Rapid detection, Neurologia i Neurochirurgia Polska od 2012

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ORIGINAL PAPER/ARTYKU£ ORYGINALNY
Rapid detection of large expansions in progressive myoclonus epilepsy type 1,
myotonic dystrophy type 2 and spinocerebellar ataxia type 8
Szybkie wykrywanie du¿ych ekspansji wpostêpuj¹cej padaczce mioklonicznej typu 1,
dystrofii miotonicznej typu 2 iataksji rdzeniowo-mó¿d¿kowej typu 8
Wioletta Krysa, Marta Rajkiewicz, Anna Su³ek
Zak³ad Genetyki, Instytut Psychiatrii i Neurologii w Warszawie
Neurologia i Neurochirurgia Polska 2012; 46, 2: 113-120
DOI: 10.5114/ninp.2012.28253
Abstract
Streszczenie
Background and purpose: Human genetic disorders associat-
ed with multiple unstable repeats resulting in long DNA
expansions are difficult to identify by conventional polymerase
chain reaction (PCR) in routine molecular testing, and there-
fore require time-consuming hybridisation. To improve and
expedite the diagnostic methods for progressive myoclonus
epilepsy (EPM1), myotonic dystrophy 2 (DM2) and spino-
cerebellar ataxia 8 (SCA8) caused by dynamic mutations, we
adapted a repeat primed PCR (RP-PCR) assay which was pre-
viously developed for testing of other triplet repeat disorders.
Material and methods: The new algorithm for molecular
analysis was to run a standard PCR to yield alleles in an
amplifiable range and then run a RP-PCR to detect larger
expansions. Electrophoresis and visualisation of PCR pro-
ducts on an automatic sequencer were applied to determine
normal and pathogenic alleles comprising (C
4
GC
4
GCG)
n
in
EPM1 in 44 subjects, (CCTG)
n
in DM2 in 76 individuals
and (CTG)
n
in SCA8 in 378 patients.
Results: The protocol combining conventional PCR and
RP-PCR proved to be a rapid and reliable test to diagnose
the above named disorders. Among 44 individuals tested for
EPM1, two expanded alleles were identified in 7 patients.
Out of 76 apparently homozygous subjects, RP-PCR allowed
us to detect 56 expansions specific to DM2, and out of 378 ata-
Wstêp i cel pracy: Oparta na konwencjonalnej reakcji ³añcu-
chowej polimerazy (PCR) diagnostyka molekularna chorób
zwi¹zanych z niestabilnymi sekwencjami powtórzonymi bywa
niewystarczaj¹ca do wykrycia ogromnych ekspansji, wówczas
konieczne jest stosowanie hybrydyzacji. W celu usprawnie-
nia analizy molekularnej trzech chorób wywo³ywanych muta-
cjami dynamicznymi: postêpuj¹cej padaczki mioklonicznej
(EPM1), dystrofii miotonicznej typu 2 (DM2) i ataksji rdze-
niowo-mó¿d¿kowej typu 8 (SCA8) zaadaptowano technikê
RP-PCR (
repeat primed PCR
), któr¹ uprzednio opracowano
dla chorób powodowanych ekspansjami sekwencji trójnukleo-
tydowych.
Materia³ i metody: Oprócz standardowej reakcji PCR, w któ-
rej uzyskiwano allele w zakresie mo¿liwym do amplifikacji,
celem detekcji wiêkszych ekspansji stosowano RP-PCR.
Elektroforetyczny rozdzia³ produktów PCR na automa-
tycznym sekwenatorze umo¿liwia³ analizê alleli z zakresu
prawid³owego oraz patogennego, które zawiera³y dwunasto-
nukleotyd (C
4
GC
4
GCG)
n
w EPM1 u 44 osób badanych,
czte ro nukleotyd (CCTG)
n
w DM2 u 76 osób i trójnukleo-
tyd (CTG)
n
w SCA8 u 378 pacjentów.
Wyniki: Zastosowany protokó³ diagnostyczny oparty na kon-
wencjonalnej reakcji PCR i RP-PCR pozwoli³ na szybkie
otrzymanie wiarygodnych wyników testu genetycznego w kie-
Correspondence address: dr Wioletta Krysa, Zak³ad Genetyki, Instytut Psychiatrii i Neurologii w Warszawie, ul. Sobieskiego 9, 02-957 Warszawa,
e-mail: krysa@ipin.edu.pl
Received: 17.02.2011; accepted: 22.12.2011
113
Neurologia i Neurochirurgia Polska 2012; 46, 2
Wioletta Krysa, Marta Rajkiewicz, Anna Su³ek
xia patients, a large allele of the
ATXN8OS
gene (SCA8) was
found in 25 subjects.
Conclusions: Here, for the first time, we report detection of
large expansions in EPM1 and SCA8 patients. This RP-PCR
assay is high throughput, reproducible and sensitive enough
to be successfully used for diagnostic purposes.
Key words: dynamic mutations, neurodegenerative disorders,
repeat primed PCR (RP-PCR).
runku wy¿ej wymienionych chorób. Spoœród 44 osób pod-
danych analizie w kierunku EPM1 wyodrêbniono 7 przy-
padków patogennej ekspansji. Wœród 76 osób z dystrofi¹ mio-
toniczn¹, których wyniki po standardowej reakcji PCR
wskazywa³y na genotyp prawid³owy (homozygoty) zastoso-
wanie reakcji RP-PCR umo¿liwi³o identyfikacjê 56 osób
z ekspansj¹ powoduj¹c¹ DM2. Analogicznie w grupie 378
pacjentów z ataksj¹, w 25 przypadkach wykryto patogenne
allele genu
ATXN8OS
(SCA8).
Wnioski: W pracy przedstawiono po raz pierwszy zastoso-
wanie RP-PCR w diagnostyce molekularnej do wykrywania
du¿ych ekspansji u pacjentów z podejrzeniem EPM1
i SCA8. Ze wzglêdu na wysok¹ wydajnoœæ, powtarzalnoœæ
i czu³oœæ techniki RP-PCR mo¿e byæ stosowana do celów dia-
g no stycznych.
S³owa kluczowe: mutacje dynamiczne, choroby neurodege-
neracyjne, reakcja RP-PCR.
Introduction
hybridisation steps, and requires large amounts of
DNA. Another method used to detect long expansions
is called RP-PCR (repeat primed PCR). In a previous
study, RP-PCR was developed as TP-PCR (triplet
primed PCR) for the molecular analysis of DM1 [2]
and was later successfully applied in other repeat-asso-
ciated diseases such as Friedreich ataxia (FRDA),
SCA2, SCA7 and SCA10 [3,4]. RP-PCR was recent-
ly used to detect tetranucleotide expansions in DM2
patients [5,6].
Progressive myoclonus epilepsy type 1 (EPM1,
Unverricht-Lundborg disease, OMIM 254800) is an
autosomal recessive neurodegenerative disorder caused
by expansions of the (C
4
GC
4
GCG)
n
minisatellite
sequence in the promoter of the
CSTB
gene that causes
a defect in cystatin B, a cysteine protease inhibitor [7].
Normal alleles contain two or three copies of the dode-
camer repeat, whereas a majority of pathogenic expand-
ed alleles contain roughly 45-70 repeats. About 10% of
EPM1 cases are caused by point mutations. Conven-
tional PCR is able to detect products of about 950 bp,
but amplification of these CG-rich sequences is known
to be problematic and requires several modifications.
The onset of clinical symptoms in EPM1 ranges from
6 to 15 years of age. Affected individuals present with
stimulus-sensitive myoclonus and tonic-clonic epileptic
seizures with variable progression; mental retardation
or dementia as well as ataxia are also observed [8].
Myotonic dystrophy type 2 (DM2, OMIM
602668) is a progressive multisystem disorder affecting
skeletal and smooth muscles and is inherited as an auto-
Over 40 neurodegenerative disorders are known to
result from unstable DNA repeat expansion in both cod-
ing and non-coding regions of implicated genes. This
genetic instability, called dynamic mutation, manifests
as a change in copy number (expansion and sometimes
contraction) with a rate that depends to some extent on
the tract length of initial repeats, tissue specificity or
generational age [1]. Disorders associated with repeat
multiplication include the tri-nucleotide expansion dis-
orders, such as Huntington disease (HD), eight spin-
ocerebellar ataxias (SCAs), myotonic dystrophy type 1
(DM1) and others. Disorders such as myotonic dys-
trophy type 2 (DM2), caused by tetranucleotide expan-
sions; spinocerebellar ataxia type 10 (SCA10), caused
by pentanucleotide multiplication; and progressive
myoclonus epilepsy 1 (EPM1), caused by minisatellite
(dodecamer) expansions, belong to the same class of
human genetic diseases.
Dynamic mutations resulting in long expansions
(for example, there are over 2,000 CTG repeats in the
congenital form of DM1) are difficult or sometimes
impossible to detect by the genotyping of PCR prod-
ucts. For example, testing for DM2 by routine PCR
only allows detection of repeat fragments up to 300 base
pairs (bp) in the
ZNF9
locus. Thus, Southern blot
analysis has been used to detect large expanded alleles
even though the size of the expansion can be only
approximated using this method. Moreover, this tech-
nique often involves radioactive labelling and laborious
114
Neurologia i Neurochirurgia Polska 2012; 46, 2
 Rapid detection of large expansions in EPM1, DM2, and SCA-8
somal dominant trait. The DM2 corresponding gene
ZNF9
(3q13.3-q24) codes for cellular nucleic acid-bind-
ing protein (zinc finger protein 9) and contains the com-
plex repeat motif (TG)
n
(TCTG)
n
(CCTG)
n
in the first
intron. The size in normal alleles ranges from 104 to
176 bp and is usually reported in base pair length due
to highly polymorphic TG and TCTG repeat tracts [9].
Moreover, the CCTG tract in normal alleles contains
one or more tetranucleotide interruptions (TCTG or
GCTG) [10]. A characteristic feature in DM2 patients
is the loss of interruptions and a (CCTG)
n
repeat expan-
sion that ranges from 75 up to 11,000 repeats; amplifi-
cation of this expansion is impossible in a routine PCR.
Additionally, the sensitivity of Southern blotting was esti-
mated to be about 70% and can miss expansions con-
taining over 5,000 CCTG repeats [11].
Spinocerebellar ataxia type 8 (SCA8, OMIM
608768) is a slowly progressive ataxia in which patients
present with dysarthria, gait instability, nystagmus and
other neurological signs. Patients with SCA8 were
reported to have an abnormal number of CTA/CTG
repeats in the
ATXN8OS
gene. The non-pathogenic
range of this microsatellite region is 15–50 repeats; the
size usually associated with ataxia ranges from 80 to 250
repeats, but alleles with up to 800 CTA/CTG repeats
have also been observed. The mode of inheritance is
autosomal dominant with a reduced penetrance, as alle-
les of more than 80 repeats are also observed in healthy
individuals [12].
Due to high demand for molecular diagnosis in the
above named neurological diseases, a reliable and rapid
detection methodology must be developed. As the appli-
cation of RP-PCR in testing for EPM1 and SCA8 has
not previously been reported, the purpose of this study
was to evaluate RP-PCR as a screening and diagnostic
technique in affected individuals suspected of having
these disorders.
genotype in a routine PCR was determined, and 378
ataxia patients in whom expansions specific for SCA1,
SCA2, SCA3, SCA17 were not found. All ataxic sub-
jects had only one
ATXN8OS
normal allele detected,
suggesting either the status of normal homozygote or
the presence of large allele unamplifiable in a routine
PCR. Moreover, apart from 76 affected subjects, for
4 individuals at 50% risk of DM2 molecular analysis
was performed.
The patients included in the study were referred for
genetic testing mainly by neurologists to confirm or to
exclude clinical diagnosis of DM, SCA or EPM1. After
the proband’s molecular evaluation, unaffected individ-
uals at risk were given genetic counselling and had test-
ing done at their request.
The study was approved by the Bioethical Commis-
sion of the Institute of Psychiatry and Neurology, Warsaw.
The general principle in the RP-PCR protocol is to
use three primers: a fluorescently labelled primer adja-
cent to the polymorphic sequence specific to the gene;
a second primer consisting of several repeats, for exam-
ple trinucleotide (DM1), tetranucleotide (DM2) or one
dodecamer repeat (EPM1); and a 5’ tail sequence com-
plementary to the third universal primer P3R (artifi-
cially produced random DNA).
In the early amplification rounds, the PCR assay
uses a locus-specific forward primer together with the
reverse 5’ tailed primer consisting of several microsatel-
lite repeats to amplify a mixture of products. The mix-
ture is due to priming within different sites of the repeat-
ed sequence. Then, after exhausting the first reverse
primer with the 5’ tail sequence (due to a 10 : 1 molar
ratio of P3R to 5’ tailed reverse primer), the third
primer, P3R, which comprises the sequence comple-
mentary to the tail, preferentially binds to the end of
amplicons from previous amplification cycles. In visu-
alisation of RP-PCR products, a characteristic ladder
can be observed which results from the mixture of
amplicons of different length.
The molecular diagnosis of EPM1, DM2 and
SCA8 was performed in two steps: (1) following the
standard PCR reaction, the procedure was finished
when two normal size alleles were observed; (2) the RP-
PCR reaction was performed for DNA samples that
showed only one visible signal in the electropherogram
(suggesting possible homozygosity or presence of an
abnormal allele) or in samples with no signal in the nor-
mal range (in cases of recessive EPM1).
Primer sequences used in PCR and RP-PCR are
shown in Table 1; PCR thermal conditions and reac-
Material and methods
DNA samples were extracted as described elsewhere
[13] or by automated isolation on a Roche MagNA
Pure Compact (Japan) device. Informed consent was
obtained from all patients participating in the study. The
following numbers of tested patients’ DNA samples
were analysed: 44 individuals suspected of having
EPM1 and 17 family members (parents), 76 patients
with clinical diagnosis of myotonic dystrophy in whom
DM1 was previously excluded and a homozygous DM2
115
Neurologia i Neurochirurgia Polska 2012; 46, 2
 Wioletta Krysa, Marta Rajkiewicz, Anna Su³ek
tions mixes are shown in Table 2 and Table 3, respec-
tively.
The GeneAmp 9700 (Applied Biosystems, Foster City,
CA USA) thermal cycler was used to perform the PCR
reactions. Analysis of C
4
GC
4
GCG (EPM1) repeats was
done by conventional PCR with primers as previously
described [14]. The problematic amplification of the CG-
rich region of the
CSTB
gene required the following mod-
ifications to thermal cycling conditions: 0.9°C/s RAMP
and use of 150 ng/μL of genomic DNA.
The standard PCR and RP-PCR amplicons were
separated on ABI PRISM 377 (Applied Biosystems,
Foster City, CA USA) with the internal size marker
FraX or TAMRA2500 (Applied Biosystems, UK) in
a 4% denaturing gel (EPM1) and in a 5% gel (DM2)
or a 4% gel (SCA8) with the internal size marker TAM-
RA500 (Applied Biosystems, UK). Sizing analyses were
performed with Gene Scan v. 3.1.2 software. Alterna-
tively, analysis of fluorescent RP-PCR products was
done using an ABI3130 automatic sequencer (Applied
Biosystems, Foster City, CA USA) on a 4 capillary array
with the universal POP7 polymer and a ROX-GS500
size standard marker (Applied Biosystems, UK). Gene
Mapper v. 4.0 software was used for amplicon sizing.
Results
Progressive myoclonus epilepsy type 1
The diagnostic protocol established by combining
standard PCR and RP-PCR assays correctly identified
the allelic sizes of the
CSTB
gene in all 44 symptomatic
individuals tested: 7 subjects with two expanded alleles
and 37 individuals with normal-sized alleles. Moreover,
Table 1. Routine polymerase chain reaction (PCR) and repeat primed PCR (RP-PCR) primer sequences
Disease
Primers flanking the polymorphic sequence, specific to the gene – routine PCR
EPM1
F 5’-AGC CTG CGG CGA GTG GTG-3’
R 5’-FAM-GGC CGG GGA GGA GGC ACT-3’
DM2
F 5’-GCC TAG GGG ACA AAG TGA GA-3’
R 5’-FAM GGC CTT ATA ACC ATG CAA ATG-3’
SCA8
F 5’-GTA AGA GAT AAG CAG TAT GAG GAA GTA TG-3’
R 5’-FAM GGT CCT TCA TGT TAG AAA ACC TGG CT-3’
Primers specific to the repeats – RP-PCR
EPM1
F 5’-TAC GCA TCC CAG TTT GAG ACG CCC CGC CCC GCG CCC CGC CCC GCG-3’
DM2
F DM2A 5’-TAC GCA TCC CAG TTT GAG ACG CCT GCC TGC CTG-3’
R DM2B 5’-FAM TGA GCC GGA ATC ATA CCA GT-3’
SCA8
P4CAG 5’-TAC GCA TCC CAG TTT GAG ACG CAG CAG CAG CAG CAG CA-3’
Universal primer
P3R
P3R 5’-TAC GCA TCC CAG TTT GAG ACG-3’
EPM1 – progressive myoclonus epilepsy type 1, DM2 – myotonic dystrophy type 2, SCA8 – spinocerebellar ataxia type 8, F – forward primer (starter forward), R – reverse primer (starter
reverse)
Table 2. Polymerase chain reaction (PCR) conditions for EPM1, DM2, SCA8 and RP-PCR
EPM1/EPM1 RP-PCR
DM2
DM2 RP-PCR
SCA8/SCA8 RP-PCR
Initial denaturation
98°C/5 min
95°C/5 min
95°C/5 min
95°C/5 min
Denaturation
98°C/1 min
95°C/45 s
95°C/1 min
95°C/1 min
Annealing
65°C/1 min
57°C/45 s
57°C/1 min
56°C/1 min
Elongation
78°C/3 min
72°C/1 min
72°C/2 min
72°C/2 min
Final elongation
78°C/10 min
72°C/10 min
72°C/10 min
72°C/10 min
Number of replication rounds
32
28
32
32
RP-PCR – repeat primed PCR, EPM1 – progressive myoclonus epilepsy type 1, DM2 – myotonic dystrophy type 2, SCA8 – spinocerebellar ataxia type 8
116
Neurologia i Neurochirurgia Polska 2012; 46, 2
 Rapid detection of large expansions in EPM1, DM2, and SCA-8
Table 3. Polymerase chain reaction (PCR) reaction mixes
EPM1
DM2
SCA8
PCR/RP-PCR
PCR RP-PCR
PCR/RP-PCR
Polymerase
1.25 U Pfu TURBO
Ampli Tag Gold + II buffer
Ampli Tag Gold + II buffer
Hot Start + buffer (Stratagene)
(Applied Biosystems)
1.25 U
5 U
5 U
dNTPs
dNTPs mix + 7-deaze (2 mM)
dNTPs 2.5 mM each
dNTPs 2.5 mM each
(mix of dATP, dTTP, dCTP,
without dGTP)
Others
DMSO 10%
Triton X 10%
DMSO 10%
DMSO 10%
(BDH England)
Gelatine 0.1%
(Sigma)
Primers forward/reverse
10 μM
10 μM
10 μM
5’ tailed reverse primer
1 μM
1 μM
1 μM
Total volume
20 μL
20 μL
20 μL
RP-PCR – repeat primed PCR, EPM1 – progressive myoclonus epilepsy type 1, DM2 – myotonic dystrophy type 2, SCA8 – spinocerebellar ataxia type 8
additional testing of 17 DNA samples of the affected chil-
dren’s parents revealed 8 carriers (heterozygotes) with
one expanded allele. A range of 60-79 detectable dode-
camer repeats in DNA samples from symptomatic indi-
viduals and carriers was established. In RP-PCR a sig-
nal consisting of a ladder with 12 bp periodicity
corresponding to the dodecamer (C
4
GC
4
GCG)
n
was
obtained (Fig. 1). This ladder confirmed the presence of
the expansion. In cases in which only one allele of normal
range was detected in the electropherogram, suggesting
homozygosity or carrier status, re-evaluation by RP-PCR
was performed to detect or to exclude the expansion.
Within the group of 7 patients mentioned above (with
two expanded alleles), prior testing in 2 children by a con-
ventional PCR yielded no products and failed to detect
expansions even though testing in their parents revealed
A
120
180
240
300
360
420
480
540
600
660
720
780
840
900
Expanded alleles
70 repeats
180
90
0
68 repeats
bp
B
120
150
180
210
240
270
1440
960
480
0
bp
Fig. 1. Electropherograms of progressive myoclonus epilepsy type 1 (EPM1) conventional polymerase chain reaction (PCR) and repeat primed PCR
(RP-PCR) products. A) Two expanded alleles (peaks) indicated by the arrows: 910 bp (68 repeats) and 934 bp (70 repeats) of CSTBgene in EPM1 patient. The other
peaks visible in the electropherogram correspond to the internal size standard FraX from Applied Biosystems. B) Full mutation of CSTBgene confirmed by RP-PCR –
the ladder of peaks, with 12 base pairs (bp) periodicity is shown. Peaks visible in the electropherograms represent the PCR reaction products, the x axis shows product
size in bp, and the y axis shows the peak height measured by signal intensity of fluorescently labelled PCR products. Each electropherogram is presented with its own
scale according to different PCR products’ size and reaction efficiency
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Neurologia i Neurochirurgia Polska 2012; 46, 2
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