Download Nonisotopic method for accurate detection of (CAG

Clinical
C’hemistry42:10
1601-1603
(1996)
Nonisotopic method for accurate detection of
(CAG) repeats causing Huntington disease
MARIA
MUGLIA,
CRISTINA
OFELIA
LEONE,
GIIAzIA
ANNESI,
FRANCESCA L. CONFORTI,
GRANDINETTI,
Huntington
disease
(HD) is a neurodegenerative
disorder
caused by an expanded trinucleotide
repeat (CAG)
located
at the 5’ end of the novel 1T15 gene. Discovery
of this
expansion
allows the molecular
diagnosis
of HD by measuring
repeat length.
We applied
a simple
nomsotopic
method to detect (CAG)
repeats, avoiding both radioactive
and Southern
transfer analysis. The assay is based on direct
visualization
of electrophoresed
PCR products,
after silver
nitrate gel staining. Its accurate sizing of lID alleles allows
presymptomatic
diagnosis
of at-risk persons.
By avoiding
isotopic
manipulations,
the method
is safe and accurate,
with no radioactive
background
bands. Furthermore,
because it permits direct allele visualization
after gel staining,
the method is simple and rapid, allowing allele sizing within
hours rather than days.
INDEXING
ThItIS:
repeats . polymerase
amide gel
molecular
genetics . expanded trinucleotide
chain reaction . electrophoresis,
polyacryl-
NASO,
EMILIA
and
IMBROGNO,
CARLO
BRANCATI*
Materials and Methods
Seven families affected by HD, including
13 affected
and 20 unaffected individuals, were analyzed. Diagnosis of HD
on the basis of clinical symptoms
was made either by private
neurologists
or at the Hospital of Galabria.
Subjects.
Sperimentale e Biotecnologie,
CNR,
87100 Cosenza, Italy.
*Author for correspondence. Fax 0984/391106;
PCR assay. Genomic
DNA was extracted
with an automated
DNA extractor (Applied Biosystems, Foster Gity, GA). A double
Via Fratelli
Cervi,
PGR profile, using a total of 35 cycles, was carried out in a 9600
DNA thermal cycler (Perkin-Elmer,
Norwalk,
CT). After an
initial denaturation
of 2 mm at 96 #{176}G,
there were 12 cycles at
94 #{176}C
for 30 s, 65 #{176}G
for 30 s, and 72 #{176}G
for 2 mm, followed by
23 cycles at 92 #{176}G
for 30 s, 65 #{176}G
for 30 s, and 72 #{176}G
for 2 mm;
final extension was at 72 #{176}G
for 10 mm.
The PGR was carried out in a final volume of 25 mL with the
primers HD 1 (5 ‘-ATGAAGGGGYFGGAGTCGGTGAAGTGG’VITG-3’) and HD3 (5 ‘-GGGGGTGGGGGGTGTTGGTGGTGGTGGTGC-3’)
[14]. Reaction
mixtures
contained
2
mmolfL MgCl2, 16.6 mmoVL (NH4),S04,
67 mmoVL TrisHC1, pH 8.8, 67 .tmoVL Na,EDTA,
35 mLIL formamide,
10
mmol/L
13-mercaptoethanol,
2.5
.tmol/L
bovine serum albumin, 200 mmol/L of each dNTP (with a final ratio of 1:3 dGTP:
7-deaza-GTP),
12.5 pmol each of HD1 and HD3, 1.25 U of
Taq polymerase,
and 250-500 ng of genomic DNA. From each
I,
e-mail [email protected].
CNR.IT.
Received January
FRANCESCO
GABRIELE,
or chemiluminescent
detection of blotted PCR products [13].
We have applied a simple and rapid method for HD diagnosis avoiding both radioactivity
and Southern transfer analysis.
The system involves sample PGR, separation
of alleles on
polyacrylamide
gels, and staining with silver nitrate. The new
PGR conditions
we describe improve the yield of the product,
allowing direct visualization
of HD alleles on silver nitratestained polyacrylamide
gels.
is characterized
by involuntary
movements,
psychiatric changes,
intellectual
and cognitive decline, and dementia. The symptoms
of HD appear to be caused by marked neuronal
death, most
notably in the caudate nucleus and putamen [2].
The mutation responsible
for HD has recently been discovered as an expansion of a GAG trinucleotide
repeat located at
the 5’ end of a novel 4pi#{243}.3
gene, named 1T15 (interesting
transcript
15) [3]. The repeat is polymorphic
in the normal
population,
varying between 8 and 36 units on normal chromosomes, but is expanded to at least 37 copies on HD chromosomes [3-7]. A significant inverse correlation
between the size of
di Medicina
L.
the GAG repeat and the age of onset of symptoms
has been
observed in HD, especially when the repeat is >50 [8-10].
The discovery of the defect causing HD allows the direct
presymptomatic
diagnosis of the disease through measuring the
number of GAG repeats in the DNA of a person at risk. Until
now, the procedures
used to detect the length of this trinucleotide repeat required
radioactive
analysis-radiolabeled
polymerase chain reaction (PCR) and Southern
transfer [3, 11, 12]
Huntington
disease (HD) is a progressive
neurodegenerative
diseaseof midlifeonset, inheritedin an autosomal dominant
manner, that affects 1:10 000 individuals [1].
The clinical picture
Isututo
ANNA
23, 1996; accepted
May
14, 1996.
1601
1602
Muglia
et al.: Nonisotopic
detection
of (GAG),,
amplified DNA sample, 5 iL was tested on 3% agarose gel with
Tris-acetate-EDTA
buffer (0.04 molJL Tris-acetate,
0.001
molIL EDTA, pH 8.0) containing
0.02 g/L ethidium bromide.
After electrophoresis,
the DNA was visible under ultraviolet
light.
Allele sizing. The remaining
20 p.L of PCR product was precipitated with cold ethanol and electrophoresed
through an 8%
nondenaturing
polyacrylamide
gel (acrylamide:bisacrylamide
=
19:1) at 500 V for 17 h at 4#{176}C.
For better resolution
of normal
alleles, we used 10% gel when analyzmg DNA from normal
subjects. After electrophoresis,
the gels were stained with silver,
as follows [15]: wash in 4.607 mol/L ethanol solution for 5 mm;
oxidize in 0.6301 mol/L nitric acid solution for 3 mm; rinse in
distilled water for few seconds; incubate in 0.0 12 mol/L silver
nitrate solution
for 20 mm; rinse in distilled water for few
seconds; reduce in a solution of 0.28 mol/L anhydrous
sodium
carbonate
and 6.327 j.molJL
formaldehyde,
with several
changes of the reducing solution (each time the solution turned
brown); stop the reducing
process with 6.005 mol/L glacial
acetic acid for 10 mm; and wash in distilled water for 2 mm. The
size of the polymorphic
HD alleles was detected after silver
nitrate
staining
by comparison
with both DNA molecular
marker V (Boehringer
Mannheim,
Mannheim,
Germany)
and
previously sequenced
alleles.
Results and Discussion
Discovery of the gene responsible for HD has had a great impact
in the diagnostic field, making it possible to do presymptomatic
and prenatal diagnosis of HD by recombinant
DNA techniques.
In the first published studies on HD alleles, the DNA region
containing
the HD mutation was amplified with original primers HDI and HD2 [3], which spanned the CAG trinucleotides
as
well as an adjacent GGG repeat. When this GGG repeat was
found to be polymorphic
[16, 17], a new set of primers was
designed that selectively amplified the GAG repeat and excluded
the GGG polymorphic
region [14] (Fig. 1). However,
the high
repetitiousness
of the HD-region,
together with its high GG
repeats
in Huntington
disease
samples and allele visualization
on silver nitrate-stained
polyacrylamide gels, has proved to be very simple and rapid.
We found that certain conditions
affected the utility of the
PCR product from the HD region. Tests to improve the PCR
demonstrated
that formamide
(35 rnL/L) was necessary to have
a good product
yield but dimethyl
sulfoxide was not. The
presence
of 7-deaza-dGTP
in the ratio for dGTP:7-deazadGTP of 1:3 was crucial for specificity. With regard to the PCR
profile, we found that lowering the denaturation
temperature
by
2 #{176}C
after the initial 12 cycles greatly improved the yield of the
product [18], whereas increasing
the number of PCR cycles to
>35 gave a background
of nonspecific
bands.
We used this method to examine seven HD families, whose
members
included
13 patients
diagnosed
from their clinical
symptoms
and 20 unaffected
individuals.
Fig. 2 shows PGR
products from 3 HD patients. Both normal and expanded alleles
are clearly visible and no background
bands are present. Fig. 3
illustrates the separation of different normal (non-HD)
alleles in
three unaffected
individuals,
both homozygotes
and heterozygotes. Differences
of only one trinucleotide
can be easily
detected (lane 4).
Molecular
analysis confirmed
the clinical diagnosis in 12 of
13 cases and showed an association
between size of alleles and
age of onset. The remaining subject showing HD symptoms had
two allele repeat sizes well within the normal range; detection
of
the size of GAG repeat in this individual makes it possible to
have an alternative
diagnosis
of an “HD-like”
neurological
condition.
Trinucleotide
repeat expansion was also observed in
one asymptomatic
member of an HD family: A 23-year-old
son
of an affected 50-year-old
woman showed an expansion from 45
to 50 repeats. This study confirmed both the diagnostic value of
the (GAG)
repeat expansion of the ITI 5 gene and the appropriate use of this molecular
test in differentiating
HD from
other illnesses [19, 20].
23
]
content,
make the PCR analysis very difficult,
so that the
described amplification
procedures
often fail to detect the upper
alleles and radioactive
analysis is needed to distinguish
between
a normal individual and an affected one.
The method described here allows rapid and precise diagnosis of HD. To size the GAG repeat accurately, we used HD1 and
HD3 primers (see Fig. 1) that exclude the polymorphic
GCG
repeat, thus allowing correct HD diagnosis even in borderline
cases. The procedure,
involving
nonradioactive
PGR of the
fflH
-,
4
HD3
HD2
Fig. 1. 5’ Polymorphic region of 1T15 gene.
OriginalHD1 and HD2 primers span both CAG-repeatand CCGrepeat, whereas
HD3 primer, used together with HD1 primer, selectively amplifies the CAG
repeat.
76
26
71
25
73
26
Fig. 2. PCR analysis of trinucleotide repeats in HD patients.
PCRproductsare separatedon 8%polyacrylamidegel, stained with silver nitrate.
N. normal alleles; Exp, expanded alleles.
Clinical
Chemistiy
bp
123
4
42, No.
8.
20
18
20
15
17
Fig. 3. PCR analysis in 10% polyacrylamide gel of trinucleotide repeats
in unaffected individuals: lane 1, homozygous subject; lane 2, DNA
molecular marker V (Boehringer); lanes 3-4, heterozygous subjects.
The accurate detection of the size of GAG repeats is essential
for HD diagnosis,
and the use of HDI and HD3 primers is
necessary to avoid diagnostic
mistakes in individuals
carrying
borderline
numbers of repeats. By optimizing
PGR conditions,
one can obtain an accurate and rapid sizing of both normal and
expanded HD alleles.
9.
10.
11.
12.
13.
In summary, the method described here offers many advantages
over the published procedures
for sizing HD alleles. No isotopic
manipulations
are involved, making the method both safe and
accurate,
because of the absence of radioactive
background
bands. Previously
published
nonradioactive
assays [13, 21], as
performed
with the originally recommended
primers, were not
suitable for detection of borderline-repeat
alleles. Furthermore,
because it permits direct visualization
of alleles after gel staining,
our simple and rapid method allows allele sizing within hours
rather than days.
14.
15.
16.
17.
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