and stored at 80°C until RNA isolation was performed.
In
family
1
for microsateUite
a
analysis
tail
of 0.5
biopsy
within three
—
15
Chapter
16
Blood
2.1.3
Blood
samples
crosateUite
Methods
samples
were
taken from animals of families 2-6
From the Vena
analysis.
containing
vacutainer
a
2.
jugularis
(Table 3.4)
10.0 ml of blood
was
for mi¬
taken with
EDTA and stored at —20°C until DNA extraction.
Clinical examinations
2.2
Clinical, neurophysiological, biochemical,
macological observations
2.2.1
and
phar¬
observations, all affected and unaffected piglets from matings 3-12
(Table 3.4) were examined in the stable under supervision of Dr. E. Bürgi,
Department of Veterinary Internal Medicine, University of Zurich. The devel¬
opment and phenomenology of the movement disorder was videotaped.
For clinical
The neurophysiological studies were performed at the Institute of Animal
Neurology, University of Bern, under supervision of Dr. S. Cizinauskas. Two
affected and one healthy piglet were examined. The NeuroScreen Plus system
(TOENNIES) was used for electromyography (EMG) and motor nerve con¬
duction studies (NCS), for electroencephalography (EEG) the Medelec Profile
Multimedia EEG (Oxford) was applied. For EMG, the animals were positioned
in
lateral
right
ined: M.
recumbency.
supraspinatus,
M.
The following muscles of the left side were exam¬
infraspinatus, M. triceps brachii, M. biceps brachii,
carpi radialis, M. extensor digitalis longus, M. flexor carpi radi¬
alis, M. flexor digitalis longus, M. glutaeus, M. quadriceps femoris, M. biceps
femoris, M. semitendinosus, M. semimembranosus, M. gastrocnemius and M.
tibialis cranialis. NCS were performed of the left peroneal nerve. For EEG,
five subdermal recording needle electrodes were placed over the scalp.
M. extensor
Hematology and blood chemistry parameters were determined by the Unit
Laboratory Diagnostics, Department for Farm Animals, University of Zurich.
Blood was drawn from the jugular vein from 13 affected and 6 unaffected piglets
both at rest and after physical exercise, i.e., animation of the animals to get
up and walk around if possible.
of
In the pharmacological studies, Neostigmin, an anitcholinesterase agent and
Ethosuximide, an anticonvulsant, were evaluated. Neostigmin (Konstigmin®,
Chassot AG) is used for diagnosis and symptomatic treatment of myasthenia
gravis. In three newborn affected piglets 1.0 ml Konstigmin® was injected
subcutaneously. Ethosuximide (Suxinutin®, Parke-Davis) is a calcium current
blocker and has been indicated by calcium channel involvement in human pa¬
tients with epilepsy (Escayg et al, 1998; CapoviUa et al, 1999). A daily dose
of 15
mg/kg
for
one
week and 20
mg/kg
for another week
was
administered
2.3.
Primers
parenterally
17
in
one
affected
treated. The animals
the animals
follows.
as
were
were
piglet,
while another affected
derived from the
same
observed for at least 30 min and
After 10 min, the
piglets
were
piglet remained un¬
drug application,
litter. After
drug
effects
were
evaluated
stimulated to get up and
move
in
order to
judge the effect of treatment on locomotion. Skeletal muscle tone was
judged by palpation during recumbency and standing. All behavioral alter¬
ations observed after drug treatment were noted. The time interval between
two drug applications was at least 24 h.
Neuropathology
2.2.2
Histological examination were performed at the Institute of Veterinary Pathol¬
University of Zurich, under supervision of Dr. P. Ossent. All affected
piglets were euthanized. The brain, spinal cord, parts of M. biceps brachii, M.
triceps brachii and M. quadriceps femoris as well as N. radialis and N. ischiadicus were fixed in 10 % formalin and processed for paraffin embedding. Sections
of cerebellum, midbrain, pons, medulla, parietal cerebrum, muscles and nerves
ogy,
stained with haematoxylin and
staining, respectively.
were
(HE)
and Luxol Fast Blue for
primers used
in this
study
are
shown in Table 2.1. The forward
each microsateUite is labeled at its 5'-end with
marker FAM
*
=
myelin
Primers
2.3
All
eosin
cross
(blue),
JOE
(green),
HEX
(yellow)
one
or
primer of
of the four fluorochrome
TAMRA
(yellow).
species primer (human)
Table 2.1:
Sequence information of the primers used
Name
in this
Sequence 5'—> 3'
study.
Remarks
Microsatellites
SW1066-Y
-GCAGGATGAACCACCCTG-
SW1066-R
-CTCTTGAGGCAACCTGCTG-
S0216-F
-TCCACCACTGCCAGTCACTT-
S0216-R
-CTGGGCTTTGAACCCACA-
SW2618 -F
-GCTTTTTCCTGTAGTCACTAGATTG-
SW2618 -R
-ATGTTCTCAAAGACTCTGACAAACC-
fluorochrome marker
JOE1
FAM1
HEX1
Chapter
18
Table 2.1:
Remarks
JOE1
SW902 -F
-ATCAGTTGGAAATGATGGCC-
SW902 -R
-CTTGCCTCAAAGAGTTGTAAGG-
S0094
S0094
-F
-AGTTCTCAGGGAGTTCCCTCATGC-
-R
-CGAGCTCGCCTATCTATCAATTCC-
SW460
SW460
-F
-ATTGCACACCTATCTCTATGCG-
-R
-AATCTCCATGTGCCGCAG-
GACT-F
-CATCTTCCTCTTCCCTTCCC-
GACT-R
-TGTGGACTCAAGGCTGTAAGC-
Anchored
TAMRA1
JOE1
JOE1
transcription primer
reverse
T(17)VX-R
Methods
(continued)
Sequence 5'—> 3'
Name
2.
-T(17)VX-
V=A/C/G
X=A/C/G/T
Calcium channel
ß±
CACNB4.1
CACNB4.1
-F
-TGAAAGAATCTTTGAGTTGGCG-
-R
-AACAATAATTGGTGCTAAGGAG-
CACNB4.2
CACNB4.2
-F
-GATGAAAACCAGCTGGAGGATGAG-
-R
-AGCCCAAATTCCTTCCCAGCAGC-
CACNB4.3
CACNB4.3
-F
-GCATCTCTGCATACTGTGTCCCG-
-R
-GTTCAATTGGTGAGTTCTCTGTGG-
CACNB4.4
CACNB4.4
-F
-CAGCGAATGAGGCACAGCAACC-2
-R
-GTAAGTGTCCTGGTATGAGTCAG-
2
CACNB4.5 -F
CACNB4.5-R
-CGGAGGAGCAGGTTGAAAAGATCC-2
(rat)
-GTTTGGGCAGCCTCAAAGCCTATGTCG-2
(rat)
CA CNB4.6 -F
-CCTCCTTACGATGTTGTACCGTC-2
CA CNB4.6 -R
-TCTGCATCAAGAACAACCAGTTGC-
CA CNB4.7 -F
-
CA CNB4.7 -R
-
ATTTGGGCTCC ACGGC ACTCTC-
2
2
AG AGGGTA ATG ATCTCGGCTATGC-
2
Calcium channel
ß4
SP1
-TGTCCCAGATACCATTTCTCCAAG-2
2
cross
-
RACE
species primer (human)
2.3.
Primers
19
Table 2.1:
(continued)
Sequence 5'-
Name
SP2
-AAAGGAGATAGCTGTGCTTGGAACAGG-
SPS
-TGCTTGCTGTTCTCTCTCCTGTCG-2
Cholinergic receptor
ol\
CHRNAl. 1 -F
-AAGCGACCAGCCAGACCTGAGC-2
CHRNAl. 1 -R
-GAAGGAGAAGAGCAGGCAGGGG-2
PAC
primer
T7
-TAATACGACTCACTATAGGG-
SP6
-CGATTTAGGTGACACTATAG-
F150093-T7-F
-TGTTCCTTGCAGGCAAAAGC-
F150093-T7-R
-TTTTGAATGCAGTTCCCTCC-
F150093-SP6 -F
-GTATACCGATAGACCGACTATGGG-
F150093-SP6 -R
-CAGCAGCTAGGGATCACTGAATGG-
A78G1-T7-F
-TTGATACCCCTGCAGTACTGAGC-
A78G1-T7-R
-TTGCCTGCTGCCTACATGGCCC-
A78G1-SP6-Y
-CCAGGAGGGAGGATGGACTCGT-
A78G1-SP6-R
-CTGCACCTGGAGATCCAGGAGG-
A276A1-T7-F
-TGACCCCTAGTCTAGGAACTTCC-
A276A1-T7-R
-AGTTTCAGAATCCCTTAACCCCCC-
A276A1-SP6 -F
-CCATTGCTTTAAGAAGGATGAATCC-
A276A1-SP6 -R
-TGTCCATCCAGTCAACCATCTACC-
A340D12-T7 -F
A340D12-T7 -R
-GAGACACGGATTGACATACGGGG-
A340D12-SP6
A340D12-SP6
-F
-AACAAACACACTAAGTATAAACACCC-
-R
-ACCCCTGTATGTGTGCTTATTAGC-
-AAGTGACCCTATAAGTGGAAATAGG-
D60036-T7-F
-AAAAATCTGTGCGAAGTCTTCCC-
D60036-T7-R
-GAGGGTTTCCTAATGGTTCACCC-
D60036-SP6 -F
-GATCCTGGGAGGAATCTGGGC-
D60036-SP6 -R
-AACTGAGGCTCAAGGAGTGGC-
Potassium channel J3
KCNJ3.1 -F
-CACTCCGAAGGAAATTTGGGGACG-2
KCNJ3.1 -R
-TTGATGAACATGCAGCCGATGAGG-2
Remarks
Chapter
20
Table 2.1:
(continued)
Sequence 5'-
Name
Methods
2.
Remarks
KCNJ3.2 -F
-CTCTCGGACCTCTTCACCACC-
KCNJ3.2 -R
-TAGACATTGGCCACGCAGGGC-
Sodium channel la
SCN2A.1 -F
-AAATTACAACCTCTGCTGGCTGGG-2
SCN2A.1 -R
-ATGTACATGTTCACCACCACCAGG-2
SCN2A.2 -F
-TCTGCTGGCTGGGATGGACTGC-
SCN2A.2 -R
-AAAATCCCAACAGATGGGTTCCCG-
Spastin
SPG4.1
SPG4.1
-F
-TGCTGTGGCTCGAGAACTTCAGCC-2
-R
-TACAAGCCCTGTGTCATCTCCAGC-2
SPG4.2
SPG4.2
-F
-TGCTGTGGCTCGAGAACTTCAGCC-2
-R
-AAAGGCCAAGCTATATGAGTCACC-
SPG4.3
SPG4.3
-F
-ACACACTAGTAATTCACTGCCTCG-2
-R
-CTTATGAGTGGTAGGAGCAGGACC-2
SPG4-4
SPG4.4
-F
-AACAGGCCTTCGAGTACATC-
-R
-TCTTCTACTCTTTGCTGTCTCTGG-
SPG4.5
SPG4.5
-F
-GCTTCGCGCTGCTGCGTTTGG-
-R
-CTTTCTCGTCCTCGTCGATGCG-
SPG4.6
SPG4.6
-F
-TGATATAGCTGGTCAAGAATTGGC-
-R
-GGCCTCAGAGAAGGAAGAATGAC-
SPG4.7
SPG4.7
-F
-TGCTACATTACATAGAACTTAGAG-
-R
-AACTTTTCTTCACTGCCACTATGG-
2.4
2.4.1
The tail
DNA methods
Isolation of
biopsy
was
genomic
biopsies
immediately after collection to 0.5 ml of lysis
agitation at 55°C overnight the tubes were centrifuged
transferred
buffer. After continuous
DNA from tail
DNA methods
2.4-
21
a firm pellet. The supernatant was then poured into prelatubes, each containing 0.5 ml isopropanol. The samples were mixed until
precipitation was completed. The DNA was recovered by lifting the precipitate
with a pipette, excess liquid was dabbed off and the DNA was dispersed in an
Eppendorf tube containing 0.5 ml 10 mM TE, pH 8.0.
for 10 min to obtain
beled
Isolation of
2.4.2
For
of the
hemolysis
35.0 ml of 10 mM
leukocytes
had
was
and incubated with 50.0
Proteinase K
into
a
/A
(20 mg/ml)
color.
at 50°C for 12-14 h.
4°C.
fil
500.0
2.4.3
10 mM
same
The tube
The DNA supernatant
precipitate the DNA, washed
mixed with
was
250.0 ml 10% SDS and 50.0 ml
(10 mg/ml),
RNase
milky suspension appeared.
at
prewarmed blood
centrifugation at l'300g for 10 min at
This step was repeated until the pellet of
The pellet was resuspended in 5.0 ml TNE
PLG tube and mixed with the
a
samples
After
removed.
white/beige
a
5.0 ml of
erythrocytes,
NaCl/EDTA.
4°C the supernatant
DNA from blood
genomic
was
The solution
volume of
centrifuged
was
at
4'000g
until
for 15 min
100% ethanol
in 20.0 ml of
poured
two times in
transferred
was
phenol/chloroform
to
70% ethanol and resuspended in
TE, pH 8.0.
Isolation of PAC DNA
B.
Phage artificial chromosome (PAC) clones were obtained from Prof.
DNA
The
extracted
the
was
QiagenBrenig (Göttingen, Germany).
using
The protocol is based on
tip 500 Plasmid Purification Kit (Qiagen®).
modified alkaline lysis procedure, followed by binding of PAC DNA
a
to
an
anion-exchange resin under appropriate low-salt and pH condi¬
tions.
RNA, proteins, dyes and low-molecular-weight impurities are re¬
moved
by
buffer
and
The
a
medium-salt
then
isolation
was
wash.
concentrated
and
PAC
DNA
eluted
high-salt
by isopropanol precipitation.
the manual provided with the
is
in
a
desalted
performed according
to
(http://www.qiagen.com/literature/handbooks/plk/VeryLowCopyPlasmid
Cosmid.pdf). Nevertheless, before centrifugation the cells were divided into
kit
two 250 ml tubes instead of
sion.
centrifuging the whole 500.0 ml of cell
100.0 /xl
was resuspended in 70.0
After precipitation the DNA
Tris-HCl, pH
2.4.4
-
suspen¬
10 mM
8.5.
Isolation of
A-phage
DNA
A-phage DNA was obtained with a pig cDNA brain library from Prof. B.
Brenig. The DNA was extracted using the Stratagene Lambda DNA Purifica¬
tion Kit (Stratagene®). The procedure uses diethylaminoethyl (DEAE) resin
Chapter
22
2.
Methods
contaminating polyanions prior to phage particle disruption. The
lysed with ethylenediaminetetraacetic acid (EDTA) and pronase
and the lambda DNA was then selectively precipitated with the cationic de¬
to
remove
phages
were
(CTAB). Following an exchange re¬
chloride, the highly purified A-DNA was precipitated with
ethanol. The isolation was performed with plate lysate according to the manual
provided with the kit (http://www.stratagene.com/manuals/200391.pdf).
tergent cetyltrimethylammonium bromide
action with sodium
2.4.5
Quantification
of DNA
DNA concentration was determined with a spectrophotometer measuring the
optical density (OD) at a wavelength of 260 nm and 280 nm. An OD26o of
1 corresponds to 50 /xg/ml of double stranded DNA (Sambrook et al, 1989).
The OD260 and OD280 of the diluted DNA sample (2.5 /A DNA, diluted 1:200)
was
measured.
Pure DNA has
The DNA concentration
DNA concentration
was
(/xg/ml)
a
ratio of
calculated
=
OD26o/OD28o
as
measured
of
approximately
1.8.
follows:
OD26o
x
x
1
dilution factor
OD26o
or very small sample volumes, DNA concentration
using the ethidium bromide (EtBr) plate assay. Diluted A-DNA
standards (5, 10, 25, 50, 75 and 100 ng//d) and the unknown samples were
spotted on the surface of an EtBr plate. The brightness of the spots of the
unknown samples and the diluted A-DNA were compared using an UV-light
With diluted solutions
was
estimated
box and the DNA concentration
2.4.6
was
estimated.
Polymerase chain reaction
Polymerase chain reaction (PCR) was performed in a final reaction volume of
25.0 iA. The reaction medium consisted of 10 200 ng DNA, lx PCR buffer, 200
/xM dNTP's, 0.4 /xM upstream and downstream primers and a variable amount
of DNA polymerase, as indicated in the respective product descriptions. After a
denaturation step of 95°C for 5 min, the PCR was performed in 25
40 cycles
with denaturation at 95°C for 30
45 sec, annealing at the primer specific
-
-
-
temperature
followed
2.4.7
by
a
(56
—
final
66°C)
for 30
-
45
elongation step
sec
and
elongation
at 72°C for 30
-
90
sec
at 72°C for 7 min.
Restriction enzyme
digestion
Digestion was carried out according to the suppliers recommendations. For the
digestion of 1 ßg of DNA 10 U of the respective enzyme were used. The volume
of the enzyme should not exceed
more
than 10% of the total reaction volume.
DNA methods
2.4-
Gel
2.4.8
23
electrophoresis
of DNA
According to the expected size of the DNA fragments, gels were poured con¬
taining between 0.8% (w/v) agarose in 0.5x TBE for genomic DNA or A-DNA
and 1.5
2% (w/v) for PCR fragments. Staining was performed in ddH20
-
containing 0.4 /xg/ml
UV-light box.
EtBr for 20 min.
The DNA could be detected
mapping of the porcine CHRNAl
used. For this gel SYBRGold® (1:10'000)
For
2.4.9
gene,
was
a
used
DNA extraction from agarose
Fragments
were
cut from the
gel
and extracted
Spreadex
as
®
using
EL 600 gel
staining solution.
an
was
gel
using the Qiagen
II Gel Extrac¬
Qiagen® (http://www.qiagen.com/literature/handbooks/qexII/
qiaexII_agarose.pdf). Purification of DNA fragments with the QIAEXII system
tion Kit from
on solubilization of agarose and selective adsorption of nucleic acids
QIAEX II silica-gel particles in the presence of chaotropic salt. DNA was
quantified using the ethidium bromide plate assay.
is based
onto
Genescan
2.4.10
To map the CPA
analysis
SW2618, S0094, SW902, SW1066,
PCR amplified and their size was analyzed
SW460, GACT,
using the ABI PRISM 377 DNA Sequencer, the GeneScan and Genotyper
software. The forward primer of each microsateUite was labeled with either
phenotype,
microsatellites
and S0216
were
FAM
(blue),
dye.
The color red
Standard
(green), HEX (yellow) or TAMRA (yellow) color fluorescent
(ROX) was reserved for the GeneScan Internal Lane Size
350-P
(GS
ROX). This size standard is used for precise size calling.
JOE
Sequencer automatically analyses DNA molecules labeled with multiple
dyes. After samples are loaded onto the system's vertical gel, they
undergo electrophoresis, laser detection, and computer analysis.
The
fluorescent
2.4.11
Sequencing
Automated
sequencing was carried out according to the manual supplied with
sequencing kit. A total of 1.0 fig of A-DNA (1.5-2.5 fig of PACDNA) was added to 4.0 fi\ (8.0 fil) of Big Dye Mix, 10 pmoles (30 pmoles) of
primer in an 10.0 fA (11.0 /A) reaction volume. After a 3 min denaturation step
at 95°C, templates were subjected to 35 (99) cycles of 30 sec at 95°C, 20 sec at
55°C, and 4 min at 60°C. The amount of target DNA in ng derived from PCR
products was determined by calculating for every 100 bp of fragment length 5
ng of template.
the ABI PRISM
Chapter
24
Methods
2.
Sequence analysis
2.4.12
A
systematic search of homologous sequences was performed by using the pro¬
gram BLAST 2.0 (Altschul et al, 1997) for screening the NCBI nucleotide
database
(http://www.ncbi.nlm.nih.gov).
cessed with the GCG sequence
Gene
2.4.13
For
the
The sequences
were
analysis package (Devereux
mapping by using
et
somatic cell
further pro¬
al, 1984).
hybrids
mapping of the porcine sodium channel a subunit type II (SCN2A) gene,
porcine potassium channel subfamily J, member 3 (KCNJ3) gene, the nico¬
cholinergic receptor a\ (CHRNAl) gene, and the spastin SPG4 gene
as the sequence tagged sites (STSs) obtained from the PAC library,
the INRA somatic cell hybrid panel, containing 27 pig x rodent cell hybrids
was used (Yerle et al,
1996). For the candidate genes, porcine specific or
The gene specific annealing
cross species primers in exons were designed.
tinic
as
well
temperatures, the fragment length and the GenBank Accession numbers
are
performed on genomic DNA ex¬
As a positive con¬
tracted from each of the 27 hybrid cells of the panel.
trol, genomic DNA from porcine spleen cells was used. Genomic DNA from
mouse and Chinese hamster parental cells were used as negative controls.
The amplified products were separated on a 2% agarose gel and stained with
shown in Table 2.2.
ethidium bromide.
PCR reactions
PCR results
were
evaluated
were
using the interpreting web
http://www.toulouse.inra.fr/lgc/pig/hybrid.htm
al, 1997).
page
at
INRA
(Chevalet
et
mapping of the porcine SCN2A gene, the primer pair SCN2A.1, which
amplifies a 190 bp fragment corresponding to nucleotides 23 to 212 of human
For
SCN2A
(GenBank
mouse
To map the
a
Accession
M55662)
screening. As with pig,
hamster,
bp
detected, each fragment
fragment
was purified from the gel, sequenced and new pig specific primers for SCN2A
(SCN2A.2) were designed.
and
DNA
porcine KCNJ3
fragment corresponding
was
used for
of 190
a
gene,
was
primer pair KCNJ3.1
was
used to
amplify
to nucleotides 1368 to 1909 of the human sequence
(GenBank Accession U39196). A fragment of 541 bp was obtained with pig,
hamster, and mouse DNA. After sequencing of these fragments, pig specific
primers (primer pair KCNJ3.2) were derived.
Primer
pair CHRNAl.1 amplified
corresponding
Accession
For
a
199
bp fragment only with pig DNA
to nucleotides 591 to 789 of the human CHRNAl gene
(GenBank
S77094).
mapping of the porcine SPG4 gene, primer pair SPG4-1 was designed
primer in exon 10 and the reverse primer in exon 12 according
with the forward
to the human sequence
(GenBank
Accession
AJ246001).
A
fragment
of ~1'200
DNA methods
2.4-
Table
25
Primer pairs,
2.2:
GenBank Accession
of
annealing temperature T„(°C), fragment size and
for mapping with the somatic cell hybrid
the genes used
panel.
Primer
bp
was
SPG4-2)
Ta(°C)
size
(bp)
Ace. No
SCN2A.1
60
190
M55662
SCN2A.2
62
125
AF540390
KCNJ3.1
58
541
U39196
KCNJ3.2
66
182
AF540391
CHRNAl
66
199
S77094
SPG4.1
64
~1'200
AJ246003
SPG4.2
64
253
AF540392
obtained and
was
primer
pair
sequenced. Within the forward
sequence,
an
intron
reverse
previous forward primer (primer pair
253 bp product, which amplified from only porcine genomic
created and used with the
to obtain
a
DNA.
Fluorescence in situ
2.4.14
hybridization
mapping of the porcine calcium channel ß\ subunit gene ( CA CNB/) with the
hybrid panel was not possible, the fluorescence in situ hybridiza¬
A A-genomic DNA fragment carrying the
tion (FISH) technique was used.
was
CACNB4 gene (chapter 2.4.15)
subjected to FISH on porcine metaphases.
As
somatic cell
Q-banded and photographed to define the chromosomes and chro¬
hybridization. The genomic probe was labeled with
biotin-16-dUTP by random priming. Signal detection and amplification were
performed using the complex avidin-FITC and biotinylated anti-avidin. The
These
were
mosomal segments before
chromosomes
relative
were
positions
counter-stained with
of the
signals
were
4,6-diamidino-2-phenylindole, and the
as described by Toldo et al.
determined
(1993).
2.4.15
For
FISH,
Library screening
a
porcine liver A-genomic library
derived from
was
screened with the PCR
product
primer pair CACNB4-1.
For the first
screening
in each of the 20 falcon tubes 50'000
pfu
were
incu-
Chapter
26
2.
Methods
fi\ XLl-BLue MRA (OD600=0.5 in 10 mM MgS04) at 37°C
Immediately, 7.0 ml of melted LB top agarose was added to the
cell suspension and the mixture was poured onto a 150 mm LB agar plate.
After incubation at 37°C for 7 h and chilling at 4°C for at least 1 h, the
bated with 600.0
for 15 min.
phages
were
transferred to
a
nylon
immersed afterwards for 5 min in
tion.
After
rinsing with
membrane for 2 min. Each membrane
denaturing
2x SSC the DNA
at 80°C for 2 h.
solution and
was
was
solu¬
crosslinked to the membrane
then prehybridized in a 150
hybridization solution (3.0 ml/membrane) at 50°C for 4
h. Meanwhile PCR products (25.0 ng) were randomly primed with [a—32P]dATP using the Prime-It II Random Primer Labeling Kit from Stratagene®
(http://www.stratagene.com/manuals/300385.pdf). Hybridization was carried
out with 2.0 ml hybridization solution per membrane for 20 h. The membranes
were washed with 2x SSC, 1% SDS, starting from r.t.
up to 65°C until the
ratio signal to background was acceptable. X-ray films were exposed to the
membranes at —80°C at least o/n until positive clones could be identified.
The plaques which gave a positive signal were picked with the thicker end of
For the
a Pasteur pipette and transferred into 1.0 ml of lx A-dilution buffer.
secondary screening 200 pfu and 20'000 pfu of each isolate were replated on 90
The positive clones were picked
mm plates and screened as described above.
from the agar plate with the thinner end of a Pasteur pipette and transferred
into 1.0 ml of lx A- dilution buffer. For isolation of DNA 30'000 pfu were plated
on a 90 mm plate as described above. After incubation at 37°C o/n the plates
by baking
mm petri
were
The membranes
neutralizing
were
dish with
overlayed
The buffer
with 3.0 ml of lx A-dilution buffer and incubated at r.t. for 4 h.
was
removed, 1/50 volume of chloroform was added, vortexed for
for 10 min. After centrifugation the supernatant
1 min and incubated at r.t.
was
stored at 4°C.
screening of the cDNA library, Y1090r~ cells instead of XLl-Blue MRA
were
plates, each containing 30'000 pfu, two replicate filters were
which
were hybridized with labeled fragments derived from primer
prepared
and
CACNB4.4 (Table 2.1), respectively. After DNA purifica¬
pairs SPG4.3
For
used. From 12
tion
(chapter 2.4.4)
restriction enzyme
2.4.16
isolated clones
digestion
and
were
characterized
by
means
of
sequencing,
subcloning.
Ligation
Ligation of PCR products was performed with the pGEM(r)-T Easy Vector
System (Promega) according to the supplier's manual. Vector DNA (25.0 ng)
was incubated with 2x Rapid Ligation Buffer, 3 Weiss units of T4 DNA Ligase
and the prepared insert (molar ratio insert:vector
3:1) at 4°C o/n. Addi¬
tionally, control DNA provided with the Kit was used to determine whether
=
the
ligation
was
proceeding efficiently,
while vectors without inserts
were
used
DNA methods
2.4-
27
to determine the number of
For
blue colonies.
Transformation
2.4.17
fi\
background
transformation, the TOP10 One Shot
of One Shot cells thawn
mixed
gently
and incubated
on
on
(Invitrogen)
Kit
was
ice, 5.0 fi\ of ligation reaction
After
ice for 30 min.
used. To 50.0
was
heating
at
pipetted,
42°C for
pre-warmed SOC medium was added and the vials were
shaken at 37°C for 1 h at 225 rpm in a shaking incubator. From each vial,
20.0 /A and 200.0 fA were spread on agar plates containing the appropriate
antibiotic and incubated at 37°C o/n. For blue-white selection, the agar plates
30 sec, 250.0
were
fi\
of
covered with 4
mg/ml
plasmids
appear white
while the
background
as
X-Gal before
the lac-Z gene
plating
the cells.
The recombinant
expression is disturbed by the insert,
colonies appear blue due to the
from the lac-Z gene.
The white colonies
were
/3-galactosidase expression
picked and incubated in 5.0
o/n at 225 rpm in a shaking incubator. DNA was extracted
using the NucleoSpin® Plasmid Kit (Macherey Nagel) according to the protocol
(http : / /www. macherey-nagel. de).
ml LB at 37°C
Organization
2.4.18
The
of the PAC
library
porcine Pl-derived artificial chromosome (PAC) library of
Landrace
consists of 90'240 clones with
described
pig
by Al-Bayati
(1999),
a
male German
average insert size of 120 kb. As
the PAC
organized as follows:
plates and 95 384-well
microtiter plates. From each microtiter plate, DNA is pooled into ten plate
pools (PP). Ten PPs are combined in one super pool (SP). Five SPs are pooled
into one super/super pool (SSP). The identification of a positive PAC in a
microtiter plate is done by screening 8-row and 12-column pools.
The 90'240 clones
2.4.19
are
Library screening
were
was
library
is
stored in 560 96-well microtiter
Screening
the 13 SSPs
et al
an
of the PAC
library
performed by PCR
as
described in
chapter
screened with microsateUite SW902 which showed
2.4.6.
no
First,
recombi¬
nation with CPA. Then the five SPs
to determine the
round of
comprising the positive SSP were screened
positive PPs. Once the appropriate PP was identified, a final
amplification of the
intersection of the
8-row and 12-column
appropriate
row
and column
fied the correct clone. Genomic DNA of
control.
control.
a
on
pools
was
performed. The
plate identi¬
the microtiter
Large White pig
A PCR reaction mixture without any DNA
was
was
used
used
as
as
a
positive
negative
Chapter
28
Table 2.3:
:
Methods
Components of the SSCP gel.
Quantity (ml)
Components
Acrylamid
2.
bisacrylamide (49:1)
End concentration
8.75
10 %
3.5
10 %
3.5
1%
40% stock
Glycerol (100%,
water
free)
(10x)
TBE
H2Q
fill to 35 ml
Total volume
35
positive PAC clone
The
was
isolated
79%
100%
as
described in
of the 5' and 3' ends of the PACs
done
chapter
DNA
2.4.3.
described in
chap¬
sequencing
using the standard sequencing primers SP6 and T7 (Table 2.1). After
obtaining approximately 500 bp of each end sequence, primers were designed
for each end of the clone in order to amplify sequence tagged sites (STS). These
PCR primers were used to rescreen the PAC library to find overlapping PAC
clones. This strategy was repeated several times. The amplified STSs were
screened for SNPs using the SSCP technique (chapter 2.4.20) and mapped us¬
ing the somatic cell hybrid panel (chapter 2.4.13).
was
as
ter 2.4.11
2.4.20
Single
ysis
stranded conformation
polymorphism
anal¬
Single stranded conformation polymorphism (SSCP) analysis was performed to
identify single nucleotide polymorphisms (SNPs) in the sequence tagged sites
(STSs) obtained from the PAC contig harboring microsateUite SW902. The
amplified STSs were purified with spin columns (Millipore) and eluted in 10
mM Tris-HCl (pH
8.0). Before loading the samples on the SSCP gel, 5.0 fA
of DNA were mixed with 8.0 fi\ of formamide and denatured at 95°C for 5 min.
The gel was prepared according to the instructions in Table 2.3. The polymer¬
ization of the gel was initiated by adding 128.0 fA APS (10 % stock) and 32.0 fA
TEMED. After 1 h the gel was fixed into the gel apparatus (DCode TM, Univer¬
sal Mutation Detection System) and the samples were loaded. Electrophoresis
was performed in lx TBE buffer at 23 V/cm at 20°C for 8 h. Afterwards, the
=
gel
was
stained in 25.0 ml 1:10'000 diluted SYBRGold® solution. Pictures
taken with
a
Polaroid apparatus
exposure time with
f-stop
using
at 5.6 and
a
a
667 black and white
yellow
filter.
film,
one
were
second
RNA methods
2.5.
29
RNA methods
2.5
RNA extraction
2.5.1
Total RNA
was
isolated from 0.5 g of
cerebellum, heart, muscle and fat
tissue
using the Qiagen RNeasy Maxi Kit (Qiagen®). This kit combines the selec¬
tive binding properties of a silica-gel-based membrane with the speed of spin
technology. A specialized high-salt buffer system allows up to 6.0 mg of RNA
longer than 200 bases to adsorb to the RNeasy silica-gel membrane. The tissue
samples are first lysed and homogenized in the presence of a highly denaturing
guanidine isothiocyanate (GITC) containing buffer, which immediately inacti¬
vates RNases to ensure isolation of intact RNA. Ethanol is added to provide
appropriate binding conditions, and the sample is then applied to the RNeasy
column where the total RNA binds and contaminants
away.
High-quality RNA
Diluted RNA
tometer at
(10.0 fA RNA,
OD26o and OD280.
diluted
An
single
stranded RNA
DNA concentration
Pure RNA has
2.5.3
Gel
Before
loading
a
(Birren
1:50)
OD26o
was
of 1
(/xg/ml)
ratio of
=
et
measured
OD26o/OD2go
electrophoresis
on
a
measured in the
corresponds
al, 1997).
calculated using the following equation:
of
washed
efficiently
of RNA
Quantification
2.5.2
are
is then eluted in RNase-free water.
OD26o
to 40
spectropho¬
/zg/ml
solution
The RNA concentration
x
x
1
was
dilution factor
OD26o
between 1.9 and 2.1.
of RNA
1.25% formaldehyde-agarose gel, 3.0 fig of RNA
were
de¬
containing MOPS, formamide and
formaldehyde, and cooled on ice. The RNA was separated by electrophoresis
in lx MOPS. In order to visualize the 18S and 28S rRNAs, the gel was stained
natured at 60°C for 15 min in
with
methylene
a
mixture
blue and destained in H20 until the rRNA bands
28S ribosomal RNA bands should be present with
an
were
visible.
intensity approximately
twice that of the 18S RNA band.
2.5.4
Reverse
transcription
fA. A mixture
containing 2.5 fig RNA, 3.0 fiM Poly-A specific T(17)VX primer (Table 2.1)
and DEPC treated ddH20 up to 12.7 fA was incubated at 70°C for 5 min
Reverse
transcription
was
carried out in
a
total volume of 25.0
Chapter
30
and cooled down to
Methods
2.
The following substances
/xM dNTP's, 25 U of RNasin, 4 mM
Na-Pyrophosphate and 15 U of AMV-reverse transcriptase. The mixture was
incubated for 1 h at 42°C, 10 min at 55°C and stopped for 10 min at 72°C.
For the following PCR, 5.0 /xl were used as template.
were
added:
room
temperature for 10 min.
5x RT reaction
buffer,
250
Rapid amplification
2.5.5
of cDNA ends
Rapid amplification of the cDNA end (RACE) of the CACNB4 gene was per¬
using a 5' RACE Kit (Roche Molecular Biochemicals). First strand
cDNA is synthesized from total or poly(A)+ RNA using a gene specific primer
SP1 (Table 2.1), AMV reverse transcriptase and the deoxynucleotide mixture.
The cDNA is purified from unincorporated nucleotides and primers by the
High Pure PCR Product Purification Kit. Terminal transferase is used to add
a homopolymeric A-tail to the 3' end of the cDNA. The tailed cDNA is then
amplified by PCR using the gene specific primer SP2 (Table 2.1) and the oligo
dT-anchor primer, followed by a second PCR using the nested, specific primer
SPS (Table 2.1) and the PCR anchor primer.
The obtained fragment was
subcloned and sequenced.
formed
Northern blot
2.5.6
For the Northern blot
cerebellum of
trophoresed
as
healthy
analysis
analysis,
20.0 /xg of total RNA from
heart, fat and
animals and cerebellum of affected animals
described in
chapter
To bind the RNA the membrane
2.5.3 and transferred to
a
nylon
were
elec-
membrane.
baked at 80°C for 2 h. The blot
was hy¬
product of pig cerebellum, specific for
CACNB4 (primer pair CACNB4-7). In Figure 2.1, the alignment of deduced
amino acid sequences of four neuronal ß subunits (ßi-n) is shown. Primer pair
CACNB4-7, amplifying a fragment corresponding to nucleotides 1228 to 1437
of the human CACNB4 gene (GenBank Accession AF038852) was chosen in a
was
bridized with the radio-labeled RT-PCR
region where the
The
probe
amino acids showed
was
stripped
of O.lx SSC and 1%
sisting
porcine 18S ribo probe.
2.5.7
For
and
the
Slot blot
a
low
similarity.
from the membrane with
SDS,
and the membrane
boiling solution con¬
was rehybridized with a
a
analysis
quantifying mRNA expression of the CACNB4
healthy animals, the slot-blotting technique was
gene of several affected
used.
The total RNA of
following tissues was chosen: cerebellum of six affected and two healthy
animals, M. quadriceps of an affected and a healthy animal and as negative
RNA methods
2.5.
31
Cab4hum
KVTEHIP
Cab3rat
-QA--V-
Cab2Arat
PFF-K
T-
CablBrat
-S
V-
Cab4hum
GRISITRVTA
Cab3rat
PYDVVPSMRP
VVLVGPSLKG
YEVTDMMQKA
LFDFLKHRFD
243
2 09
E
II
DISLAKRSVL
261
NNPSKRAIIE
-L
258
G--T
RSNTRSSLAE
--SA
VQSEIERIFE
1--
2 93
259
Cab2Arat
H
308
CablBrat
HI
311
Cab4hum
LARSLQLWL
Cab3rat
--K
Cab2Arat
T
CablBrat
T
DADTINHPAQ
A-
Cab4hum
SQSKHLNVQL
VAADKLAQCP
Cab3rat
--M
T--M
M-Y
Cab2Arat
--A
M
CablBrat
1
Cab4hum
ATHTTSSTPM
LIKTSLAPII
VHVKVSSPKV
-A
-F
-S
-Y--I
358
-S
-YI-IT
361
P
EMFDVILD
V
R
ENQLED
-S
D-
-Q-S
A-SE
1
-
TPLLGR
N
L
G
S
LQRLIKSRGK
TA
343
309
LGEYLEAYWR
392
-A
358
V
-AD
K
408
-A
K
410
PYPTAIS
423
S
AGGA
~
Cab3rat
-S--P
377
Cab2Arat
HPAPGPP--NLP
N
S-
T
-ATS
P
LS--LA-
439
CablBrat
PP-R--P
N
N-
T
M
A
VS-APV-
441
Cab4hum
GLQSQR
Cab3rat
N-Q
GM--
_
A
T
A
DENYHNERAR
KSRNRLSSSS
470
-ASE
S--QAWTG--
418
MRH
SNHSTENSPI
ERRSL
MTS
LL
GERGE-H--L
--D--
-P-
-
Cab2Arat
NS-GSQGDQR TDR-APR-AS
QAEEEPCLEP
VKKSQHRSSS
ATHQNHR-GT
489
CablBrat
N--GPY
EHA-VHEYPG
ELGQPPGLYP
SNHPPGRAGT
490
Cab4hum
Q
LVS
H
S
GDQPLDRATG
R
D
H
Y
P
L
VEE
DYPDSYQ
489
CA
Cab3rat
-
S
R
-
L--
--A-A--
434
Cab2Arat
G
R
G
L
S
R
Q
E
T
FDS
ETQE-RD
508
CablBrat
L
W
A
L
S
R
Q
D
T
FDA
-T-G-RN
509
-
Alignment of
deduced ammo acid sequences of four neuronal cal¬
ß subunits. Shown is the partial alignment of the deduced ammo
acid sequence of human brain ß± (Cab4hum, GenBank Accession AF038852),
rat brain ß% (CabSrat, GenBank Accession M88751), rat brain /52a (Cab2Arat,
GenBank Accession M80545) and rat brain ßn> (CablBrat, GenBank Accession
X61394). Regions of sequence identity are indicated by dashes, dots represent
Figure
cium
gaps
and
2.1:
channel
m
the sequence. Shaded nucleotides
471-479 indicate the specific
primer
corresponding to ammo acids 409-416
pair for CACNB4 (CACNB4-7).
Chapter
32
control heart and fat tissue of
a
healthy
animal.
with 5 M NaOH and DEPC-treated water. The
of Whatman paper
paper
was
placed
were
on
soaked in 20x SSC for
The manifold
nylon
a
Methods
2.
was
membrane and
was
closed and the slots
a
sheet
few minutes. The Whatman
the lower part of the manifold and the membrane
onto it. The manifold
cleaned
were
was
laid
rinsed twice with 500.0
of lOx SSC. Of each tissue 1.0 fig, 3.0 fig, 6.0 fig and 9.0 /xg
were
fi\
denatured
SSC, 7% formaldehyde (vol/vol) and 50 %
The samples were put on ice, 60.0
/xl of 20x SSC were added and the samples were loaded into the slots of the
filtration manifold. Using a vacuum pump the sample load was pulled through
until no liquid was in the wells. After the slots were rinsed twice with 500.0 fi\
lOx SSC, the membrane was baked at 80°C for 2 h and hybridized as described
in chapter 2.5.6.
in
a
final volume of 30.0
formamide
2.6
(vol/vol)
/xl
in lx
at 68°C for 15 min.
Statistics
Linkage analysis
was performed by sequential insertion
FIX, and FLIP options.
sis
were performed
al, 1990). Multipoint analy¬
of the microsatellites with the BUILD,
and calculation of recombination fractions
with the CRI-MAP version 2.4 program
(Green
et
Chapter
3
Results
Phenotype approach
3.1
Congenital progressive ataxia (CPA) and spastic paresis in pigs is a new
hereditary movement disorder, recently identified in Switzerland. To charac¬
terize the phenotype of affected pigs more precisely, clinical, pharmacological,
neurophysiological, biochemical, and pathological studies were carried out.
The
Clinical examination
3.1.1
The movement disorder
birth
or
birth
require
can
be observed in both
within the first three
days.
Those
sexes
piglets
either
affected
immediately
immediately
after
after
prolonged time to get up and reach the udder. Characteristics
of affected piglets are lying on one side, paddling, and trying to rise. If they
are standing they show a spastic gait and incoordination.
Usually, they are
willing
and able to suckle.
If the
ataxia
a
can
piglets develop symptoms
after the first
day, slight weakness and
drifting to one side after walking
and remain in lateral recumbency,
be observed in the hind limbs with
for
a while. Finally, they fall on their sides
attempting to rise and raising their heads. After rest their condition is strik¬
ingly better than after walking or suckling. These animals are eager to drink
and always go to suckle with the normal siblings.
When the
piglets
were
Their average life span
unable to stand up anymore,
was
8.7 ± 8.3
were
euthanized.
=
animals) days
in
there
differences in other clinical features between affected and unaf¬
fected
were no
pigs.
family
All animals
1
(see
they
35
(mean ± standard deviation for n
3.4). Despite these severe impairments,
were
Table
alert and
33
reflexes, skin sensitivity and muscle
Chapter
34
3.
Results
Table 3.1: Quantitative Electromyography (EMG) and Electroencephalogra¬
phy (EEG) of two affected and one unaffected pig. Duration, amplitude, and
frequency was equal m all three animals. For EMG, no frequency and for EEG
no
duration
was
measured.
Amplitude
Duration
EMG
1-5
EEG
tone
were
The
imide
20-200
ms
15-95
-
normal. Tilted
heads, nystagmus,
Frequency
/xV
fiX
or
15-35 Hz
convulsions
were
not observed.
therapeutic application of Neostigmin (Konstigmin®) and Ethosux¬
(Suxinutin®)
exerted
no
effect in the affected animals
on
their movement
disorder.
3.1.2
Consanguinity
by CPA were compared. All families had
boar, which generated the K7 line. This boar was
imported from the United Kingdom in 1978. A close relationship was found
between families 1, 3 and 4 (Table 3.4). In the pedigree of family 1, the mother
of the boar of matings 1 and 2 is a sister of a boar which is grandfather of
the sows of family 3. Moreover, this boar is a great-grandfather of the sow of
family 4.
The
the
pedigree
of the families affected
same common
3.1.3
ancestor
Neurophysiological
Neurophysiological
piglet.
studies
were
studies
performed with
two affected and
one
healthy
Electromyography (EMG) of the muscles of the left front and hind limb re¬
prolonged insertion activity which outlasted the cessation of the needle
movement (no data available). The fibrillation potentials ranged from 20-200
/xV with durations of 1-5 ms (Table 3.1, Fig. 3.1), which is in the range con¬
sidered normal (S. Cizinauskas, personal communication). The motor nerve
conduction (NCS) of the left peroneal nerve was examined (Table 3.2). No
vealed
difference
The
latency between affected and unaffected animals.
amplitude of the recorded compound muscle action potential seemed to be
was
found in the
lower in the affected animals. Nerve conduction
velocity
was
not determined.
Phenotype approach
3.1.
35
(\*4?A<-r~f*~
100
200 U.V
•4—1
ms
Figure 3.1: Needle electromyography (EMG) of the infraspinatus muscle of
Fibrillation potentials are in the range considered normal.
an affected pig.
Table
nerve
Motor
3.2:
of
two
conduction
nerve
affected
and
one
latency
unaffected piglet.
Animal
and
Amplitude
Latency
unaffected
pig
amplitude of the peroneal
3.7
ms
19.1 mV
affected
pig
2.9
ms
5.7 mV
affected
pig
3.7
ms
10.9 mV
Electroencephalography (EEG) recorded a high voltage fast activity pattern
fiV and 95 fiV in all three animals (Table 3.1).
between 15
3.1.4
Hematology
and chemical parameters
Hematology parameters were in the range considered normal in pigs (BauerPham et al., 2001). In blood samples taken at rest, levels of LDH, CK, and
ASAT in 6 unaffected pigs (Table 3.3) were in the range considered normal
in swine (Bauer-Pham et al, 2001). In 13 affected pigs, the levels of CK and
ASAT
were
not increased
(Table 3.3).
animals than in unaffected
in
pigs (Bauer-Pham
et
LDH levels
pigs, but are
al., 2001). Physical
seem
to be
higher
in affected
still in the range considered normal
exercise resulted in
comparable
Chapter
36
Table 3.3:
Clinical
and 13
fected
affected
Unaffected
(U/liter)
ASAT
CK
m
Affected
pigs
6
unaf¬
pigs
L353.33 ± 226.06
93 ± 26.22
38.46 ± 10.61
445 ± 209.83
198.76 ± 112.04
(U/liter)
increases in lactate levels in both groups, while CK
activity
was
not altered
by
pigs (not illustrated).
Pathological
3.1.5
plasma of
1'046.23 ± 221.68
(U/liter)
exercise in affected
venous
Results
pigs at rest.
Parameter
LDH
data determined
chemistry
S.
histological
and
examination
brain, spinal cord, muscles and
staining methods (HE
and Luxol Fast Blue) were carried out. No macroscopical or histopathological
alterations were detected in any brain region, spinal cord or nerve tissue. In
two affected, but also in one healthy animal chronic-inflammatory alterations
Detailed
nerves
neuropathological examinations
animals, including
of the blood vessels in the muscle
be confirmed in the
The CPA
can
be
diagnosed,
sexes are
were
are
if the
two different
observed. These observations could not
13 affected
diagnosis
clinical symptoms
both
•
remaining
Clinical
3.1.6
•
of the
of 15 affected
piglets.
of CPA
following symptoms
can
be observed:
observed within the first three
days
after
birth;
involved,
the affected animals show
ataxia in the hind
spastic gait, incoordination, and progressive
limbs,
observed,
•
no
healing
•
no
pathological changes
process is
in other organs
a
can
in the
be found.
brain, spinal cord,
nerves, and
muscle,
nor
Assignment of
3 2
37
of CPA
Assignment
3.2
Mode of inheritance
3.2.1
The disease
was
ings 1 and
2),
The dams
were
(69 6%)
first observed
two litters of pigs
in
derived from two dams and
cousins
Of the 23
generations
16
CPA
were
(Table
and not related to the sire,
offspring,
normal
seven
3
4, family 1,
mat¬
sire, all of
one
(30 4%)
were
Large White origin
referring to the last two
found to be affected and
The observed ratio of
approximately 1 3 suggested
allele Therefore, affected ani¬
that the disease may be controlled
by a recessive
homozygous for the recessive allele ( cpa/cpa), and normal
animals either heterozygous for the recessive allele (CPA/cpa), or homozygous
(CPA/CPA) To confirm the autosomal recessive inheritance, two males and
mals
considered
were
five females of the 16
produced
animals
(19 4%)
phenotypically normal animals were mated Each dam
Of these 144 descendants, 28
3 4, matings 3-15)
(Table
two litters
showed signs of ataxia and paresis
The x2-test, calculated
data, showed that the observed ratios of the cpa vs CPA
2 37,
deviate significantly from the expected 1 3 ratio (\2
from the segregation
alleles did not
0 1<P<0
2,
1
=
df)
The ratio of 1 3 of cpa
which CPA occurred
Based
on
autosomal
3.2.2
these
recessive
Genetic
vs
(Table
3
CPA could also be observed
in
other families
in
4, mating 16, 19, 20)
observations, the CPA
is
assumed to be inherited
as
an
disease
mapping
of the CPA
phenotype
A
panel of 38 microsatelhtes was screened for linkage with the unknown gene
responsible for CPA in family 1, matings 1 and 2 (Table 3 4) From each
chromosome one to three microsatelhtes according to the length of the chro¬
The genome scan revealed a not significant but rela¬
mosome were analyzed
tively high linkage (Z=l 81, 6=0 00) between CPA and SW1066 (Gmur, 1997)
locus
phenotype more precisely, six additional markers (S0216,
SW2618, SW902, S0094, SW460 and G ACT, Table 2 1) m close proximity to
SW1066 were selected for further genotypmg in order to generate a multipoint
size in bp), located
map The analysis revealed that the SW902189 allele (189
on pig (Sus scrofa) Chromosome 3 (SSC3), co-segregated 100% with the reces¬
sive allele involved in the disease, while the SW902197, SW902204, or SW902214
To map the CPA
=
co-segregated 100%
study have already
(Kratzsch et al, 1999)
alleles
with the normal allele
of this
been
published based
(Table
on
a
3
4, Fig 3 3)
smaller
family
Parts
material
Chapter
38
Table 3.4:
SW902,
3.
Results
Transmission patterns of CPA
located
on
SSC3 in
phenotype and marker
offspring, produced by different mating
pairs.
No
Deduced
Family
Mating
No
No
1
1
CPA,SW902
haplotypes
F
x
2
cpa,l89/CPA,204
M
Affected
(CPA/CPA,
(cpa/cpa)
cpa/CPA)
4(189/189)2
5(189/204)
cpa,189/CPA,204
cpa,189/ CPA, 204
x
Not affected
Total
10
1(204/204)
3(189/189)
cpa,189/ CPA, 197
4(189/197)
13
5(189/204)
1(197/204)
3
cpa,189/C/M,204(Ml)
x
5(189/189)
cpa,189/C/M,197(M2)
2(189/197)
13
2(189/204)
4(197/204)
4
cpa,189/ CPA, 204(M2)
x
5
2(189/204)
CPA,204/ CPA,204(M1)
4(204/204)
cpa,189/C/M,204(Ml)
x
1(189/204)
CPA, 197/CPA,204(M2)
3(197/204)
2(204/204)
6
cpa,189/ CPA, 204(M2)
x
7
cpa,189/C/M,204(Ml)
x
8
9
3(189/189)
3(189/204)
3(189/189)
cpa,189/C/M,204(M2)
cpal89/C/M,197(M2)
5(189/204)
10
2(204/204)
5(189/204)
CPA,204/CPA,204(M1)
cpa,189/C/M,204(Ml)
x
13
7(204/204)
cpa,189/C/M,204(M2)
x
10
7(189/204)
cpa,189/C/M,204(Ml)
cpa,189/C/M,204(M2)
x
2(189/189)
cpa,189/C/M,204(M2)
4(204/204)
2(189/189)
5(189/197)
14
6(189/204)
1(197/204)
11
cpa,189/C/M,204(Ml)
x
CPA,197/CPA,204(M2)
4(189/197)
14
5(189/204)
4(197/204)
1(204/204)
alleles CPA and
alleles 189, 197, 204, 214; F, paternal haplotype; M,
cpa; SW902:
haplotype; (Ml): offspring from mating 1; (M2): offspring of mating 2, ?: unknown
2
Genotype of SW902
maternal
3 2.
Assignment of
CPA
39
Table 3.4:
(continued)
No
Deduced
Family
No
Matmg
No
12
CPA,SW902
haplotypes
cpa,189/C/M,204(Ml)
x
13
14
(CPA/CPA,
cpa/CPA)
2(189/189
8(189/204)
Total
13
3(204/204)
3(189/189
cpa,189/C/M,204(Ml)
cpa,189/C/M,204(M2)
x
Affected
(cpa/cpa)
cpa,189/C/M,204(Ml)
cpa,189/C/M,204(M2)
x
Not affected
7(189/204)
11
1(204/204)
4(189/189
cpa,189/C/M,197(M2)
2(189/197)
11
3(189/204)
2(197/204)
15
cpa,189/C/M,204(M2)
x
2
16
cpa,189/C/M,204(M7)
cpa,189/CPA,214
x
4(189/189
9(189/204)
15
2(204/204)
3(189/189)
cpa,189/CPA,204
2(189/204)
8
1(189/214)
2(204/214)
3
17
x
18
19
20
6
21
22
Total
?
1
2(189/189)
4(189/204)
7
1(204/204)
3(189/189)
6(189/204)
11
2(204/204)
2(189/189)
cpa,189/CPA,204
1(189/204)
3
?
1(189/189)
?
x
1(189/189)
cpa,189/CPA,204
cpa,189/CPA,204
x
8
2(204/204)
cpa,189/CPA,204
cpa,189/CPA,204
x
3(189/204)
cpa,189/CPA,204
cpa,189/CPA,204
x
5
cpa,189/CPA,204
?
x
4
3(189/189)
?
?
1
cpa,189/CPA,204
50
156
206
Chapter
40
8
Marker
-SIJ2618
cM
cM1
0.0
0.0
-IL1-E
3.
Results
AlogL
2.9
15.3
0.02
0.02
-80094
13.4
7.0
-SU902, CPA
15.2
7.6
-SU1066
17.0
9.7
-SW460
19.3
10.2
-GRCT
20. S
11.8
0.02
0.02
4.0
6.4
9.7
3.4
27.5
q2
3-2 5
SSC3
1-50216
Figure
3.2:
mapping
of
Physical
CPA gene
and genetic
(cM:
by
inversion
Rohrer et
map
35.3
(sex-averaged) of
of adjacent loci).
estimated genetic distances
SSC3 and
cM; 9:
cM1
:
multipoint
from
analysis;
genetic dis¬
al. (1996); A log L: differences m likelihood against the
estimated recombination rates
tances
linkage
46.0
the
m
Kosambi
3.2.
Assignment of
Heterozygous boar,
CPA
earner
41
(189/204)
-6000
-4000
-2000
„^X"1
Heterozygous
sow,
earner
„A--
(189/204)
-6000
-4000
-2000
Homozygous offspnng, affected (189/189)
-6000
-4000
-2000
Homozygous offspnng, healthy (204/204)
-4000
-2000
„A-J
Figure 3.3: Electropherogram of microsateUite SW902 alleles on SSC3 of a
family affected by CPA. The size of the alleles is given m bp. The numbers at
the vertical scale display the fragment quantity m terms of peak height.
Chapter
42
Pairwise lod
scores
3.
Results
CPA and the
and recombination fractions for
seven
presented in Table 3.5. High lod scores of 23.18, 16.91, and
11.40 were obtained for linkage of CPA with markers SW902, SW1066, and
S0094, respectively. The markers achieve a lod score of 66.50 for SW902 and
SW1066 and 40.12 for SW902 and S0094. Recombination was estimated to
be 0.03 between SW1066 and CPA, 0.02 between S0094 and CPA, while no
marker loci
are
recombination occurred between SW902 and CPA.
computationally not feasible to perform a multipoint linkage analysis
considering all eight loci jointly with n!/2 possible locus orders. Thus, the
order SW2618-SW902-GACT-S0216 was fixed according to the genetic map of
Rohrer et al (1996), and the loci SW460and SW1066 were inserted sequentially
with the CRI-MAP "build" option. The most likely order SW2618-SW902SW1066-SW460-GACT-S0216 fitted the data best, in accordance with Rohrer
It
(1996).
et al
a
was
factor of
The likelihood of six other loci orders did not differ
1000,
and
Similar results
they
were
were,
therefore,
not considered
obtained when other loci
were
by more than
significantly different.
assumed to be in
a
fixed
order, and subsequently two additional loci were inserted. The marker or¬
der described by Rohrer et al (1996), that is SW2618-S0094-SW902-SW1066SW460-GACT-S0216, was never rejected by our data. Therefore, this order
subsequent analyses. As expected, the estimated genetic distances
are not completely in accordance with the data of
al (1996) (Fig. 3.2), probably owing to the different family material
used in
was
and recombination rates
Rohrer et
and limited number of meiosis.
The two orders of CPA in
In both cases, CPA showed
gle
best
fitting
order
no
adjacent
intervals to SW902 fit the data
recombination with
coinciding
equally.
SW902, thus revealing a sin¬
with SW902 and CPA
of CPA in the SW902 chromosomal
(Fig. 3.2).
3.2.3
CPA
was
reinforced
The location
significant al¬
lelic association found between CPA and SW902. The genotype SW902189/189
was found in all 50 affected animals (Table 3.4), whereas no healthy animal
possessed this marker genotype.
region
by
the
diagnostics
currently the genotype SW902189/189 has been found
no healthy animal possessed this genotype. There¬
we put forward the hypothesis that a positive test result (genotype
SW902189/189) leads to clinical CPA. Now CPA should be diagnosed by means
of the clinical phenotype (chapter 3.1.6) and analysis of the genotype. Still, it
Our studies revealed that
only
fore,
in affected animals and
=
has to be taken into consideration that all animals examined in this
related.
Therefore, the hypothesis
can
be valid
only
in this
Moreover, the knowledge of the genotype makes it also possible
guish
between
homozygote healthy
and carrier animals in
study
are
family.
our
to distin¬
family. Fig.
3.3
3.2.
Assignment of
Table 3.5:
cific
CPA
Two-point linkage analysis of CPA and
marker loci used
and lod
43
scores
(Z).
Locus
pair
for
estimation
of
sex
averaged
seven
chromosome
recombination
3-spefraction (9)
6
Z
S 0216
SW1066
0.27
5.34
SW2618
S W1066
0.11
13.81
SW2618
S 0216
0.25
2.56
SW902
S W1066
0.02
66.50
SW902
S 0216
0.26
4.70
SW902
SW2618
0.13
9.08
SW460
S W1066
0.04
17.04
SW460
S 0216
0.25
1.14
SW460
SW2618
0.14
2.53
SW460
SW902
0.03
20.12
S0094
S W1066
0.03
42.51
S0094
S 0216
0.22
4.42
S0094
SW2618
0.12
12.09
S0094
SW902
0.02
40.12
S0094
SW460
0.03
19.62
GACT
S W1066
0.03
41.81
GACT
S 0216
0.22
4.16
GACT
SW2618
0.13
10.95
GACT
SW902
0.04
33.65
GACT
SW460
0.02
21.99
GACT
S0094
0.07
35.79
CPA
S W1066
0.03
16.91
CPA
S 0216
0.24
0.57
CPA
SW2618
0.17
1.44
CPA
SW902
0.00
23.18
CPA
SW460
0.05
3.35
CPA
S0094
0.02
11.40
CPA
G ACT
0.04
9.84
Chapter
44
shows
3.
Results
electropherogram of a family with two heterozygous parents, a ho¬
homozygous healthy offspring (mating 1, Table 3.4).
The affected animal showed the genotype SW902189/189, while the healthy an¬
imal had the genotype SW902204/204. This healthy sow was mated to a carrier
boar twice, but never gave birth to affected piglets (Table 3.4, matings 4 and
9) as anticipated.
an
mozygous affected and
SW902189/189
To determine the distribution of the
boars which derived from the K7 line and
SUISAG,
SW902189/204.
at the
were
tested.
Only
one
are
allele in
Switzerland, the
used for artificial insemination
boar out of 83 revealed the genotype
Candidate genes
3.3
physically and genetically mapped
ql3-q21
(Fig. 3.2). This region is likely to correspond
to human 2ql-q2 region (Rettenberger et al, 1995; Pinton et al, 2000), where
ion channel genes (Ca2+, Na+, K+) and a cholinergic receptor gene are mapped.
SW902 is
mapped
in close
ILl locus in band
to the
proximity
of SSC3
These ion channels
epilepsy and ataxia in humans.
Furthermore, we mapped
SSC3q21-q27 using a somatic
cell hybrid panel. This region corresponds to human chromosome 2pl3-p24
(Pinton et al, 2000). To this region, the gene spastin (SPG4) was mapped
(Hazan et al, 1994; Hentati et al, 1994). Mutations in the SPG4 gene may be
responsible for Spastic Paraplegia (Hazan et al, 1999). Epilepsy, ataxia and
Spastic Paraplegia resemble the phenotypical appearance of CPA in the pig.
seem
to
be involved in
the marker SW902 to
3.3.1
3.3.1.1
Calcium channel
Regional
/?4
subunit
localization
mapping of the porcine calcium channel /?4 subunit (CACNB4) gene,
hybrid panel was used. Primers were designed based on
the corresponding human sequence (GenBank Accession AF038852).
The
panel was screened with three different primer pairs (CACNB4-1, CACNB4-2,
CACNB4-3). All three primer pairs showed positive signals of the expected size
For
the somatic cell
in cell lines
sults
by
fluorescence
the
shown). The reliability of these re¬
high error risk ( > 5%). Therefore, the
hybridization technique was additionally used for mapping
5, 12, 22, 23, and
24
(data
statistical evaluation showed
m
CACNB4
situ
not
a
gene.
Representative, Q-banded metaphase spreads from a normal pig were
probed with a A-genomic DNA fragment carrying the CACNB4 gene. After hy¬
bridization, the chromosomes were compared with the previous photographed
Candidate genes
3.3.
45
t
«t»
t
1»
#
1**1/
##
••
*
ait
JHP
tl&B
A
Figure 3.4: Fluorescent in situ hybridization (FISH) analysis of porcine
CACNB4-' QFQ-banded metaphase (A) prior to FISH (B) with the fragment
containing the porcine CACNB4 gene. Arrows indicate the hybridization region
at SSC3ql4-q21.
metaphases to determine the location of the signals. In Fig. 3.4A, a porcine
metaphase q-band painting is indicated, with dark and light bands defining
chromosomal segments. Fig. 3.4B shows the fluorescence signal, mapped to
the ql4-q21 region of chromosome 3.
3.3.1.2
Characterization
of the
cDNA
of affected
and
unaffected
pigs
CACNB4 cDNA was amplified from cerebellar RNA using primer pair
CACNB4-5. A fragment of about 1'400 bp was obtained which corresponded to
the predicted length of 1'491 bp in the rat. The fragment was sequenced using
primer pairs CACNB4-5 and CACNB4-6. An open reading frame (ORF) of
1'491 bp was confirmed which corresponded exactly to nucleotides 284-F774 of
the rat CACNB4 sequence (GenBank Accession L02315). As neither the start
nor the stop codon were found in this sequence, a porcine brain cDNA was
screened using primer pair CACNB4-4- Positive clones were sequenced and the
3' UTR could be identified. To determine the 5' UTR of the porcine CACNB4
gene, a rapid amplification of the cDNA end (RACE) was performed. Thus,
the missing start codon was obtained. Finally, an ORF of 1'509 bp was iden¬
Porcine
tified.
The sequence has been submitted to GenBank
The nucleotide and amino acid sequence with the
Fig.
3.5.
exon
(Accession AF540878).
boundaries is shown in
Chapter
46
3.
ATGGATGTGGTGGCCCAAGAAACCA.CGACCCA.GAAGAGCA.GGTTGAAAAGATATGATGGC
Results
6 0
MDVVAQETTTQKSRLKRYDG
\Exon2
AGCACCACTTCGACCAGCTTCATTCTCA.GACA.GbGTTCAGCGGATTCCTACA.CGAGCAGG
12 0
STTSTSFILRQGSADSYTSR
CCGTCTGACTCCGATGTCTCTTTGGAAGAGGATCGGGAAGCGATTCGGCAGGAGAGAGAG
180
PSDSDVSLEEDREAIRQERE
\Exon3
CAGCA.GGCCGCCA.TCCA.GCTTGAGAGAGCAAAGbcCAAACCCGTAGCA.TTTGCTGTGAAG
QQAAI
24 0
QLERAKSKPVAFAVK
ACGAATGTGAGCTACTGTGGTGCCTTGGACGAGGATGTCCCTGTTCCAAGCA.CGGCCATC
300
TNVSYCGALDEDVPVPSTAI
\Exon4
TCCTTCGACGCCAAGGACTTTCTCCACA.TTAAAGAgIaAATATAACAATGATTGGTGGATA
36 0
SFDAKDFLHIKEKYNNDWWI
GGAAGGCTGGTAAAAGAAGGCTGCGAGATTGGCTTCATCCCAAGTCCA.CTTAGGTTGGAG
42 0
GRLVKEGCEIGFIPSPLRLE
\Exon5
AACATACGGATTCAGCAAGAACAAAAGAGAGGACGTTTTCACGGAGGbAAATCCAGTGGA
480
NIRIQQEQKRGRFHGGKSSG
AATTCTTCTTCAAGCCTTGGAGAAATGGTATCAGGAACATTTCGAGCAACTCCCACATCG
54 0
NSSSSLGEMVSGTFRATPTS
I Exon 6
\Exon7
ACAQCAAAACAGAAGCAAAAAGTGkcGGAGCA.CATCCCCCCTTACGATGTTGTGCCATCG
TAKQKQKVTEHI
600
PPYDVVPS
I Exon 8
ATGCGTCCA.GTGGTGTTAGTGGGGCCGTCACTGAAGGGTTATGAGGTCACAGACATGATG
66 0
MRPVVLVGPSLKGYEVTDMM
\Exon9
CAGAAAGCCCTCTTTGATTTCCTGAAGCACAGGTTTGATGGGAGbATATCAATAACGAGA
72 0
QKALFDFLKHRFDGRISITR
GTGACAGCTGACA.TTTCTCTTGCTAAGAGGTCTGTCCTAAATAATCCCAGCAAGAGAGCA.
VTADI
780
SLAKRSVLNNPSKRA
\Exon10
ATAATTGAACGTTCGAACACTCGGTCCA.GCTTAGbGGAAGTACAAAGTGAAATTGAAAGA
84 0
IIERSNTRSSLAEVQSEIER
ATCTTTGAGTTGGCGAGATCTTTGCAACTGGTAGTTCTTGATGCAGACACCA.TCAATCAC
I
900
FELARSLQLVVLDADTINH
CCAGCACAACTTATAAAGACCTCCTTAGCACCAATTATTGTTCA.TGTAAAAGTCTCATCT
PAQLI
KTSLAPI
96 0
IVHVKVSS
\Exon11
ccaaag|gttttacagcggttgattaaatctagaggaaagtcccaaagtaaacacttaaat
102 0
pkvlqrliksrgksqskhln
\Exon12
GTTCAACTGGTGGCAGCTGACAAACTCGCACAATGTCCCCCcbAGATGTTTGATGTTATA
1080
VQLVAADKLAQCPPEMFDVI
CTAGATGAAAACCAGCTGGAGGATGCGTGTGAACATTTGGGAGAGTACCTGGAGGCCTAC
114 0
LDENQLEDACEHLGEYLEAY
TGGCGTGCCACTCACACGGCCAGCAGCA.CCCCCATGACCCCGCTGCTGGGAAGGAATTTG
1200
WRATHTASSTPMTPLLGRNL
I Exon13
GGCTCAACA.GCACTCTCGCCA.TATCCCA.CAGCCATTTCTGGGTTACAGkGTCAACGCATG
GSTALSPYPTAISGLQSQRM
126 0
Candidate genes
3.3.
47
AGGCACGGCAACCA.CTCCACAGAGAACTCA.CCAATTGAACGACGAAGTCTAATGACCGCC 1320
RHGNHSTENSPI
ERRSLMTA
GATGAAAATTATCA.CAATGAAAGGGCGCGAAAGAGTAGGAACCGCTTGTCTTCCAGTTCC
13 80
DENYHNERARKSRNRLSSSS
CAGCATAGCCGAGATCACTACCCTCTGGTGGAAGAAGATTACCCTGATTCCTACCAGGAC
1440
QHSRDHYPLVEEDYPDSYQD
ACTTACAAACCCCA.CAGGAACCGAGGATCGCCCGGGGGATACAGCCATGACTCTCGACAT
1500
TYKPHRNRGSPGGYSHDSRH
AGGCTTTGA
R
L
1509
*
Figure 3.5: Nucleotide and deduced ammo acid sequence of the porcine
CACNB4 gene. The initiator ATG codon and the stop codon TGA are shown
m
bold letters.
The
The 12 vertical lines represent the
porcine CACNB4
sequence
boundaries.
exon
analyzed using
was
the BLAST 2.0 pro¬
(Altschul et al, 1997) of the National Center of Biotechnology Infor¬
mation (http://www.ncbi.nlm.nih.gov) as well as the Pileup program of the
GCG sequence analysis package (Devereux et al, 1984). Figure 3.6 shows the
gram
comparison between the porcine CACNB4 amino acid
sequence
displaying 99%
(GenBank Accession AF038852), 98% simi¬
rat sequence (GenBank Accession L02315), 96% similarity to the
bovine sequence (GenBank Accession AF273332) and 96% similarity to the
mouse sequence (GenBank Accession AF039417).
similarity to
larity to the
the human sequence
screening of the cDNA of three affected animals was performed by
RT-PCR, subcloning and sequencing. A transition of cytosine (C) to thymine
(T) at position 1'398 (termed M1398) in exon 13 (Fig. 3.5) of the CACNB4
Mutation
ORF
was
identified in
resulted in the
of this
same
of the animals.
one
amino acid histidine
SNP, primer pair CACNB4-4
animals of different CPA genotypes
was
This alteration of the DNA codon
(H).
To determine the
used to
amplify
(CPA/CPA, CPA/cpa, cpa/cpa). Sequenc¬
ing of these sequences did not confirm the M1398 silent
including the affected, were homozygous M1398GIG.
3.3.1.3
carried out. Northern
while Slot
mutation. All
animals,
Expression study
To characterize the mRNA
were
polymorphism
the target sequence in
blotting
was
tissues of affected and
expression, Northern blot and Slot blot analyses
blotting
used to
was
performed to determine the RNA size,
the CACNB4 expression in different
measure
healthy pigs.
The Northern blot with
polyA+
RNA from
heart, fat, and cerebellum of
a
Chapter
48
MDV
pig
VAQETTTQKS
RLKRYDGSTT
human
NGT
ADGPHSPTSQ
--RG
RR-
S
rat
MSSSYAKNGA
ADGPHSPSSQ
--RG
RR-
S
Results
STSFILRQGS
bovine
pig
3.
RA
ADSYTSRPSD
SDVSLEEDRE
AIRQEREQQA AIQLERAKSK
PVAFAVKTNV
SYCGALDEDV
PVPSTAISFD
AKDFLHIKEK
YNNDWWIGRL
VKEGCEIGFI
150
PSPLRLENIR
IQQEQKRGRF
HGGKSSGNSS
SSLGEMVSGT
FRATPTSTAK
200
human
rat
bovine
pig
SH
human
rat
bovine
pig
human
rat
T
bovine
pig
QKQKVTEHIP
PYDVVPSMRP
VVLVGPSLKG
YEVTDMMQKA LFDFLKHRFD
250
GRISITRVTA
DISLAKRSVL
NNPSKRAIIE
RSNTRSSLAE
VQSEIERIFE
300
LARSLQLVVL
DADTINHPAQ
LIKTSLAPII
VHVKVSSPKV
LQRLIKSRGK
350
SQSKHLNVQL
VAADKLAQCP
PEMFDVILDE
NQLEDACEHL
GEYLEAYWRA
THTASSTPMT
PLLGRNLGST
ALSPYPTAIS
GLQSQRMRHG
NHSTENSPIE
450
YPDSYQDTYK
500
human
rat
pig
human
rat
pig
human
rat
mouse
pig
human
rat
pig
human
T
rat
S
V
mouse
S
V
pig
S
RRSLMTADEN
human
S
rat
S
mouse
S
pig
PHRNRGSPGG
S
YHNERARKSR
YSHDSRHRL*
F
S
NRLSSSSQHS
RDHYPLVEED
519
human
rat
mouse
3.6: A comparison of the porcine (GenBank Accession AF540878),
(GenBank Accession AF038852), rat (GenBank Accession L02315),
bovine (GenBank Accession AF27332)
and mouse (GenBank Accession
AF039417) calcium channel ßn subunit (CACNB4) ammo acid sequence. Iden¬
Figure
human
tities
m
ammo
(M) represent
acid sequences
the start
codon,
are
indicated
asterixes
(*)
by
dashes.
The bold methionines
indicate stop codons.
3 3
Candidate genes
49
28S
18S
<
CACNB4
-4
28S
-4
18S
(~9
0
kb)
18S
Figure
3.7:
Northern Blot
Tissues
were
collected
analysis for
from
and from
porcine
CACNB4 mRNA
(lane 1),
cerebellum (lane 3) of
heart
cerebellum
(lane 2)
expression
and
fat (lane 4)
an affected animal
healthy animal
RNA
blue
stained
separated electrophoretically on a 1 25%
methylene
formaldehyde agarose gel before hybridization
B Northern blot analysis with a cDNA radiolabeled probe specific for CACNB4
shows a transcript of ~P 0 kb for CACNB4 m cerebellum, none m heart nor
fat
C hybridization with an 18S probe as correction for the variation m the total
of
a
A
RNA amount
The
arrows
indicate the
size
of
the 18S
(~1
9
kb)
and 28S
(~4
7
kb)
rRNA
Chapter
50
3.
Results
analysis of porcine CACNB4 mRNA expression m cere¬
of affected and healthy pigs
A- Hybridization with a radiolabeled porcine CACNB4 cDNA probe
B- Hybridization with a radiolabeled porcine ß-actme probe as internal control.
Lanes 1-8, cerebellum; lane 9-10, M quadriceps; lane 11, fat; lane 12, heart
Affected animals are indicated by asterixes (*)
Figure
3.8:
Slot blot
bellum and muscle
healthy animal as well as polyA+ RNA from cerebellum of an affected animal
was hybridized to a labeled CACNB4 specific cDNA probe. A single transcript
of approximately 9.0 kb was only present in the cerebellum (Fig. 3.7 B). No
transcripts were detected in heart and fat tissue.
expression of CACNB4
healthy animals,
blot, containing polyA+ mRNA
tissue (Fig. 3.8). Affected and healthy
animals showed a hybridization signal only with cerebellum. The signal inten¬
sity corresponding to CACNB4 mRNA was corrected for the amount of RNA
loaded by hybridization with the housekeeping gene /5-actin.
Hybridization
signals were equal in affected and healthy pigs.
To determine the
the cDNA
probe
hybridized
from cerebellum, muscle, heart, and fat
was
Drug
3.3.1.4
also
were
seizures
treated with
a
day
in affected and
Slot
calcium current blocker, which is indicated in
al, 1998; CapoviUa et al, 1999). Affected animals
dose of 15 mg/kg twice a day parenteral for one week. As
as
(Escayg
a
the condition of the
twice
a
treatment
Ethosuximide is known
myoclonic
to
piglets
a
et
improve, the dose was increased to 20 mg/kg
Nevertheless, no improvement in comparison to
did not
for another week.
affected untreated animals could be observed.
3 3.
Candidate genes
51
•
4*
Regional localization of SCN2A by PCR analysis on
125 bp) could
hybrids Specific PCR fragments (size
lines 2, 4, 5, 6, 12, 16 and 23. Pig (P), hamster (H) and
Figure
3.9:
matic cell
m
cell
DNA,
=
(W)
and water
were
used
as
positive and negative controls
so¬
porcine
be detected
mouse
m-
(M)
50
bp
marker
Chromosomal
3.3.2
assignment
of
ion
other
channel
genes
3.3.2.1
Chromosomal
assignment
of
a
sodium channel
alpha
subunit
cluster
porcine SCN2A gene, primers were designed (SCN2A 1)
corresponding human sequence (GenBank Accession M55662).
The resulting PCR product was sequenced and the obtained sequence, exclud¬
ing the primer sequence (GenBank Accession AF540389) was submitted to the
In order to map the
based
on
the
BLAST program. An identity of 92% was found with the human SCN2A2 and
SCN3A cDNA, while identities of 88% and 89% were found with the human
SCN2A1 and SCN1A sequence,
termine which
respectively.
From
our
porcine sodium channel alpha subunit
data
was
we
could not de¬
sequenced. As the
clustered (Malo et. al, 1991), we presume that these isoby the same chromosome in the pig. Thereupon, pig specific
primers (primer pair SCN2A 2) were designed and a fragment of the expected
size of 125 bp was obtained. PCR revealed amplification products in cell lines
2, 4, 5, 6, 12, 16, and 23 of the somatic cell hybrid panel (Figure 3.9). Statis¬
tical evaluation predicted the gene to map to chromosome 15 with a maximal
correlation (1.0) and a probability of 0.88 for region ql5-q22. Probabilities for
murine subunits
forms
are
are
encoded
Chapter
52
250bp—
m
250bp-».f|
m
^
Results
^#B-<—182bp
Üt
iDemm
3.
m
p
^_182bp
Figure 3.10: Chromosomal assignment of porcine KCNJ3 using the somatic
cell hybrid panel. Fragments of the expected size of 182 bp could be found in cell
lines 2, 4, 5, 6, 12, 16 and 23. Pig (P), hamster (H) and mouse (M) DNAs
and water
controls,
other
(W)
m:
regions
50
on
were
run
in the
corresponding
porcine chromosome 15
less than 0.011 for
were
below P
qll and q23-q26, respectively.
porcine sodium channel alpha subunit cluster
3.3.2.2
lanes
as
positive and negative
bp marker.
Chromosomal
assignment
on
on
ql2-ql4 and
We therefore
mapped the
=
0.097
SSC15ql5-q22.
of KCNJ3
porcine KCNJ3 gene, primers (KCNJ3.Î) were designed based
corresponding human sequence (GenBank Accession U39196). A PCR
product of 541 bp was obtained with these primers with porcine DNA, hamster
and mouse genomic DNA. The fragments were sequenced and submitted to
the BLAST program. The porcine sequence (excluding the primer sequences,
GenBank Accession AF540391) showed 96% identity with the human, 93% with
the rat, and 92% with the mouse KCNJ3 gene. Moreover, an identity of 71%
was found with another porcine sequence, representing a cardiac-type inwardly
rectifying K+ channel. A pig specific primer pair was designed (KCNJ3.2),
with which the hybrid panel was screened (Figure 3.10). Only hybrid cell lines
0.88, error risk < 0.1%) showed
harboring the region SSC 15ql5-q22 (P
amplification products of the expected size of 182 bp. Conclusively, the gene
could be mapped to this segment.
To map the
on
the
=
Candidate genes
3.3.
M3
201
H
1
«
bp
201
4
5
6
7
8
9
M
20
21
22
23
24
25
26
27
P
Figure
3.11:
somatic cell
m
10
11
12
13
14
15
16
17
18
19
of porcine CHRNAl using the INRA
fragments of the expected size of 199 bp were
MS: marker; H: hamster background cell line
Chromosomal assignment
hybrid panel.
2, 4, 5 and
lanes
PCR
16.
mouse background
(negative control);
genomic DNA (positive control).
M:
3.3.2.3
M3
W-
«?
bp
found
3
j||
fit
M3
2
53
Chromosomal
assignment
cell line
(negative control);
P: porcine
of CHRNAl
mapping of the porcine CHRNAl gene, primer pair CHRNAl.1 was used
was designed based on the corresponding human sequence (GenBank
Accession S77094). A fragment with the expected size of 199 bp was only
obtained with pig genomic DNA (no amplification with hamster and mouse
For
which
genomic DNA). The porcine sequence (GenBank Accession AF540391), ex¬
cluding the primer sequences, showed 93% identity with the bovine, 92% with
the dog and 91% identity with the corresponding mouse and human CHRNAl
The PCR results from the rodent-porcine hybrid cell lines (Fig¬
sequences.
ure 3.11) were submitted to the statistical analysis program supplied for the
hybrid panel. The probability of CHRNAl being located on the q23-q26 re¬
gion of chromosome 15 was high (P=0.97) with an error risk lower than 0.5%.
Therefore, we mapped the porcine CHRNAl gene to SSC15q23-q26.
3.3.3
3.3.3.1
Spastic paraplegia
Mapping
of
SPG4 by
4
SPG4
somatic cell
hybrids
mapping of the porcine SPG4 gene the somatic cell hybrid panel was used.
Corresponding to the human sequence (GenBank Accession AJ246001) primer
For
Chapter
54
3.
Results
PI
0'<-CMco^rincD„t-
<-
CT-cNco'srm<oh-coo)'«-T-T-T-T-T-T-a.
250bp—
*wn*#
»
<
.,
mm, «•*
mm
Et-t-t-C\|C\|C\|C\|C\ICM<MC\|Q-IS>
250bp—
-
3.12:
E
4—253bp
PI
«tif
Figure
*»
£
*—
*
Chromosomal
253bp
assignment of porcine SPG4 using the INRA
so¬
hybrid panel.
Fragments of the expected size of 253 bp were
detected in lane 1, 3, 6, 7, 8, 9, 10, 14, 16, 19 and 23. m: 50 bp marker; P:
porcine genomic DNA, positive control; H: hamster background cell line; M:
mouse background cell line; W: water, negative control.
matic cell
PCR
pair SPG4-1
designed with the forward primer in exon 10 and the reverse
A fragment of ~1'100 bp was obtained and sequenced.
Within the forward sequence an intron reverse primer was created and using
this primer with the previous forward primer (primer pair SPG4-2) for PCR, a
product of 253 bp was obtained with porcine genomic DNA, but not with ham¬
ster and mouse DNA. The porcine sequence (GenBank Accession AF540392)
excluding the primer sequences, showed an identity of 87.5 % with the human
SPG4 gene (GenBank Accession AJ246003). Screening the somatic cell hybrid
panel showed amplification products in cell lines 1, 3, 6, 7, 8, 9, 10, 14, 16, 19,
and 23 (Figure 3.12). Statistical analysis revealed an error risk lower than 0.1%
with a probability of P
0.8 for a chromosomal location on SSC3q21-q27.
primer
in
was
exon
12.
,
=
3.3.3.2
Characterization of the
fected
SPG4
cDNA of affected and unaf¬
pigs
screening of the porcine brain cDNA library, a primer pair (SPG4-3) in exon
corresponding human SPG4 sequence (GenBank Accession AJ246001)
A fragment with the expected size of 151 bp was obtained.
was designed.
Analysis of the sequence without the primer sequences using the BLAST pro¬
gram (Altschul et al, 1997) showed high similarity to the human and mouse
SPG4 gene, 85% and 77%, respectively. This fragment was used for screening
For
5 of the
Candidate genes
ATGGCCGCCAAGAGGAGCTCCCGGGCTGCGCCGGCCCCGGCCTCGGCCTCGCCCCCGGCG
60
MAAKRSSRAAPAPASASPPA
CCGGTGCCAGGCGGGGAGGTCGAACGAGTACGAGCCTTCCACAAACAGGCCTTCGAGTAC
12 0
PVPGGEVERVRAFHKQAFEY
\Exon2
ATCTCCGTTGCCCTGCGCATCGACGAGGACGAGAAAgItAGGACAAAAGGAGCAAGCTGTG
18 0
ISVALRIDEDEKVGQKEQAV
GAATGGTATAAGAAAGGTATTGAAGAACTAGAAAAAGGAATTGCCGTTGTAGTTACAGGA
24 0
EWYKKGIEELEKGIAVVVTG
\Exon3
CAAGGTGAACAGTGTGAAAGAGCCAGACGCCTTCAAGCTAAAATGATGACTAATTTGGTT
300
QGEQCERARRLQAKMMTNLV
\Exon4
ATGGCAAAGGACCGTTTACAGCTATTAGIAGAAGCTGCAACCAGTTTTGCAATTTTCCAAG
360
MAKDRLQLLEKLQPVLQFSK
TCACAGATGGACGTCTATAATGATAGTACTAACTTGACATGCCGCAACGGACATCTCCAG
42 0
SQMDVYNDSTNLTCRNGHLQ
\Exon5
TCAQAAAGTGGAGCTGTTCCTAAAAGAAAAGACCCCTTAACACACCCTAGTAATTCACTG
48 0
SESGAVPKRKDPLTHPSNSL
CCTCGTTCAAAAGCGATTATGAAAACTGGATCCACAGGTCTTTCAGGCCACCACAGAGCA
54 0
PRSKAIMKTGSTGLSGHHRA
CCTAGCTGCAGCGGTTTATCCATTGTTTCTGGAATGAGACAGGGGCCTGGTCCTACAACT
60 0
PSCSGLSIVSGMRQGPGPTT
\Exon6
GCCACTCATAAgIaGTACACCAAAAACAAATAGAACAAATAAACCTTCCACTCCTACAACT
66 0
ATHKSTPKTNRTNKPSTPTT
GCTCCCCGTAAAAAGAAAGACTTGAAGAATTTTAGGAATGTGGACAGCAACCTTGCTAAC
72 0
APRKKKDLKNFRNVDSNLAN
\Exon7
TTTATAATGAACGAAATTGTGGACAATGGAACAGCTGTTAAATTTGATGATATAGCTGGT
78 0
FIMNEIVDNGTAVKFDDIAG
CAAGAATTGGCAAAACAAGCATTGCAAGAAATTGTCATTCTTCCTTCTCTGAGGCCTGAG
84 0
QELAKQALQEIVILPSLRPE
\Exon8
FTGTTCACAGGTCTTAGAGCTCCTGCCAGAGGATTGTTACTCTTTGGTCCACCTGGAAAT
90 0
LFTGLRAPARGLLLFGPPGN
\Exon9
GGGAAAACAATGCTGbcTAAAGCAGTAGCTGCAGAATCTAATGCAACCTTCTTTAATATA
96 0
GKTMLAKAVAAESNATFFNI
\Exon10
AGTGCTGCAAGTCTAACTTCAAAATATGTAGGAGAAGGAGAAAAATTGGTGAGAGCTCTT
102 0
SAASLTSKYVGEGEKLVRAL
\Exon11
TTTGCTGTGGCTCGAGAACTTCAGCCTTCTATAATTTTTATAGATGAAGTTGATAGCCTT
108 0
FAVARELQPSIIFIDEVDSL
TTGCGTGAAAGAAGAGAAGGAGAACATGATGCCAGTAGACGTCTAAAAACTGAATTTTTA
114 0
LRERREGEHDASRRLKTEFL
\Exon12
ATAGAATTTGATGGTIGTACAATCTGCTGGAGATGACAGAGTGCTTGTAATGGGTGCAACT
12 0 0
IEFDGVQSAGDDRVLVMGAT
lExon 13
AACAGGCCACAAGAGCTTGATGAGGCTGTTCTCAGbcGTTTCATCAAACGGGTATATGTG
NRPQELDEAVLRRFIKRVYV
12 6 0
Chapter
56
3.
Results
\Exon 14
TCTTTGCCAAATGAGGAGkcACGACTACTTTTATTAAAAAATCTATTATGTAAACAAGGA
13 2 0
SLPNEETRLLLLKNLLCKQG
\Exon 15
AGCCCACTGACCCAAAAGGAACTGGCACAACTTGCTAGkTTGACCGACGGATACTCAGGA
13 80
SPLTQKELAQLARLTDGYSG
\Exon16
AGTGATCTAACAGCTTTGGCAAAAGATGCAGCCCTGGGTCCTATCCGAgIaACTGAAACCA
1440
SDLTALAKDAALGPIRELKP
\Exon17
GAACAAGTGAAGAATATGTCTGCCAGTGAgIaTGAGAAATATTCGATTATCTGACTTCACT
15 00
EQVKNMSASEMRNIRLSDFT
GAATCCTTAAAAAAGATAAAACGCAGCGTGAGCCCTCAGACCTTAGAAGCATACATACGT
15 60
ESLKKIKRSVSPQTLEAYIR
TGGAACAAGGACTTTGGAGACACCACTGTTTAA
1593
WNKDFGDTTV*
Figure
gene.
3.13: Nucleotide and deduced
ammo
acid sequence
of the
The initiator ATG codon and the stop codon TA A
letters.
The 16 vertical lines represent the
exon
are
porcine
shown
SPG4
m
bold
boundaries.
phages of the cDNA library by m situ hybridization.
clones, three were sequenced with the 5' and 3' insert
screening amplimer of the A-gtll vector as well as gene specific primers until
of
approximately
400'000
Of the twelve identified
the sequence
was
determined in both strands
(Figure 3.13).
SPG4 sequence was submitted to GenBank (Accession
compared to the human homolog using the GCG sequence
analysis package (Devereux et al., 1984). Figure 3.14 shows a comparison be¬
tween the porcine SPG4 amino acid sequence displaying 96% similarity to the
The
porcine
AF540879)
and
human sequence
mouse
sequence
(GenBank Accession AJ246001)
(GenBank Accession AK00793) in
and 95%
the
similarity
coding region.
to the
For the characterization of the cDNA of affected animals, RT-PCR was
performed with mRNA of the cerebellum of three affected animals using primer
pair SPG4-4 and SPG4-5. The two fragments were subcloned and sequenced.
A transition of thymine (T) to cytosine (C) at position 802 (termed M802)
in exon 7 (Fig. 3.13) of the SPG4 ORF was identified in one animal. This
change of the triplet TTG to CTG results in the same amino acid leucine
(L). To determine the polymorphism of this SNP, a PAC clone containing
the SPG4 gene was sequenced with primer pair SPG4-6. A new primer pair
(SPG4-7) was designed, the forward primer in intron 6, the reverse primer in
intron 7, harboring exon 7. The target sequence was amplified in animals of
different CPA genotypes (CPA/CPA, CPA/cpa, cpa/cpa). Sequence analysis
did not confirm the M802 mutation. All animals, including the affected, were
homozygous M802TIT.
3.3.
Candidate genes
57
PGGRGKK
pig
human
•
KGSGGPSSPV
NS
PPRPPPPCLA
A-N--
SSRPAPRPAP
PPQSPHKRNL
PAP--AG
--E
RFSRAL-AAK
RSSRAAPAPA
51
100
pig
YYFSYPLFLG
human
V-
pig
SASPPAPVPG
human
A
FALLRLVAFH
KGIEELEKGI
GEVERVRAFH
A-
A-S--V--
1-
E--A-
CERARRLQAK
MMTNLVMAKD
AWVTGQGEQ
mouse
--I
VLQFSKSQMD
--P
T-
pig
AIMKTGSTGL
human
TV
mouse
TVL-S--A--
100
QKEQAVEWYK
150
RLQLLEKLQP
200
M-Y
VYNDSTNLTC
T-
mouse
LRIDEDEKVG
I-
--I
pig
KQAFEYISVA
V--
human
human
LGLLFVWLCQ
G
--A
mouse
pig
50
RNGHLQSESG
AVPKRKDPLT
A-
HPSNSLPRSK
250
-T
E
-A
SGHHRAPSCS
A--
KKDLKNFRNV
GPGPTTATHK
STPKTNRTNK
M
VK-
-S--APT
G
M
A-P
AAT
G
GLSIVSGMRQ
Y-
DSNLANFIMN
EIVDNGTAVK
300
P
pig
PSTPTTAPRK
human
T--
L
mouse
V--
L
pig
KQALQEIVIL
PSLRPELFTG
LRAPARGLLL
FGPPGNGKTM
LAKAVAAESN
400
ATFFNISAAS
LTSKYVGEGE
KLVRALFAVA
RELQPSIIFI
DEVDSLLRER
450
FDDIAGQELA
350
D--
human
mouse
pig
human
C
mouse
C--
pig
-
-
REGEHDASRR
LKTEFLIEFD
GVQSAGDDRV
LVMGATNRPQ
ELDEAVLRRF
500
IKRVYVSLPN
EETRLLLLKN
LLCKQGSPLT
QKELAQLARL
TDGYSGSDLT
550
KIKRSVSPQT
600
human
mouse
pig
human
M
M
mouse
pig
ALAKDAALGP
IRELKPEQVK
LEAYIRWNKD
FGDTTV
NMSASEMRNI
RLSDFTESLK
human
mouse
pig
human
•
mouse
A comparison
Figure
3.14:
human
(AJ246001)
quence.
Identities
methionines
617
•
m
and
of
mouse
ammo
(M) represent
(GenBank Accession AF540879),
(AK007793) spastin (SPG4) ammo acid se¬
the porcine
acid sequences
start
codons,
are
indicated
asterixes
(*)
by
dashes.
The bold
indicate stop codons.
Chapter
58
F150093-T7
SW902
57
57
57
A340D12-T7
A276A1-SP6
57
57
Results
F150093-!
57
57
3.
A276A1-T7
^7__
57
„
A340D12-SP6
D60036-T7
57
57
„
D60036-SP6
Figure 3.15: The sequence tagged site (STS) contig map for the chromosomal
region deriving from microsateUite SW902. All STSs were localized to SSC3
except F150093-SP6 which was mapped to SSC4- Therefore, the assembly of
the
contig
at this side
was
Histological
3.3.3.3
not continued.
examination
As described by Reid (1999) the main characteristic in hereditary spastic para¬
plegia (HSP) families is an axonal degeneration involving the terminal ends of
the longest fibers of the corticospinal tracts and dorsal columns. The spinocere¬
bellar tracts are involved to a lesser degree. In all piglets histologically exam¬
ined no axonal degeneration could be found.
3.4
PAC
contig
Contig
3.4.1
PAC
library with microsateUite SW902 identified clone
starting point of the contig (Fig. 3.15). Both ends
of the clone were sequenced with SP6 and T7 primers and new primers were
designed (Table 2.1). This primers were named after the clone of which they
descend from and after the primer of which the clone was sequenced. The se¬
quence tagged sites (STS) amplified with these primers were named F150093SP6 and F150093-T7, respectively. F150093-T7 determined the clone A78G1.
Both STSs of this clone, A78G1-SP6 and A78G1-T7, respectively, could not be
Screening
F150093,
of the
around microsatellite SW902
which is the
identified in clone F150093.
is
larger
This could
mean
than the insert of clone F150093.
could not be identified in clone
A78G1,
it is
that the insert of clone A78G1
But
as
more
SW902 and F150093-SP6
likely
that clone F150093 is
PAC contig
3.4-
59
recombinant.
Screening
of the PAC
clones A340D12 and
library with A78G1-SP6
A276A1, respectively. The STS
and A78G1-T7 identified
A276A1-SP6, A276A1A78G1, while the frag¬
ment A340D12-SP6 of clone A340D12 was identified in clone A78G1. Screening
with primer pair A340D12-T7determined clone D60036, the STS D60036-SP6
of
T7 and A340D12-T7 could not be determined in clone
being
also localized in clone A340D12.
The boundaries of this
S0094, which show
and
contig
are
marked
by
the two microsatelhtes SW1066
0.03 and 0.02 recombination with
CPA, respectively
(Table 3.5, Fig. 3.2).
As the gene responsible for CPA lies between these two
all clones analyzed so far were screened with these two microsatel¬
markers,
htes to determine the
flanking
clones.
Yet,
in
none
of the clones
a
signal
was
detected.
SW902 and all STSs
chromosomally assigned using the somatic cell
are located on SSC3q21-q27, while
STS F150093-SP6 has been assigned to SSC4q21-q24. This indicates that clone
F150093 is a chimeric clone with two co-ligated inserts deriving from SSC3 and
SSC4. Therefore, the PAC library was not screened further with STS F150093SP6. Chromosomal assignment of all other STSs revealed location on SSC3ql2.
hybrid panel.
were
SW902 and STS F150093-T7
far, the assumed arrangement of PAC clones F150093, A78G1, A276A1,
A340D12, and D60036 is shown in Fig. 3.15.
So
Single
3.4.2
nucleotide
polymorphism
For the detection and identification of SNPs in the STSs of the PAC
contig,
polymorphism (SSCP) technique was used. This
method allows to detect mutations in DNA fragments as the mobility of singlestranded DNA in which mutations occur varies considerably in nondenaturing
Polyacrylamide gel electrophoresis. Each STS was analyzed with the animals
of mating 7 (Table 3.4). Polymorphism was found in STSs A340D12-SP6 and
the
single
strand conformation
A78G1-SP6.
SNP in A340D12-SP6
3.4.2.1
Primer
pair A340D12-SP6 (Table 2.1) amplified
STS A340D12-SP6.
different
In this STS
one
SNP
was
a
fragment of 254 bp called
Fig. 3.16A shows the
found.
migration pattern of STS A340D12-SP6 of four animals in the SSCP
fragments were designated A and B, respectively.
The slow and fast
gel.
Sequencing of these fragments revealed a transition of adenine (A) to
(G) at position 115 (termed A115G, Fig. 3.16B). All 206 animals
nine
gua¬
were
Chapter
60
A
1
2
3
4
AB
AB
AA
BB
xx.GG'xxGCI
3.
Results
II"1
(a)
Homozygous
animal
rTTGGas.TTGCTATTAa.rt
Homozygous
animal
rr rcT ga t tac tattaaa
! I ! I I I I I I I I ! I I I I !
I I I I I I I I I I I I I I I I I
mAACCTAACGATAATTT
AAAC C TA AÏ GA T AATT T
Heterozygous
animal
TTIGG^TTGCTATTÄÄ-
I I I I I I I I I I I I I I I I I
mAA ^ATAACGATAATTT
Figure 3.16: PCR-SSCP pattern of STS A340D12-SP6 of four pigs and
electropherogram of STS A340D12-SP6 flanking the identified mutation. (A)
PCR-SSCP pattern of STS A340D12-SP6 of four pigs. Fast and slow frag¬
Lane 1: father,
ments were designated as A and B alleles, respectively.
CPA carrier, AB; lane 2: mother, CPA carrier, AB; lane 3: offspring, CPA
healthy, AA; lane 4: offspring, CPA affected, BB. (B) Electropherogram of
STS A340D12-SP6 flanking the identified mutation. The nucleotide sequence
from two healthy (CPA/CPA and CPA/cpa,) and one affected animal (cpa/cpa,)
The arrow indicates the location of the SNP. a: homozygous (G)
are presented.
animal, healthy; b: homozygous (A) animal, affected; c: heterozygous (A/G)
animal, healthy.
PAC contig
3.4-
61
Assignment of
A340D12-SP6.
Table
3.6:
the
SSCP alleles
change
polymorphism
m
STS
SNP
A115A
BB
homozygous G/G (C/C)
G115G
AB
heterozygous A/G (T/C)
A115G
A78G1-SP6.
were
revealed
segregation analysis. The assigned binary codes
termed A340D12J3P6-M115.
was
a
In this STS two SNPs
visible but
they
fragment
(A)
to
a
of 282
bp called STS
On the SSCP
reliably interpreted.
guanine (G) at position
(M143), respectively (Fig. 3.17A).
and 143
fragments
were
a
found.
could not be
transition of adenine
143 eliminates
SP6
115
pair A78G1-SP6 (Table 2.1) amplified
Primer
M131)
bp
at
the
SNPs in A78G1-SP6
3.4.2.2
bands
for
homozygous A/A (T/T)
shown in Table 3.6. This SNP
are
code
AA
for this transition for
typed
binary
gel several
Sequencing
131
(termed
This transition at
position
restriction site for BsiRKAI.
resulted in
a
restriction
Digestion of amplified A78G1fragment length polymorphism (RFLP).
3.17B shows the RFLP pattern obtained from four animals of
mating 7
137-bp
282-,
heterozygous parents (M143A/G)
(Table 3.4).
fragments. The homozygous healthy offspring (M143A/A) shows only a 282-bp
fragment, while the affected homozygous offspring (M143G/G) shows 145- and
137-bp fragments. The M143 mutation was typed by PCR-RFLP in all 206
animals for segregation analysis. This SNP was termed A78G1-SP6JVI143.
Fig.
The
reveal
Due to the small distance of 12
that
no
M143
was
analyzed
Linkage analysis
Pairwise
M131 and M143 it
recombination occurred between these two loci in
Therefore, only
3.4.3
bp between
linkage analysis
in
our
145- and
our
was
assumed
206 animals.
family.
and fine
to estimate the lod
mapping
scores
and recombination fractions
for A340D12J3P6-M115 and
A78G1_SP6_M143, respectively, with the seven
chromosome 3 microsatelhtes (SW2618, S0094, SW902, SW1066, SW460,
As the lod
G ACT, S0216) and the disease locus (CPA) were performed.
score
values and recombination fractions of SNPs A340D12_SP6_M115 and
A78G1_SP6_M143
shown in the
were
identical, only the SNP A78G1_SP6_M143 results
two-point linkage analysis
in Table 3.7.
are
Chapter
62
(A)
121
OGTQCCCÄGG
'iTTGGGCCl
GJLCACCCAC
3.
Results
150
(B)
2S2
145
_
m»
^
mm»
137
Figure
Partial
3.17:
SNPs M131 and
BsiHKAI.
to
(A)
guanine (G)
sequence
of
STS
A78G1-SP6
harboring
the
two
M143 and digestion of amplified A78G1-SP6 fragments with
Partial sequence
transitions at
of
STS A78G1-SP6 revealed two adenine
position 131 bp and 143 bp, respectively. The
(A)
se¬
of DNA at which BsiHKAI cuts the DNA (GAGCA7 C) is underlined.
(B) Digestion of amplified A78G1-SP6 fragments with BsiHKAI results in a
The M143A'G genotype results in 282-,
restriction fragment polymorphism.
145-, and 137-bp restriction fragments (lane 1 and 2, parents), while the two
homozygous M143A'A (lane 3, healthy offspring) and M143G'G (lane 4, affected
offspring) genotypes generate a 282-bp fragment, and two 145- and 137-bp frag¬
ments, respectively. M: 50-bp ladder.
quence
PAC contig
3.4-
Table
3.7:
63
Two-point linkage analysis of A78G1SP6-M143,
marker loci
and
the
mosome
3-specific
sex-averaged recombination fractions (0)
Locus
CPA
and lod
locus
used
scores
pair
for
seven
chro¬
estimation
of
(Z).
9
Z
A78G1SP6-M143
SW2618
0.10
6.50
A78G1.SP6.M143
S0094
0.06
18.56
A78G1.SP6.M143
SW902
0.03
52.03
A78G1.SP6.M143
CPA
0.02
19.01
A78G1SP6-M143
SW1066
0.04
46.47
A78G1SP6-M143
SW460
0.07
5.79
A78G1.SP6.M143
G ACT
0.05
20.41
A78G1.SP6.M143
S0216
0.22
3.46
According to the contig assembly, both SNPs (A340D12.SP6-M115 and
A78Gl_Sp6_M143) were expected to be located in close distance (<1%) to
SW902. Instead, a recombination frequency of 3% between A78G1-SP6JVI143
and SW902 was calculated (Table 3.7).
It
was
not feasible to insert the SNP
computationally
with the CRIMAP "build"
A78G1-SP6JVI143
option into the fixed order SW2618-S0094-SW902-
SW1066-SW460-GACT-S0216 or S0094-SW902-SW1066, respectively. There¬
fore, three of the seven microsatelhtes were fixed and SNP A78G1_SP6_M143
This revealed the following orders: SW2618-S0094-SW1066was inserted.
A78G1.SP6.M143A78G1_SP6_M143,
SW902-A78G1SP6MU3-SW460,
SW460-GACT-S0216,
SW2618-S0094-SW902-A7&G1SP6-MU3-SW460.
The order SW2618-S0094-SW902-SW1066-A7&G1SP6JAU3-SW460-GACTIn Fig. 3.18, the relative genetic
S0216 was never rejected by our data.
position of A78G1.SP6_M143 between SW1066 and SW460 is shown. This
alignment
of the loci
STS A78G1_SP6_M143
differs from the
expected
between SW1066 and SW460.
was
alignment
to lie between
shown in
The
Fig 3.2.
S0094 and SW1066, not
Chapter
64
Marker
-SW1066
3.
Results
cM
0.0
0.04
—FÏ78G1_SP6_M143
3.9
0.05
-SN460
Figure
some
3
8.5
of SNP A78G1SP6JÂ143 on porcine chromo¬
by 3-pomt analysis. Sex-averaged map distances are given m Kosambi
3.18:
Genetic mapping
cM. 0: estimated recombination rate.
Chapter
4
Discussion
In the search for genes associated with CPA
we have mapped the CPA pheno¬
region of microsateUite SW902, which is located on
SSC3. Comparative mapping revealed the candidate genes CACNB4, SCN2A,
CHRNAl, KCNJ3 and SPG4, as mutations in these genes lead to diseases,
type
to the chromosomal
phenotype of CPA. Only the genes
SSC3ql4-q21 and SSC3q21-27, re¬
spectively, while the other three ion channel genes were mapped to SSC15.
Mutation screening of the ORF and the RNA expression study of the porcine
CA CNB4 gene did not reveal any differences between healthy and affected ani¬
mals. Mutation screening of the SPG4 ORF did not reveal any modifications in
affected piglets. Moreover, axonal degeneration in the corticospinal tract which
whose clinical appearance resembles the
CACNB4
and
SPG4 could be mapped
to
found in humans suffering from HSP could not be observed in affected pigs.
Therefore, the hypothesis that CACNB4 or SPG4 is identical with the CPA
gene was rejected. To isolate the region harboring the gene responsible for CPA
the creation of a PAC contig around SW902 was started. Altogether, five clones
were arranged. However, the starting clone containing SW902 was a chimeric
clone with two co-ligated inserts deriving from SSC3 and SSC4. Additionally
it was confirmed through linkage analysis, genetical, and physical mapping of
was
STSs and SNPs found in the
subsequent clone
4.1
4.1.1
contig that
Diagnosis
gap between the
starting and the
The clinical
of CPA
picture
of CPA
picture was defined by clinical examinations, therapeutic applica¬
drugs, neurophysiological, electromyographical, electroencephalograph-
The clinical
tions of
a
exists.
65
Chapter 4-
66
ical,
well
as
clinical
pathological
as
findings
and
discussed and
are
by spastic gait
most
severe cases
a
movement disorder which is character¬
and ataxia in the hind limbs with
the
piglets
first disease symptoms
can
Here, the
histopathological examinations.
compared to splayleg disease.
The clinical examinations revealed
ized
Discussion
drifting
to
fall down and remain in lateral
one
side. In the
recumbency.
The
be observed within the first three weeks after birth.
The
congenital splayleg disease is also characterized by incoordination within
days after birth, but this incoordination is due to splayed hind limbs
(Bergmann, 1976). In comparison to CPA, animals with weak symptoms will
the first
while
recover
affected
seriously
piglets
may be treated with
between the hind limbs to prevent the limbs
1984).
For
CPA,
no
enzymes
controversial
a
loose
outwards
coupling
(Bickhardt,
treatment is known.
In animals affected
was
splaying
by CPA,
activity of muscle specific and further
pigs while for splayleg animals
described. Kolb et al. (1981) reported an increase
the
in the range considered normal for
findings were
splayleg pigs in contrast to Tucek et al. (1985) who found no
the activity of CK in the blood plasma of healthy and affected
in CK levels in
difference in
pigs.
Histological
brain, nerves and
splayleg pigs incomplete development of mus¬
legs could be observed (Bergmann, 1976; Curvers
examinations revealed
alterations in the
no
muscles of CPA animals while in
cle fibers in the front and
et
rear
al, 1989).
On the basis of these results
genital splayleg
we
concluded that CPA is different from
con¬
disease in swine.
To exclude an involvement of the neurotransmitter acetylcholine, a thera¬
peutical dose of Konstigmin® was applied to three affected animals. As the
condition of the pigs did not improve an involvement of acetylcholine could be
excluded (Forth et al, 1992).
To
measure
NCS, and EEG
the electrical
were
activity
in the
muscle, brain and
nerves,
EMG,
carried out.
Electromyography (EMG) is the science concerned with the study of electri¬
activity in muscle. The basic physiological unit of a normal skeletal muscle
function is the motor unit, which consists of a lower motor neuron (LMN)
and a finite number of muscle cells (fibers). In the EMG, prolonged insertion
potentials were found in piglets affected by CPA. When a needle electrode is
inserted into a normal healthy muscle, electrical activity called insertion poten¬
tial is induced. This is caused by mechanical stimulation of the muscle fibers.
Insertion potentials may be prolonged, if the muscle is denervated, inflamed or
degenerated (Vandevelde and Fankhauser, 1987).
cal
The
nerve
conduction
the rate at which
an
study (NCS) is
impulse
electrical
a
useful
moves
diagnostic tool
along a nerve.
that
measures
It is used to
Diagnosis of CPA
4-1.
diagnose
67
disorders of the
peripheral
and muscles. The
nerves
between stimulation and appearance of muscle
potentials)
was
latency (the time
equal in all three
animals, while the amplitude seemed to be lower in the affected animals.
amplitude suggests that axons may be damaged.
A
low
The
electrophysiological deviations measured in muscle and nerves sug¬
gested a myopathy and/or a nerve/nerve cell disorder which could not be
confirmed by the pathological examinations. It has to be mentioned that the
patient sample was too small to detect the statistic significance of deviations.
Moreover, in the literature normal values for EMG, NCS, and EEG for pigs
are missing, as these techniques are reserved to humans and dogs so far.
Brain
waves
are
attributed to electrical activities of the brain which
alternating potential
quired through scalp electrodes,
manifest
differences at the
as
scalp surface.
When
are
ac¬
potential differences result in timecontinuous signals termed electroencephalogram (EEG). In the human, brain
waves show a characteristic pattern of development from infancy and early
childhood through adulthood with regard to EEG maturation (Alvarez-Amador
et al, 1989). In the three animals examined, a high voltage fast activity pattern
was recorded. This pattern differed from records in humans and in dogs. Data
from pigs were not found in the literature. Moreover, the interpretation of this
finding
was
difficult
examined animals
as
was
such
the stage of maturation of the cerebral cortex in the
not known.
To
our
knowledge
pigs.
there
are no
publications
about the different stages of maturation in
4.1.2
MicrosateUite SW902 and CPA
revealed that the SW902189 allele
co-segregated 100% with the
beginning
study, the disease was
in
observed
our
experimental family (Table 3.4, family 1) and therefore it
only
remains to be determined whether this association was due only to a founder
effect in this family or whether a linkage disequilibrium could also be observed
in other populations.
Linkage analysis
recessive allele involved in CPA. In the
Due to the collaboration with the
of this
Department of Farm Animals, Univer¬
sity of Zurich and the Institute of Animal Neurology, University of Berne the
knowledge about the existence and the phenotype of CPA was more propa¬
gated. Thus, further litters affected by a neuromuscular disorder which could
not be assigned to any disease known until this day, were examined in our in¬
stitute. In five out of six cases CPA could be diagnosed according to the typical
phenotype and the genotypic test (Table 3.4, families 2-5).
Are these five affected families
rium
can
be found in other
families affected
analyzed
in this
now an
populations
as
linkage disequilib¬
pedigree of the
Thus, all the animals
indication that the
well?
Analyses
of the
by CPA revealed a common ancestor.
study belong to one family. To date, the genotypic
test is
only
Chapter 4-
68
significant
in these animals. It is
which exhibit another genotype
Discussion
possible that there might be affected animals
healthy animals showing the SW902189/189
or
genotype in unrelated families.
4.2
Chromosomal
assignment
channel
subunit
alpha
SCN3A), KCNJ3,
of
a
cluster
sodium
(SCN1A-
and CHRNAl
alpha subunit genes (SCNA) is lo¬
(1991) mapped the SCN2A1 gene to
HSA2q22-q23, Lu et. al. (1992) mapped the SCN2A2 gene to HSA2q23-q24, the
SCN1A gene was localized to HSA2q24 by Malo et. al (1994a) and the SCN3A
gene was mapped to HAS2q24-q31 by Malo et. al. (1994b). In the mouse, Malo
et. al (1991) demonstrated that the SCN2A and SCN3A are physically linked
and separated by a maximum distance of 600 kb. Furthermore, also the genes
SCN1A and SCN2A are tightly linked in the mouse and separated by a distance
of 0.7 cM. Thus, the 3 isoforms of the brain sodium channel alpha subunit are
In the
cated
human,
on
a
cluster of sodium channel
human chromosome 2. Han et al.
encoded
by
both
conservation of amino acid sequence similarities and
by
3 distinct genes that share
a common
ancestral
origin as revealed
by chromosomal
encoded by the same
presumed that these isoforms are
pig. The porcine sodium alpha channel subunit cluster was
SSC15 with a high probability for region ql5-q22. Comparative
location. We therefore
chromosome in the
mapped
to
mapping data showed conserved synteny groups of human chromosome 2 on
porcine chromosomes 3 and 15 (Rettenberger et al, 1995), more precisely the
human region 2q22-q24 corresponding to SSC15q21-q22 (Pinton et al., 2000).
We therefore mapped the porcine sodium channel alpha subunit cluster on
SSC15ql5-q22.
Only cell hybrids harboring the region SSC15ql5-q22 showed amplification
products with the primer pair used for mapping the porcine KCNJ3 gene.
In the human, the KCNJ3 gene was mapped to HSA2q24.1 by Stoffel et al.
(1994). Pinton et al. (2000) showed correspondence between the human 2q24q37 and the porcine 15q21-q26 region. Thus, there is additional evidence that
the KCNJ3 gene maps to SSC15ql5-q22.
The human CHRNAl gene
was mapped to human chromosome 2q24-q32 by
al., 1990). In this study, the porcine CHRNAl gene was localized to
SSC15q23-q26. This location on porcine chromosome 15 is in agreement with
Rettenberger et al (1995) and Pinton et al (2000), who showed correspondence
(Beeson
et
between human
2q24-q37 and porcine 15q21-q26 region.
As the porcine sodium channel
KCNJ3 gene, and the
alpha subunit cluster genes, the porcine
porcine CHRNAl gene are located on SSC15, they were
The candidate gene CACNB4
4-3.
excluded
as
candidate genes and
69
therefore,
The candidate gene
4.3
The four calcium channel
tivity
of
subunit
no
investigations
were
made.
CACNB4
ß subunits (CACNB1-CACNB4) modulate the
calcium channels in
voltage-gated
further
proteins is highly conserved with
neurons
>
ac¬
and muscles. Each of the
95% amino acid
orthologs in different mammalian species (Williams et
al., 1994; Escayg et al., 1998). Yet, no calcium channel ß subunit
characterized for the pig.
between the
Collin et
4.3.1
Chromosomal
In the mouse,
ß
identity
al., 1992;
sequence
is
assignment
encoding the voltage-gated Ca2+ channel ß\ subproximal mouse chromosome 2 by Chin et
CACNB4 gene was mapped to chromosome 2q22-q23
the gene
al
(CACNB4)
(1995). The
by
Taviaux et al
on
HSA2 is divided into two groups in the
unit
localized to
was
human
(1997)
and
Escayg
et al
(1998).
A
syntenic group located
pig located on SSC3ql3-q27 and
SSC15qll-ql4/q22-q26 as described by Pinton et al (2000) and
al (1996). The porcine CACNB4 gene was therefore expected to
Goureau et
be localized
pig chromosome 3 or 15. The A-genomic DNA fragment carrying the
porcine CACNB4 gene was mapped to SSC3ql4-q21 by FISH. We can clearly
exclude CACNB4 being located on chromosome 15, as the porcine chromosome
15 is telocentric in comparison to the sub-metacentric SSC3.
on
either
4.3.2
Mutation
Analysis
a
of
protein
the
of
screening
porcine
502
amino
of the cDNA
CACNB4
acids
gene
with
(http://www.expasy.ch/tools/pi_tool.html).
a
revealed
molecular
In
the
rat,
that
it
encodes
mass
of
63-kDa
the
ß±
subunit
contains an open reading frame encoding for a 519-amino acid protein, with a
predicted molecular mass of 58-kDa (Castellano et al, 1993), while the human
ß\ subunit encodes a 512-amino acid protein, which predicts also a 58-kDa
protein (Escayg et al., 1998). The difference to the human and rat ß± protein,
respectively,
to
does not alter the
al., 1993; Walker
CACNB4 gene is
genes contain 13
In the mouse,
the
protein phosphorylation sites that are thought
regulation of calcium channel function (Castellano et
et al, 1998). The intron/exon organization of the porcine
similar to that of the human (Escayg et al, 1998). Both
contribute to the
/?4 subunit
is
coding
exons, all of them identical in
length.
Burgess et al (1997) demonstrated that a null mutation in
responsible for an autosomal recessive neurological disorder
Chapter 4-
70
Discussion
lethargic. Escayg et al (2000b) identified a prematurea patient with juvenile myoclonic epilepsy as well as
a missense mutation both in a German family with generalized epilepsy and
praxis-induced seizures and in a French Canadian family with episodic ataxia.
in the
mouse
mutant
termination mutation in
In the
pig, only
one
of the twelve
C to G transition in
exon
determine if this transition is
(Saiki
et
al, 1988; Bracho
animals
(8.3%)
exhibited
a
a
silent
possible
to
misincorporation that occurred during PCR
al, 1998)
or a
genetic
mutation.
CACNB4 expression
4.3.3
The
et
analyzed
From this limited data it is not
13.
expression of the CANB4
gene in various
porcine tissues is
in
agreement
findings
(1993):
brain, while no expression can be detected in
script of approximately 9.0 kb is similar in length to the previously described
transcripts in rat, mouse and human brain (Castellano et al., 1993; Burgess et
al, 1997; Escayg et al, 1998).
with the
Burgess
in brain of
et al.
(1997)
lethargic
the
CACNB4 mRNA expression is reduced
Comparison of the expression of this gene in the
described that
mice.
cerebellum of affected and
4.3.4
ß± subunit is expressed in the
muscle, heart or fat. The tran¬
of Castellano et al
healthy piglets
did not show any differences.
Comparison of CPA with
lethargic mice and epilepsy
the
clinical
picture
of
and ataxia in humans
Comparison of the phenotype of lethargic mice (Burgess et al, 1997) and pigs
suffering from CPA revealed both a few similarities and some differences. In
contrast to the lethargic mice (1) no spontaneous focal motor seizures; and (2)
increasing age could be observed in the CPA pigs. In agree¬
ataxia, the lethargic behavior and the finding that no pathological
changes (Dung and Swigart, 1972) can be observed.
no
recovery with
ment
are
the
Escayg
gene with
et al
(2000b)
episodic
showed
ataxia and
an
association of
a
in humans.
epilepsy
brief spells
mutation in the
The
CACNB4
patients experienced
of loss of consciousness, ataxia and
sporadic typical absences with
in
the
shoulders
and
after
arms
myoclonic jerks
awakening. In comparison, the
CPA pigs never had absences and their condition was always better after rest.
Above these differences the
ethosuximide
provoked
it
et
al, 1998; CapoviUa
et
therapeutic application of the antiepileptic drug
in the condition of the affected piglets
(Tsakiridou et al, 1995) and the human (Escayg
improvement
described for the rat
as
was
no
al, 1999).
The candidate gene SPG4
4-4-
71
SPG4
The candidate gene
4.4
SPG4
The
(1994)
assignment
Chromosomal
4.4.1
was
gene
mapped
to human chromosome
(1994).
high probability (P
and Hentati et al
SSC3 with
a
0.8) being
=
q-arm. This observation coincides with the
al
(2000).
The human chromosome
end of the q-arm of
Mutation
4.4.2
The
porcine SPG4
mass
porcine
spastin
porcine spastin
located
et al
gene
on
the distal end of the
on
gene map of Pinton et
comparative
2pl3-p24 region corresponds
to the distal
chromosome 3.
screening
gene encodes
a
of the cDNA
protein of 530 amino acids with
(http://www.expasy.ch/tools/pi_tool.html).
of 58-kDa
human
2pl3-p24 by Hazan
We localized the
gene contains 616 amino acids with
a
a
molecular
The ORF of the
molecular
mass
of 67-
(Hazan et al, 1999). Apart from a length difference of 86 amino acids
(14%) in exon 1, the porcine amino acid sequence differs only 4% from the
kDa
human sequence.
Most
important, this difference does
cassette between human amino acids 342 and 599
conserved ATPase
minimal consensus,
domains, including Walker motifs A and
were
The association between
ATPase
not
alter the AAA
(Beyer, 1997).
The three
B and the AAA
(Hazan et al, 1999).
spastin and microtubules is regulated through the
located in this AAA cassette
activity of the AAA domain (Errico
et
al, 2002). All spastin
missense
mutations located in the AAA domain bind to microtubules and lead to
a
redistribution of the microtubule
cytoskeleton which impairs the microtubule
dynamics in long axons (Errico et al, 2002). Also, the exon organization of
porcine SPG4 is similar to that of human spastin: both contain 17 exons,
similar in
length.
Until now, 88 mutations in the
spastin
located in the AAA cassette
gene have been described.
(Hazan
Most of
al, 1999; Burger et al, 2000;
Fonknechten et al, 2000; Hentati et al, 2000; Lindsey et al, 2000; McMonagle
et al, 2000; Santorelh et al, 2000; White et al, 2000; Svenson et al, 2001;
Sauter et al, 2002a). The mutation we identified in 1 (8.3%) out of 12 analyzed
them
are
animals lies in the AAA cassette, but
(leucine)
it is
a
silent mutation.
As
as
the amino acid remains the
already
is difficult to determine if this transition is
during
PCR
(Saiki
et
al, 1988; Bracho
et
et
a
same
chapter 4.3.2, it
misincorporation that occurred
mentioned in
al, 1998)
or a
genetic
mutation.
Chapter 4-
72
hereditary spastic paraplegia (HSP)
The pure
with "clumsiness"
of 5
of CPA with pure HSP
Comparison
4.4.3
years)
or
(25%
of
cases
may
in HSP families
may not appear until adult life
observed in newborn
Discussion
begin in early
symptomatic
are
(Reid, 1999),
childhood
at
an
while CPA is
age
only
The severity of the symptoms varies in both
HSP, piglets affected by CPA have an increased
tone of the lower limb and weakness of the limbs simultaneously. Hyperreflexia
1
of the limbs and a positive Babinski reflex
are another diagnostic hint (Sauter
et al., 2002b). In the pig, neurological examinations and their interpretations
are difficult as pigs are not used to be handled.
piglets.
diseases. As described for pure
spinal cord atrophy, particularly
regions (Reid, 1999). The major neuropathological
feature is axonal degeneration in the terminal portions of the longest descending
(corticospinal tracts) and ascending (dorsal column pathways) tracts within the
spinal cord (McDermott et al, 2000). Demyelination and gliosis can accompany
the axonal loss. A connection between the mutations in the spastin gene and
Pathological reports
for pure HSP describe
in the cervical and thoracic
the
degeneration of motor axons has currently been shown by Errico et al
As this degeneration of motor axons could not be found in the pigs
affected by CPA, we assume HSP does not seem to be similar to CPA.
(2002).
The PAC
4.5
contig
General aspects of
4.5.1
generating
a
contig
sequencing results were obtained when the PAC DNA was extracted with
Qiagen-tip 500 Plasmid Purification Kit as described in chapter 2.4.3.
Though with minipreparation of plasmid DNA as described in Sambrook et
al. (1989) high concentrations of DNA were obtained, this yield is ascribed
Best
the
to the
contaminating bacterial DNA. Usually, from
a
single
inoculated in 500 ml of LB medium 70.0 fig DNA
colony
template
amount of 1.5
sufficient. If
into the
more
gel.
cycles are
500 bp of
not
2.0 fig of pure PAC DNA per
-
DNA is
taken,
the
amplified
isolated bacterial
can
be
expected. A
sequencing
reaction is
DNA is too viscous for
loading
cycles of amplification, as the usual 30
amplification. Following the here described protocol,
We also recommend 99
enough
for
the insert sequence should be readable.
1The Babinski reflex
is
where the great toe flexes toward the top of the foot and the other
toes fan out when the sole of the foot
It is normal in children under 2 years
is firmly stroked
old, but it disappears as the child ages and the nervous system becomes more developed In
people more than 2 years old, the presence of a Babinski reflex indicates damage to the nerve
paths connecting the spinal cord and the bram (the corticospinal tract)
The PAC contig
4-5.
73
Discrepancies
4.5.2
in the
contig
The chromosomal
assignment of the STSs of the clones of the contig already
discrepancy. In clone F150093, STS F150093-T7 and microsatellite SW902 have been localized on SSC3q21-q27, while STS F150093-SP6 was
mapped to SSC4q21-q24. Moreover, the adjacent clones seemed to be located
more proximal, on SSC3ql2.
According to Robic et al (1996), porcine chro¬
mosome 3 is divided into nine small regions in the somatic cell hybrid panel,
which can be discriminated only by one or two PCR results. Therefore, the
localizations have to be considered very carefully. To verify these results, the
showed
contig
some
was
analyzed
for SNPs.
No recombination
was
and A78G1_SP6_M143.
found between the two SNPs A340D12_SP6_M115
According
between the two SNPs and
to the recombination
SW902, there
seems
frequency
to be
a
of 3% found
gap between clone
F150093 and A78G1. If the two loci would be 1 cM apart, there would be
chance of recombination between these loci. On the average, 1 cM
to 1 million base
pairs (Brem
two clones would have
a
et
al, 1991).
In
our
size of about 3 million
case
bp.
a
1%
corresponds
the gap between the
Additionally,
from
our
expected SNP A340D12_SP6_M115 to be either localized
between microsatelhtes S0094 and SW902 or between microsatelhtes SW902
contig assembly
and SW1066
we
(Fig. 3.2). Yet,
SNP A340D12_SP6_M115
was
localized between
SW460. Thus, it is assumed that the contig assembly is not
correct. From our analyses, clone F150093 seems to be chimeric and is therefore
not to be considered as a good starting clone.
SW1066 and
False-positive and false-negative PCR screening results in the PAC library
obtained them for clone A78G1 are a common problem (Hall et al, 2001).
Although Al-Bayati et al (1999) concluded that the PAC library harbors no
or at least negligible amounts of chimeric clones, clone F150093 seemed to
be chimeric. Its T7 end was mapped to SSC3q21-q27 while its SP6 end was
mapped to SSC4q21-q24. Therefore, this clone is not considered as a good
as we
starting clone for generating a contig. Unfortunately, microsateUite SW902
only found in clone F150093 of the PAC library. It is advisable to obtain
a clone containing SW902 from another library.
was
Chapter
5
Conclusions and further
perspectives
The present study investigated the phenotypic appearance of "Congenital pro¬
gressive ataxia and spastic paresis in pigs" (CPA), mapped the disease locus
to the porcine genome, analyzed several candidate genes, and as no candidate
gene was found to be the causal gene for the disease, explored the possibility of
a positional cloning approach to identify the gene causing CPA. The following
conclusions could be made:
•
CPA and
•
to characterize the disease
as
splayleg
electron
disease in
microscopy
pigs
are
two different diseases.
additional examinations such
precisely
more
and immunohistochemical methods should be
en¬
forced.
•
it remains to be determined if
only CPA
affected animals exhibit the
SW902189'189 genotype.
•
the genes
were
•
the
fore
SCN2A,
therefore
KCNJ3 and CHRNAl
ineligible
porcine CACNB4
a
as
gene
were
mapped
to SSC15 and
candidate genes.
was
candidate gene for CPA.
mapped to SSC3ql4-q21 and was there¬
However, mutation screening, expression
studies, and drug treatment did not confirm the assumption, that
porcine CACNB4 gene may be responsible for CPA.
a mu¬
tation in the
•
the
was
porcine spastin
mapped
to
gene
(SPG4)
SSC3q21-q27.
was
a
Mutation
75
candidate gene for CPA
screening did
not
as
it
reveal any
Chapter
76
differences between affected and
rophy,
which is
diagnostic
Conclusions and
5.
•
•
in the
The spinal cord at¬
by mutations in the spastin
affected pigs. Therefore, no further
healthy
animals.
for HSP caused
gene, could not be confirmed in the
investigations
spastin
gene
were
made.
as no other suitable candidate genes for CPA could be found, a pure
positional cloning approach was started by generating a contig around
microsateUite SW902. Unfortunately, the generated contig showed some
irregularities. A new clone harboring SW902 should be obtained from an¬
other library. To avoid new irregularities, each clone should be mapped to
the porcine chromosome by somatic cell hybrids. If the result is doubtful
the assumed localization should be proven by FISH.
to
speed
be used
The
up
contig
as new
construction microsatelhtes
comparative candidate
tify disease-causing
of the pathogenic mechanism
and
proach
no
S0094 and SW1066 should
entry points.
gene
approach
is
a
genes in animals. A successful
case
further perspectives
and
convincing way to iden¬
approach requires knowledge
very
genetic basis of the disease.
similar disease is described in humans
or
other
If this is not the
animals, this
ap¬
should not be the first choice.
Instead, the positional cloning approach should be used. Although this
approach can be a very tedious and time-consuming task, in the end the disease
gene is located between two markers. Moreover, new markers which are even
linked can be found and used for selecting animals for a special trait. Although,
this approach requires a big effort to identify a specific gene, in the end one
can be sure that the specific gene can be found between two markers.
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89
molecular cyto¬
Zhuchenko, O., J. Bailey, P. Bonnen, T. Ashizawa, D.W. Stockton, C. Amos,
W.B. Dobyns, S.H. Subramony, H.Y. Zoghbi, and C.C. Lee (1997). Autoso¬
mal dominant cerebellar ataxia (SCA6) associated with small polyglutamine
expansions in the alpha lA-voltage-dependent calcium channel. Nat. Genet.
15: 62-69.
Appendix A
Materials
A.l
Equipment
Company (Article number)
Description
A.Kühnert AG
Bacterial shaker
(Lab-Therm-Shaker)
(426.694)
Schleicher & Schuell (439.387)
Schleicher & Schuell
filter paper
Blotting
Blotting membrane,
Cellulose Nitrate
Blotting membrane,
Nylon
Blotting membrane Colony
137
82
Stratagene (420101)
lift
Duralon
(420102)
(420103)
Heraeus (Cryofuge 8000)
Heraeus (Biofuge B)
Hettich (Microliter Typ 2042)
mm
Duralon
mm
Centrifuges
DCode TM
Biorad
gel apparatus
Sequencer 377 A
DNA Thermocycler Hybaid
DNA Thermocycler
Electrophoresis Maxiphor
DNA
Electrophoresis Mighty small
Falcon polypropylene tube, 50
Falcon tube, 14 ml
Foto Developer Neutol liquid
Applied Biosystems
MWG-Biotech
Perkin Elmer Cetus
LKB
(2012-001)
(SE 245)
Fakola (9150)
Falcon (2059)
Hoefer
ml
Agfa
Agfa
B&W fixer
Foto Fixer
Agefix
B&W fixer
91
Gevaert
Gevaert
AG,
AG,
Leverkusen
Leverkusen
Appendix
92
A.
Materials
Company (Article number)
Description
Hybond-XL, nylon
membrane
Amersham Biosci.
(RPN119S)
Incubator
Heraeus
Labor shaker
Bühler
Medelec Profile Multimedie EEG
Oxford
Microcon®-PCR
centrifugal
Millipore (UFC7 RC250)
filter devices
Gilson
Micropipettes
Microsyringe,
Pasteur pipet
50
Hamilton
fi\
WU Mainz, BRD
TOENNIES, Erich Jäger GmbH
Falcon (1029)
Sarstedt (28 1184)
Falcon (1008)
Perkin Elmer (UV-VIS 550)
Omicron-Labotec (RC10)
Treff (96 1702 6 02)
Treff (96 1701 4 02)
Cambridge, MA, USA
NeuroScreen Plus
dish, 100 mm
dish, 150 mm
Petri dish, 60 mm
Photospectrometer
Petri
Petri
Pipet tips, 1-10 fi\
Pipet tips, 100-1000 fi\
Pipet tips, 20-200 fA
Polaroid
camera
Polaroid film 667
Power
Polaroid
supply (agarose gels)
Desaga Heidelberg
Bio-Rad (165-60)
PowerPac 3000
Reaction tubes
0.5
1.5
1.7
ml, Polypropylene
ml, Polypropylene
ml, chloroform-safe
Biozym (170912)
(96 7246 9 01)
Brunschwig (MCT-175-A)
Mettler (PE3600)
Amersham Biosci. (80-6095-58)
Millipore
Treff
Scale
Slot Blot manifold
Spin columns
Sterile filter unit
(0.2 fim),
150 ml
Nalgene (125-0020)
Temperature controlled
gel apparatus
Ultracentrifuge
Ultracentrifuge
Ultracentrifuge
DCode
Kontron
Kontron TST 28.38
rotor
tubes
TM, Bio-Rad
(Centnkon T-2070)
Polyallomer
13.5 ml
Beckman
38.5 ml
Kontron
UV-Transilluminator
Vacuum concentrator
Vortex
X-ray films, Kodak X-OMAT
X-ray films, Fuji New RX
(32-6814)
(9190196)
MWG-Biotech AG
Bachofer
(BA-VC-300H)
(K-550-GE)
Sigma (F-5513)
Fuji (03E250)
Scientific Industries
A. 2.
Substances/chemicals
93
Substances/chemicals
A.2
Company (Article number)
Description
/3-Mercaptoethanol
A-DNA
1 kb
Basepair ladder
5'/3'
RACE Kit
50
Basepair ladder
100 Basepair ladder
5-bromo-4-chloro-3-indolyl/5-D-galactosidase (X-Gal)
ABI Prism Dye Terminator Cycle
Sequencing Ready Reaction Kit
Acrylamide
Agar
Agarose
Ammonium peroxide sulphate (APS)
AMV reverse transcriptase
Fluka (63689)
Boehringer-Mannheim (745782)
Gibco BRL (10381-010)
Invitrogen (1734792)
Pharmacia (27-400501)
Pharmacia (27-400101)
Boehringer-Mannheim (651745)
Perkin Elmer
(401384)
Fluka
(01699)
(30391-023)
Gibco BRL (1551-027)
Fluka (09915)
Promega M (5101)
Gibco BRL
& reaction buffer
BamHI & reaction buffer
Pharmacia
Boric acid
Bovine
serum
(27-0868-04)
(15660)
Sigma (A-9647)
Fluka (18030)
BioConcept (R0570S)
Difco (0230-15-5)
Fluka (21079)
Fluka (84100)
Pharmacia (27-2025-01)
Fluka
albumin
(BSA)
Bromphenol blue
BsiHKA I & reaction buffer
Casamino acids
/
Casein
hydrolisate
Chloroform
D(+)-Sucrose
Deoxyribonucleotide
dATP/dCTP/dGTP/dTTP
sulphate
Diethylpyrocarbonate (DEPC)
Dextran
EcoRl
Ethanol
Ethidium bromide
(EtBr)
Ethylenediamintetraacetic
acid
Pharmacia
(17-034-01)
(32490)
Pharmacia (27-0854-03)
Merck (1 00983)
Sigma (E-8751)
Sigma (E-5134)
Fluka
(EDTA)
Ficoll
Formaldehyde
Formamide
Gelatine
Glycerol 100%, water free
Guanidine thiocyanate
Sigma (F-4375)
Fluka (47629)
Fluka (47670)
Merck (4078)
Fluka (49780)
Fluka (50990)
Appendix
94
A.
Materials
Company (Article number)
Description
High Fidelity Taq DNA-Polymerase
Boehringer-Mannheim (1
732
650)
& reaction buffer
HinaTlI & reaction buffer
Hydrochloric
Isopropanol
acid
(HCl 32%)
Kanamycin
Pharmacia
(27-0860-02)
(1 00319)
Fluka (59300)
Biochrom KG (A 2512)
Merck
Konstigmin®
Chassot AG
Lambda Fix® II
library
Magnesium chloride (MgCL?)
Magnesium sulphate
Morpholinopropanesulfonic
acid
Stratagene (097001b)
Fluka (63064)
Fluka (63140)
Fluka (69949)
(MOPS)
N, N'-methylene-bisacrylamide
NucleoSpin® Plasmid
Orange
(crist.)
G
Paraffin
(lOx)
PCR-buffer
pGEM®-T Easy Vector System I
Polyvinyl pyrrolidone
Phenol-chloroform-isoamylalkohol
Prime-it II Random Primer
labeling
Fluka (66669)
Macherey-Nagel (740588 250)
Chroma Gesellschaft (1A116)
Fluka (76233)
Boehringer-Mannheim
Promega (A1360)
Sigma (PVP-40)
Biosolve (16973243)
Stratagene (300-382)
Kit
Proteinase K
Qiagen II Gel Extraction Kit
Qiagen Plasmid Maxi Kit
Qiagen RNeasy Maxi Kit
RNase A
Salmon sperm DNA
Sodium acetate
(ssDNA)
trihydrate (NaAc)
Sodium chloride
(NaCl)
Sodium citrate
dodecyl sulphate (SDS)
hydroxide (NaOH)
SpreadEX®EL 600 gel
Sodium
Sodium
Standard GeneScan -350 ROX
Stratagene
Lambda DNA
Sigma (P-0390)
Qiagen (20051)
Qiagen (12263)
Qiagen (75162)
Fluka (83832)
Eurobio (017543)
Fluka (71190)
Merck (1 06404 5000)
Fluka (71497)
Fluka (71729)
Fluka (71690)
Elchrom (3428)
ABI PRISM (401735)
Stratagene (200391)
Purification Kit
Suxinutin®
SYBR Gold
Taq DNA-Polymerase
& reaction buffer
Parke-Davis
Juro
Supply (11494)
(270 799 03)
Pharmacia
A. 2.
Substances/chemicals
95
Company (Article number)
Description
TEMED
(Tetramethylendiamine)
TOP10 One Shot Kit
Trichloroacetic acid
(TCA)
Trizma base
Trizma
hydrochloride
Tryptone (Bacto tryptone)
Urea
Xylene cyanol
Yeast extract
FF Standard
(XCFF)
IBI (IE 2850)
Invitrogen (C4040-10)
Fluka (91228)
Sigma (T-1503)
Sigma (T-3253)
Difco (0123-01-1)
Fluka (51459)
Fluka (231305)
Gibco BRL (30393-029)
Appendix
3
A.
Media/Solutions
Acrylamide:bisacrylamide 49:1, 40%
Acrylamide
N, N'-methylene-bisacrylamide
39.2%
0.8%
(w/v)
(w/v)
Agarose gel
TBE
0.5x
0.75%
Agarose
Ethidium bromide
0.1
to
2%
/xg/ml
Bromphenolblue loading dye for DNA
0.25%
Bromphenolblue
40%
D(+)-Sucrose
DNA
(w/v)
(w/v)
Denaturing solution
NaCl
1.5 M
NaOH
0.5 N
lOOx Denhardt's
Ficoll
2%
Polyvinyl pyrrolidone
2%
BSA
Dextran
sulphate stock solut ion
Dextran
sulphate
(w/v)
(w/v)
2% (w/v)
20%
(w/v)
in Formamide
DNA
loading dye
XCFF
0.25%
(w/v)
(w/v)
40% (w/v)
XCFF
Orange
0.26%
G
D(+)-sucrose
Ethidium bromide
Agarose
plate
0.8%
TE
Ethidium bromide
(w/v)
lx
1
fig/ml
Materials
A. 3.
Media/Solutions
97
Hybridization
mix
SSC
4x
Denhardt's
5x
1%
SDS
Dextran
sulphate stock solution
heat to 50° C before
use
add denatured ssDNA
50%
(v/v)
and
/xg/ml
100
lOx A-dilution buffer
NaCl
100 mM
MgS04
8 mM
Tris-HCl
(pH 7.5)
Gelatin
50 mM
0.01%
(w/v)
LB medium
Tryptone
10.0
Yeast extract
5.0
NaCl
5.0
g/L
g/L
g/L
pH 7.0 with NaOH
LB
plate
LB medium add
MgS04
Agar
2.4
15.0
g/L
g/L
LB top agarose
LB medium add
MgS04
Agarose
2.4
g/L
/L
15.0 g
Lysis buffer
Tris-HCl
EDTA
pH 8.5
pH 8.0
100 mM
5 mM
0.2%
SDS
NaCl
Proteinase K
200 mM
100
/xg/ml
Appendix
A.
Methylene blue
0.02%
Methylene blue
Tris-HCl
(pH 7.5)
10 mM
lOx MOPS
MOPS
0.4 M
Sodium acetate
EDTA
0.1 M
10 mM
pH 8.0
pH 7.0 with NaOH
NaCl/EDTA
NaCl
10 mM
EDTA
10 mM
Neutralizing
solution
NaCl
1.5 M
Tris-HCl
(pH 8.0)
(stock solution)
Proteinase K
Proteinase K in
RNA
loading
0.5 M
dd^O
20
mg/ml
buffer
MOPS
lx
16.6%
(v/v)
50% (v/v)
Formaldehyde
Formamide
incubate 15 min at 60° C and cool
ice before
RNA
on
adding loading dye
loading dye
Bromophenol blue
0.04%
EDTA
1 mM
pH 8.0
Glycerol
50%
Materials
A. 3.
Media/Solutions
99
Solution D
Guanidinium
thiocyanate
Sodium acetate
4 M
25 mM
just before use add
/3-Mercaptoethanol
0.72%
(v/v)
20x SSC
NaCl
3 M
Sodium citrate
0.3 M
pH 7.0 with NaOH
lOx TBE
Trizma base
0.9 M
Boric acid
0.88 M
EDTA
20 mM
TE
pH 8.0
pH 7.5/8.0
Tris-HCl
EDTA
10 mM
pH
1 mM
8.0
adjust pH by choosing
the proper
Tris-HCl stock solution
TNE
Tris-HCl
pH 8.0
NaCl
EDTA
10 mM
100 mM
1 mM
pH 8.0
Triple loading dye
XCFF
0.25%
Bromphenol Blue
Orange G
0.25%
D(+)-Sucrose
(w/v)
(w/v)
0.25& (w/v)
40% (w/v)
Curriculum Vitae
Name
Antke Christine Kratzsch
Date of birth
December
Place of birth
Erlangen, Germany
Nationality
German
Profession
Veterinarian
1977
1981
Primary school, Wintersdorf, Germany
1990
High school, Dietrich-Bonhoeffer-Gymnasium Oberasbach, Germany
1994
Veterinary school, Justus-Liebig-University Giessen, Germany
1997
Veterinary school, University of Zurich, Switzerland
1981
1991
1994
-
-
-
-
29, 1970
Graduation June
Employee
1997
1998
-
2002
in
a
1997, Title: med.
vet.
veterinary mixed practice, Switzerland
PhD studies and research assistant in the group of
Prof. Dr. G.
at the Institute of Animal
Stranzinger
Swiss Federal Institute of
Thesis
supervisor: Prof.
101
Technology (ETH) Zurich,
Dr. P.
Vögeli
Science,
Switzerland
Acknowledgments
I
am
very
grateful
to all those
people who contributed
to the outcome of this
thesis.
all,
First of
I want to thank Prof.
"open door" for
of my thesis and his
was
P.
Many,
having
He
genetics,
encouraged
I
to work
me
am
support
grateful to my
subject with which
very
on a
to be my co-examiner I
am
many ideas to solve any
me
into the different
to Prof.
especially grateful
many thanks also to Dr. Stefan Neuenschwander who
introduced
his
Vögeli.
constant
not very familiar before.
For accepting
Bertschinger.
of
any
Stranzinger for his
kind of problems.
my interest in molecular
For arousing
supervisor, Prof.
I
G.
problem which
arose
strategies of molecular
during
biology
was never
H.U.
tired
my thesis.
and
never
He
lost
patience and enthusiasm.
I would like to thank Dr.
Bürgi for taking care of our pigs and her
good co-operation,
performing the histological examina¬
tions and the Animal Neurology Group, University of Bern for the realization
of the neurological examinations of our piglets.
Dr.
For
providing
me
Esther
Pete Ossent for
always
in time with PAC clones I
Alexandra Deppe and Prof. B.
am
very
grateful
to
Brenig.
Gerda, Dagmar, Kathelijne, Elisabeth, and Susanna for
their administrative help and the coffee.
Many thanks
Many,
library. It
had
a
to
many, many
was a
special thanks to Bozena for helping me with the PAC
long way, but together we managed! Whoopy and I
hard and
wonderful time with you in the office and
For
editing
the text of this thesis I
am
very
we
will miss
grateful
you!
to Mika.
to all (ex-) colleagues and friends here at the Institute. Adriforget our lunch walks with Whoopy in any kind of weather! Toshi
and Stefan for organizing the bowling nights
they were just great! Dasha,
Adriana, Bozena, Lara, Mika, Spela, Simone, Toshi, Pascal, Frederic, and Sem,
Special thanks
ana, I won't
-
103
Acknowledgments
104
I
enjoyed working
and
partying with
afterwards; also special thanks
Finally,
you,
we
had great times in the
to Adriana for the
library and
photo from STUZ.
I would like to thank my parents for their support in my education.
My biggest and most special thanks go to Dr. Peter Messmer. I enjoyed
DTpjX and I appreciated your constant help. Many thanks also for your friend¬
ship and support.
This
project has been financed by the Swiss Federal Institute of Technol¬
(ETH), research project No. 0-20-481-98. The contribution of the
Krämer Foundation of Department VII, ETH Zurich to the printing costs is
appreciated.
ogy Zurich