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A Unified Rapid PCR Method for Detection of Normal and Expanded

Trinucleotide Alleles of CAG Repeats in Huntington Chorea and CGG Repeats
in Fragile X Syndrome

Article in Molecular Biotechnology · March 2010

DOI: 10.1007/s12033-010-9260-y · Source: PubMed

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Mol Biotechnol (2010) 45:150–154
DOI 10.1007/s12033-010-9260-y
RESEARCH
A Unified Rapid PCR Method for Detection of Normal and
Expanded Trinucleotide Alleles of CAG Repeats in Huntington
Chorea and CGG Repeats in Fragile X Syndrome
Tihomir Todorov • Albena Todorova •
Bilyana Georgieva • Vanyo Mitev
Published online: 9 March 2010
! Springer Science+Business Media, LLC 2010
Abstract We report on a unified rapid betaine-based-
PCR protocol for amplification of the (CAG)n region in
Huntington disease (HD) and the (CGG)n region in Fragile
X syndrome (FXS), followed by an electrophoretic sepa-
ration on automated sequencer for precise determination of
the triplet numbers. The high betaine concentration (2.5 M
betaine) permits precise amplification of the CAG and
CGG repeats. Ten HD affected patients and 10 healthy
individuals from HD families were re-evaluated. For FXS
the CGG region in normal individuals and premutations of
about 100 repeats were precisely amplified by this protocol.
Ten unrelated FXS premutation carriers and 24 mentally
retarded non-FXS affected boys were re-examined by this
method. The results totally coincided with the previous
ones. This protocol is a good choice as a fast screening test.
Within 24 h we can have preliminary information on the
patient’s genetic status. Normal individuals, CGG premu-
tation carriers up to 100 repeats, as well as HD patients
carrying an expansion up to 50 CAG repeats can be easily
clarified. This accounts for a relatively large proportion
(about 90%) of the suspected HD and FXS patients,
referred to our laboratory for genetic analysis. The calcu-
lation of the repeat’s number is more accurate for the
correct interpretation of the results, screening tests and
genetic counselling.
Tihomir Todorov and Albena Todorova contributed equally to the
present study.
T. Todorov (&) ! A. Todorova ! B. Georgieva ! V. Mitev
Department of Medical Chemistry and Biochemistry, Medical
University Sofia, 2 ‘‘Zdrave’’ street, 1431 Sofia, Bulgaria
e-mail: tisho.todorov@abv.bg
Keywords Huntington disease (HD) ! IT15 (HTT) !
(CAG)n polyglutamine repeat !
Fragile X syndrome (FXS) ! FMR1 ! (CGG)n repeat
Introduction
Huntington chorea is an autosomal dominant neurodegen-
erative disease (HD; OMIM#143100) that is caused by an
expansion of a CAG triple repeat in the first exon of IT15
(HTT) gene, localized on chromosome 4p16.3 [1]. The
affected protein is called huntingtin. In normal individuals
the number of (CAG)n polyglutamine repeat ranges from
10 to 35. The affected patients have more than 36 CAG
repeats [2, 3] with different penetrance between 36 and
more than 42 repeats (from 90 to 100%) [3].
Fragile X syndrome (FXS, OMIM#300624), the most
frequent cause of X-linked mental retardation is provoked
mostly by expansion of unstable trinucleotide CGG repeat
in the 50 UTR of the FMR1 (fragile X mental retardation 1)
gene. The polymorphic CGG repeat varies between 6 and
54 ± 2 copies in normal individuals. The premutation
varies between 55 ± 2 and 200 CGG repeats, which may
expand in the next generations [4, 5]. The individuals who
possess [200 CGG copies, are missing the gene product,
because of promoter hypermethylation, and are affected by
FXS.
Until now the genetic diagnostics for unstable repeat
assessment was mainly based on classical Southern blot or
PCR, followed by hybridization with specific probes.
Moreover, there are some reports on triplet repeat-primed
PCR (TP-PCR), higher betaine concentration and ethidium
bromide or fluorescent detection of (CAG)n or (CGG)n
repeated regions [6–8].
Mol Biotechnol (2010) 45:150–154 151

Here we report on a successful application of a unified
rapid betaine-based-PCR protocol for amplification of the
(CAG)n and (CGG)n repeated regions, followed by an
electrophoretic separation on an automated sequencer for
precise determination of the triplet numbers. This ampli-
fication protocol permits easy detection of normal repeats
and pathological or premutated ones and it is suitable for
diagnostic analysis and fast carrier screening tests. This is
the first report on DNA tests for FXS carriers and HD
patients from Bulgaria.
35 s, annealing at 64 "C for 35 s, and synthesis at 68 "C
for 6 min (25 cycles).
The PCR products obtained were analysed on a 2%
agarose gel, ethidium bromide-stained. For correct sizing,
PCR products were subjected to electrophoresis on ABI
310 genetic analyzer (Applied Biosystem, Foster City,
USA), diluted in Hi-Di Formamide (Applied Biosystem,
Foster City, USA), in the presence of ROX500 size stan-
dard (Applied Biosystems, Foster City, USA).

Results and Discussion

Materials and Methods
The high betaine concentration was selected after a number
All patient blood samples taken in our laboratory were of PCR experiments using betaine concentrations from 0.5
obtained after signed informed consent and professional to 2.5 M in order to be able precisely to amplify the CAG
genetic counselling. Control DNAs from patients with or CGG repeats. The results of agarose gel electrophoresis
genetically proved diagnosis of HD or FXS premutation are given in Fig. 1a and b.
carriers were selected from our genetic register (Bulgarian
samples) or provided by other laboratories abroad (4 German
samples, kindly provided by Institute of Human Genetics,
University of Wuerzburg, Germany and Institute of Human
Genetics, University of Muenster, Germany). The patients
were chosen by means of clear genetic diagnosis and pre-
cisely determined genetic status by classical methods:
Southern blot on genomic DNA or PCR/Southern transfer
and hybridization by (CAG)5 or (CGG)5 probes. In the
majority of cases the genetic results were verified in inde-
pendent foreign laboratories. In total 20 pathological sam-
ples (10 HD affected patients and 10 unrelated FXS
premutation carriers) were chosen to set up the protocol. In
addition, 10 probands from HD families genetically diag-
nosed as non-affected and 24 mentally retarded boys with
genetically excluded FXS were re-examined by the proposed
short PCR.

Betaine-Based-PCR Protocol

The primers for CAG repeat amplification were: 6-FAM 50 -

GGC GGC TGA GGA AGC TGA GGA-30 and 50 -ATG
GCG ACC CTG GAA AGC TGA TGA A-30 (Biomers.net
GmbH, Ulm, Germany) [9]. The primers for CGG repeat
amplification were 50 -GCTCAGCTCCGTTTCGGTTT-
CACTTCCGGT-30 and 6-FAM 50 -AGCCCCGCACTTC-
CACCACCAGCTCCTCCA-30 (Biomers.net GmbH, Ulm,
Germany) [4]. The primer concentration was 0.4 lM. The
reaction was carried out in a 50 ll mixture, containing
0.2 mM dNTPs, 2.5 M Betaine (Sigma), 2% DMSO; 19
PCR reaction buffer (GENET BIO, Chungnam, Korea) and Fig. 1 a Agarose gel of PCR fragments obtained along the CAG
2.5 U Prime Taq (GENET BIO, Chungnam, Korea). The repeated region. 1 normal sample, 2 and 3 affected patients carrying
an expansion. b Agarose gel of PCR fragments obtained along the
amplification conditions were as follows: denaturation at
CGG repeated region. 1–10 Suspected premutation carrier; 3, 6, 10
97 "C for 35 s, annealing at 64 "C for 35 s, and synthesis marked by arrows—carry premutations larger than 90 CGG repeats.
at 68 "C for 4 min (10 cycles); denaturation at 97 "C for 100 bp ladder—size standard
152 Mol Biotechnol (2010) 45:150–154

The amplified constant region (i.e., without the CAG present sample), and probands with CAG expansions of
repeated fragment) is 107 bp (Table 1a); the amplified 30 about 50 repeats.
CGG repeats correspond to 302 bp (Table 1b). The simultaneous analysis of control samples is not
The amplified (CAG)n and (CGG)n repeats, separated necessary each time. The patient’s (CAG)n and (CGG)n
on automated sequencer are presented in Fig. 2a and b, repeats can be easily calculated by size (Table 1a, b).
respectively (panels 1–6). In this way, the diagnosis of HD The samples we chose to re-analyze by the betaine-
and FXS can be performed in 24 h, using a small amount of based-PCR and capillary electrophoresis method presented
DNA and the calculation of the repeat’s number is more in this paper, consisted of 10 HD patients, 10 FXS pre-
accurate, which is very important for the correct interpre- mutation carriers, 10 probands from HD families geneti-
tation of the results, screening tests, and genetic counsel- cally diagnosed as non-affected, and 24 mentally retarded
ling. This is just a preliminary fast screening step, but it can boys with genetically excluded FXS. All of these patients
clarify about 90% of HD and FXS suspected patients have been previously genetically clarified by classical
referred to our laboratory: normal cases, CGG premutation methods and most of them have been verified in indepen-
carriers (100 CGG repeats successfully detected in the dent laboratories abroad. The CAG and CGG repeats
within the normal range in all 34 (10 ? 24) samples were
precisely determined by the proposed short PCR protocol
(data not shown). The results totally coincide with the
Table 1 (a) The amplified constant region of 107 bp and CAG
repeats calculation by size. (b) The amplified CGG repeats calculated
preliminary data (variation range ±1 repeat).
by size The results from 10 CAG expansion carriers and 10
CGG premutation carriers are presented in Table 2a and b.
Size (bp) Number of
repeats Both data from classical Southern blot or PCR followed by
probe hybridization and from capillary electrophoresis
(a) method presented here are compared. One expanded CAG
155 16 allele (Table 2a, number 1) showed lack of coincidence
158 17 larger than ±1 CAG repeat. The precise repeats calculation
161 18 is extremely important at the borderline zones 35 or 36
164 19 repeats, 39 or 40 repeats. This is important for precise
167 20 disease prognosis and adequate genetic counselling.
170 21 The CGG premutated alleles were only roughly mea-
… … sured by Southern analysis. The presented here protocol
224 39 permits premutated expanded alleles to be well visualized
… … on agarose gels (Fig. 1b), which makes the method suitable
245 46 for fast carrier detection screening tests. Afterwards, the
248 47 premutation can be precisely measured by capillary elec-
251 48 trophoresis (Fig. 2b; Table 2b). The capillary electropho-
254 49 resis software 310 GeneScan v.3.1.2 for fragment analysis,
257 50 used in the present study, suffers from poor visualization of
(b) the expanded CGG repeats larger than 75 repeats. Although
287 25 in our study all cases of premutated CGG repeats larger
290 26 than 75 were clearly visible on agarose gels (Fig. 1, sam-
293 27 ples 3, 6, and 10), they are not detected by capillary
296 28 electrophoresis, because the very strong signal from the
299 29 normal allele causes the much weaker signal from the
302 30 expansion allele to be ignored as noise. The largest CGG
… …
premutation we managed to detect by capillary electro-
422 70
phoresis was 75 repeats (Fig. 2b, panel 2). The sample #7
… …
in Table 2b and Fig. 2b, panel 5, has been previously
diagnosed as premutation carrier (about 55 CGG repeats),
482 90
but after testing by the present protocol and re-evaluation
… …
by PCR/Southern hybridization it was proved to carry 50
512 100
CGG repeats.
… …
If male patient was not amplified by a betaine-based-PCR
572 120
protocol, it most probably carries larger premutation or full
Mol Biotechnol (2010) 45:150–154 153

Fig. 2 a HD patients’ CAG repeated profile. b FXS premutation carriers’ CGG repeated profiles (panels 1–6). The repeat numbers are provided
close to each peak, pointed by arrows. The remaining single peaks correspond to the ROX500 size standard

mutation and needs to be reevaluated by alternatives assessment (MRC-Holland www.mlpa.com) [10]. The same
methods like Southern analysis and multiplex ligation-depen- is true for female showing one allele by betaine-based-PCR
dent probe amplification (MLPA) for hypermethylation protocol.
154 Mol Biotechnol (2010) 45:150–154

Table 2 Comparison of the results from Southern transfer/hybrid- expansion and whether this borderline needs to change
ization and capillary electrophoresis in 10 HD patients (a) and in 10 depending on the analysis method. In our diagnostic work
FXS premutation carriers (b)
we also take into consideration the restrictions of each
Patient Number of repeats Number of repeats calculated method and the borderline depends on the methodology.
number* calculated by PCR and/or by PCR and capillary This protocol might be successfully applied for ana-
Southern analysis electrophoresis
lyzing other repeat expansions.
(a)
1 17/about 42 18/39 Acknowledgments The fellowship from Alexander von Humboldt
Foundation to Dr. Albena Todorova is gratefully acknowledged. The
2 19/about 42 20/41 study was supported by the Grants No 23/2008 and No 32/2009,
3 16/abut 43 18/42 Medical University Sofia, Bulgaria.
4 22/about 44 23/43
5 20/about 45 21/46
6 22/about 48 23/49 References
7 12/about 48 13/49
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1 About 20/about 70 21/74 ana, V., Cassiman, J.-J., et al. (1996). Phenotypic characterization
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4 30/about 60 29/70 of Human Genetics, 59(1), 16–22.
5 51/60 50/56 3. Brinkman, R. R., Mezei, M. M., Theilmann, J., Almqvist, E., &
Hayden, M. R. (1997). The likelihood of being affected with
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8 38/about 75 35/67 4. Fu, Y. H., Kuhl, D. P., Pizzuti, A., Pieretti, M., Sutcliffe, J. S.,
9 32/67 29/62 Richards, S., et al. (1991). Variation of the CGG repeat at the
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alleles marked by (*) cannot be detected by capillary electrophoresis of fragile X syndrome. Human Molecular Genetics, 9, 901–908.
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Fitzpatrick, D. R., et al. (1996). A general method for the
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