JMD 2002, Vol. 4, No. 2
Copyright © 2002 American Society for Investigative Pathology & Association for Molecular Pathology
Diagnosis of Five Spinocerebellar Ataxia Disorders by Multiplex Amplification and Capillary Electrophoresis
Michael O. Dorschner*,
Deborah Barden
and
Karen Stephens*
From the Departments of Medicine
*
and Laboratory Medicine,
University of Washington, Seattle, Washington
 |
Abstract
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|---|
The autosomal-dominant spinocerebellar ataxias (ADCA) are a
heterogeneous group of neurodegenerative disorders with variable
expression and phenotypic overlap. An accurate diagnosis relies on
detection of a mutation in a specific causative gene, which is
typically an abnormal number of CAG trinucleotide repeats. To
streamline testing in a clinical setting, we converted our
current panel of tests for the spinocerebellar ataxias (SCA) types
SCA1, SCA2, SCA3, SCA6, and SCA7 from
five independent amplification reactions analyzed by polyacrylamide gel
electrophoresis (PAGE) to a single multiplex amplification reaction
analyzed by capillary electrophoresis (CE). Multiplex amplification was
facilitated by the use of chimeric primers; different lengths and
fluorochromes distinguished the amplicons. During CE with commercially
available molecular weight standards, the SCA amplicons
migrated faster than predicted, thereby underestimating their
length compared to that determined previously by PAGE. This was
observed to varying degrees for each of the five loci, with the
greatest size differential occurring in amplicons with greater
(CAG)n. To determine accurate amplicon length, and
therefore an accurate number of CAG repeats, a size correction
formula was calculated for each locus. This multiplex semi-automated
assay has been reliable during 1 year of use in a clinical setting
during which 57 samples were tested and five positive samples were
detected.
|
Introduction
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|---|
The autosomal-dominant spinocerebellar ataxias (ADCA) are a
heterogeneous group of neurodegenerative disorders characterized by
slowly progressive cerebellar dysfunction. Affected individuals have
difficulty coordinating body movement, gait ataxia in particular, in
addition to other associated findings.1, 2
Over 15 distinct
types of hereditary spinocerebellar ataxia (SCA) disorders have been
described based on identification of different causative genes or
chromosomal loci.1
Because of variable expression and
phenotypic overlap, the SCA disorders cannot be differentiated reliably
on a clinical basis. Although algorithms may predict the likelihood of
a specific type of SCA,3, 4
an accurate diagnosis depends
on molecular testing that detects a mutation in a specific causative
gene. With few exceptions, mutations that cause SCA disorders are an
abnormally large numbers of nucleotide-repeat motifs. The most common
motif is a CAG trinucleotide repeat in the coding region of the
respective genes, which on expansion causes SCA1, SCA2, SCA3, SCA6,
SCA7, and SCA17 (Table 1)
.1, 5, 6, 7
Mutations involving expansion of other repeat
motifs in coding and noncoding regions of additional genes are known to
cause other types of SCA disease.1, 8, 9
Typically, testing for the SCA disorders employs a panel that includes
individual assays for the most prevalent types of SCA, for example
SCA1, SCA2, SCA3, SCA6, and SCA7 in the U.S. population. The number of
CAG repeats in each of the five causative genes is determined. The
detection of an abnormally large number of CAG repeats in a gene means
that the patient has, or is predisposed to develop, that type of SCA.
Testing commonly employs radiolabeled primers to individually amplify
the region containing the CAG-repeat motif from each of the five genes,
polyacrylamide gel electrophoresis (PAGE) to resolve the amplicons by
size, and autoradiography for visualization. To determine whether the
amplicons have an abnormal number of repeats, their lengths are
determined by co-migration with a DNA sequencing ladder. This method is
labor intensive, requiring both the analysis of each locus individually
and the precautions inherent in the use of radionuclides. Here, we
detail for the first time, a rapid and precise method to assay
SCA1, SCA2, MJD (SCA3),
CACNA1A (SCA6), and SCA7 by multiplex
amplification and capillary electrophoresis (CE).
|
Materials and Methods
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Single SCA Gene Amplification and PAGE Analysis
Genomic DNA was purified from peripheral blood with the Puregene
Human and Mammalian DNA Isolation kit (Gentra Systems, Inc.,
Minneapolis, MN). Segments of the genes SCA1, SCA2,
MJD (SCA3), CACNA1A (SCA6), and SCA7
harboring the CAG-repeat region were amplified in five separate
reactions in an ABI 9700 thermal cycler (Applied Biosystems, Foster
City, CA). Reactions (20 µl) contained 2 µl of 10X reaction buffer
(GeneAmp PCR Buffer II, Applied Biosystems); 200 µmol/L dNTPs
(Amersham-Pharmacia Biotech, Piscataway, NJ); 0.2 pmol
-32P-labeled forward primer; 1.5 units of
polymerase (AmpliTaq, Applied Biosystems); 0.2 to 0.8 µg genomic DNA.
For the SCA1, MJD (SCA3), and CACNA1A
(SCA6) gene assays, 12 pmols of each primer were added; the
SCA7 assay used 8 pmols of each primer; and the SCA2 assay
used 1.3 pmols of SCA2A with 15 pmols of SCA2B. In addition, the SCA7
assay contained 5% dimethylsulfoxide (DMSO). Amplification of
the SCA1, SCA2, MJD, and CACNA1A genes were
performed for one cycle at 95°C for 5 minutes, and 28 cycles at
95°C for 1 minute, 61°C for 1 minute, 72°C for 1.5 minutes, with
a final extension at 72°C for 5 minutes. Amplification of the
SCA7 gene was performed for one cycle at 95°C for 5
minutes, and 35 cycles at 94°C for 45 seconds, 55°C for 75 seconds,
and 72°C for 60 seconds, with a final extension at 72°C for 10
minutes. Samples were mixed with formamide-loading buffer (95%
formamide, 20 mmol/L EDTA, 0.05% bromophenol blue, 0.05% xylene
cyanol) and electrophoresed through a denaturing polyacrylamide gel
(GelMix6, Gibco/BRL) at 1900 V. The amplicons for each assay were
electrophoresed on separate gels, adjacent to an M13 sequencing ladder,
for a time sufficient to resolve the specific product. Amplicon lengths
(excluding the trinucleotide-repeat region) are SCA1, 124
bp; SCA2, 59 bp; MJD,162 bp; CACNA1A,
102 bp; and SCA7, 277 bp.
Multiplex SCA Gene Amplification and CE Analysis
The multiplex assay was performed using the Expand Long Template
PCR system (Roche Molecular Biochemicals, Indianapolis, IN). Chimeric
primers (Table 2)
were synthesized, labeled, and purified by high performance liquid
chromatography (HPLC) (Applied Biosystems Inc., or Sigma-Genosys, The
Woodlands, TX). A multiplex reaction of 20 µl consisted of
SCA1 primers (1.5 pmol each), SCA2 primers (1.5
pmol each), MJD primers (4.0 pmol each), CACNAIA
primers (1.5 pmol each), SCA7 primers (2.25 pmol each), 400
µmol/L dNTPs (Amersham-Pharmacia, Piscataway, NJ), 1X buffer #1 (1.75
mmol/L MgCl2), DMSO (4% v/v), Taq/Pwo
polymerase (1.4 U), and 200 to 800 ng of genomic DNA purified from
peripheral blood as described above. Reactions were performed in an ABI
9700 thermal cycler for 1 cycle at 95°C for 5 minutes, 32 cycles at
95°C for 1 minute, 60°C for 2 minutes (ramp 10% of default),
68°C for 1.5 minutes, and a final extension at 68°C for 10 minutes.
One µl of a 1:5 dilution of the reaction products was added to 12
µl of formamide (HiDye formamide, Applied Biosystems Inc.) and 0.5
µl of GS500-ROX internal molecular weight standard (Applied
Biosystems Inc.), denatured at 95°C for 2 minutes, and immediately
placed on ice for a minimum of 3 minutes. Samples were injected into an
ABI PRISM 310 Genetic analyzer (Applied Biosystems Inc.) with a 47
cm x 50 µm capillary containing Performance Optimized Polymer-4
(POP-4, Applied Biosystems Inc.) for 5 seconds with an injection kV of
15.0 and electrophoresed at 15 kV for 40 minutes at 65°C. Amplicon
length was calculated by comparison with the GS500-ROX molecular weight
standard by the Genescan program (Applied Biosystems Inc.) using the
local Southern method.
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Results
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Multiplex Assay Development and Optimization
To streamline SCA testing and shorten turn-around time, we sought
to convert our current panel of tests for SCA1, SCA2, SCA3, SCA6, and
SCA7 from five independent amplification reactions analyzed by PAGE to
a single multiplex amplification reaction analyzed by CE. For multiplex
amplification, our strategy was to employ chimeric primers, which were
shown previously to facilitate optimization of multiplex polymerase
chain reaction (PCR).10
New primers were synthesized with
10 to 20 bases of an M13 universal sequence added to the 5' end of each
SCA primer that was used in our existing individual SCA assays (Table 2)
. The number of M13 bases that was added varied to approximate a
melting temperature (Tm) of 
80°C and a
similar GC content between primer pairs and also among the five loci.
Fluorescent labels were selected based on normal allele size ranges for
each locus, making sure that loci with overlapping size ranges were
given different labels (Figure 1)
. For example, the primers for CACNAIA (SCA6) and
MJD (SCA3) were both labeled with NED because the size of
their amplicons do not overlap. Similarly, both the amplicons of
SCA2 and SCA7 were labeled with HEX because
their sizes do not overlap, with the possible exception of rare
instances of large expansions in neonatal/juvenile onset cases (Table 1)
.
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Figure 1. Capillary electrophoresis of multiplexed SCA loci. a:
Schematic showing the range of amplicon lengths for each locus. Colors
indicate fluorochromes: green, HEX; blue, 6-FAM; black, NED;
solid bar, range of normal sized alleles; hatched
bar, range of abnormally expanded alleles; dashes, the
exceptionally large expansions observed in affected neonates/children.
b–f: Electropherograms showing the test results for an
affected subject with an abnormal CAG-repeat expansion. The table
adjacent to each panel gives the amplicon length and number of CAG
repeats for each allele of the samples tested
(*, abnormal
(CAG)n).
b: Abnormal test result for SCA1. c:
Abnormal test result for SCA2. d: Abnormal test
result for SCA3 (MJD gene).
e: Abnormal test result for SCA6 (CACNAIA
gene). f: Abnormal test result for
SCA7.
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To facilitate efficient amplification of both normal and abnormal
alleles, assay parameters and polymerases that favored the
amplification of long templates were used (Materials and Methods).
Assay optimization experiments were performed with a normal control
subject and a patient with an expanded allele of known CAG-repeat
length for each locus. Assay parameters were adjusted to optimize
multiplex amplification and CE analysis. For example, the addition of
DMSO was required for robust amplification of SCA7, but too
much inhibited amplification of MJD (SCA3); 4% DMSO was
optimal. Differences in amplification efficiencies and in fluorescence
intensities of the three dyes resulted in varying peak amplitudes of
fluorescence units in the electropherograms. Therefore, primer
concentrations were adjusted to yield peaks of comparable amplitude to
ensure detection of alleles of varied length.
Examples of the results of the optimized assay are shown in Figure 1
.
Heterozygotes with normal alleles differing by one or two CAG repeat
were detectable (Figure 1
, panel c, CACNA1A and
SCA7). Expanded alleles, in the size ranges tested (Figure 2)
, were detected consistently; drop out of expanded alleles was never
observed. Somatic mosaicism and stuttering, observed routinely in PAGE
assays, was also observed in the CE electropherograms (Figure 1
, panels
d and f, MJD and SCA7). However, the dominant
peak could be clearly assigned based on amplitude or peak area.
Amplicon molecular weights calculated by Genescan based on the
GS500-ROX internal molecular weight standard were underestimated
compared to the manual PAGE assay.
View larger version (20K):
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|
Figure 2. Comparision of SCA amplicon lengths as determined by PAGE
versus CE. The graph for each locus documents that CE
results in an underestimation of length, albeit to different degrees.
The diagonal line represents the expected results if the two methods
gave equivalent lengths. N, the total number of alleles tested; A, the
number of alleles that had different
(CAG)n;
range, the range of
(CAG)n
tested. This assay had high precision of ± 1 repeat for normal
alleles and ± 2 repeats for abnormal alleles based on data from
our laboratory and from data reported by 25 other laboratories on
samples provided for proficiency testing (MGLA
1999 and MGLB 2000 surveys, College of American Pathologists,
Northfield, IL). In addition, several normal
alleles were cloned and sequenced to confirm the number of CAG repeats;
this was not possible for abnormal alleles due to their instability in
Escherichia coli. The formulae for calculation of
(CAG)n from
amplicons sized by CE for each locus are: number of
(CAG)n in
SCA1 = [CE product size
(bp) - 162]/3 x
1.0678 + 1.115; number of
(CAG)n in
SCA2 = [CE product size
(bp) - 84]/3 x
1.0121 + 0.5342; number of
(CAG)n in
SCA3/MJD = [CE product size
(bp) - 201]/3 x
1.0184 + 0.7062; number of
(CAG)n in
SCA6/CACNAIA = [CE product size
(bp) - 126]/3 x
1.0075 + 0.5572; number of
(CAG)n in
SCA7 = [CE product size
(bp) - 314]/3 x
1.1211 + 5.0958.
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To analyze the apparent discrepancy in amplicon size, samples were
assayed retrospectively by the multiplexed CE assay. Allele lengths
were compared to those determined previously by the PAGE assay. During
CE, both normal and expanded alleles for each locus migrated faster
than predicted (Figure 2)
. The degree of anomalous migration was
different at each locus. Amplicons with greater numbers of CAG repeats
tended to migrate faster, which was most pronounced at SCA1
and SCA7 (Figure 2)
. Inter-assay variability, calculated as
a 95% confidence interval, of 10 replicate assays of a normal and an
expanded allele at each SCA locus, was ± 2 bp (data not shown).
To adjust for anomalous migration of amplicons during CE, a size
correction formula was calculated by regression analysis of a pair-wise
comparison of (CAG)n values determined by PAGE
versus CE (Figure 2
legend). Because the precision of the
PAGE assay (Table 2)
was lower than the CE assay, multiple samples with
the same number of CAG repeats as determined by PAGE were included in
the analyses. To facilitate correction of amplicon length and minimize
calculation errors in the clinical laboratory, a spreadsheet was
generated in Excel (Microsoft, Redmond, WA) that converted raw amplicon
length to number of CAG repeats based on the specific correction
formula for each SCA. Fractional CAG-repeat numbers that were greater
than 0.5 were rounded up to the next whole number. An advantage of the
CE assay is that amplicons from a patient sample can be mixed with
those from an abnormal control and re-injected to confirm allele sizes.
For example, we routinely re-inject a mixture of any patient sample
that has between 18 to 22 CAG repeats at CACNA1AI (SCA6)
with the abnormal control (genotype 13, 21) to confirm that the
patient’s repeat is 
21 and therefore in the abnormal
range. Before placing the multiplex/CE assay on-line in the clinical
laboratory, 12 clinical samples were tested prospectively by both the
PAGE and multiplex/CE assays with consistent results. To date, we have
used this assay to test 57 patient samples referred for diagnostic or
carrier testing of SCA1, SCA2, SCA3, SCA6, and SCA7. Five samples were
positive, including one SCA1 case, two SCA2 cases, and two SCA3 cases.
|
Discussion
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We have developed a rapid and precise semi-automated assay that
employs multiplex PCR and CE for the molecular diagnosis of the
spinocerebellar ataxias types 1, 2, 3, 6, and 7. Relatively minimal
optimization was required for the multiplex reaction, presumably due to
the use of chimeric primers. The observation that the SCA amplicons
migrated faster than anticipated during CE, resulting in an
underestimation of their lengths, was not unexpected. Capillary
electrophoresis of fluorescent-labeled amplicons of the
trinucleotide-repeat disorders Huntington disease
(CAG)n and fragile X mental retardation
(CGG)n, resulted in faster migration and
subsequent underestimation of size.11, 12, 13, 14
Faster migration
of SCA, HD, and FMR1 amplicons compared to the internal size standards
could be attributed to significant differences in base composition
(repeat-containing versus non-repeat-containing),
particularly GC-rich sequences.13
Other factors
contributing to sizing discrepancies could be different sieving effects
of cross-linked PAGE compared to CE linear polymer, electro-osmotic
effect, or extent of denaturation.12, 13, 14
In trinucleotide-repeat disorders, an underestimation of amplicon size
could result in a misdiagnosis, particularly for alleles that are near
the threshold of the normal, intermediate, or abnormal size ranges.
Routine clinical use of CE for diagnosis of at least some
trinucleotide-repeat disorders requires a method that corrects the
sizing discrepancy. For Huntington disease, an allele-specific
molecular weight ladder was assembled using gene fragments that varied
in CAG-repeat number, thereby minimizing sequence differences between
amplicons and size markers.13, 15
This approach could not
be used in the SCA assay because it consists of amplicons of five loci
that migrate differentially during CE. For fragile X mental
retardation, a commercially-available molecular weight ladder was used
and amplicons lengths were multiplied by a migration factor to give an
accurate length.11
This approach was applied successfully
to the multiplex SCA assay, in part due to the high precision of the
instrument in calculating amplicon length and the low inter-assay
variability.
This assay has proved reliable in a clinical setting for 1 year. There
is no evidence that factors, such as capillary age or replacement, have
affected the correction formulae. Analysis of normal and abnormal
controls showed no change in amplicon length as calculated by Genescan
using the GS500-ROX molecular weight standard over this time. It is
reasonable to expect that our assay for the diagnosis of SCA types 1,
2, 3, 6, and 7 could be extended to include some additional SCA loci.
Up to 15 loci have been multiplexed successfully10
and
other unique fluorochromes are available and being developed, depending
on the instrument. It is important to note that our assay is not
expected to detect the very large CAG expansions in infantile/juvenile
onset SCA2 or SCA7. The electrophoresis ends before such large
fragments would be detected, assuming that they would amplify
sufficiently in this assay. Therefore, additional testing is needed for
at-risk SCA2 and SCA7 infants and juveniles.16
This
multiplex semi-automated assay greatly improves testing efficiency and
turn-around time thereby facilitating the diagnosis of spinocerebellar
ataxia in the clinical setting. It will also be invaluable in the
research setting for screening cohorts with autosomal-dominant
spinocerebellar ataxia and/or sporadic ataxia to determine prevalence
of specific SCA types.
|
Footnotes
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Address reprint requests to Karen Stephens, Ph.D., University of Washington, 1959 NE Pacific St., Room I-204, Medical Genetics 357720, Seattle, WA 98195-7720. E-mail: millie{at}u.washington.edu
Supported in part by Department of Defense Neurofibromatosis
Research Program grant DAMD17–00-1–0542 (to K.S.).
Accepted for publication December 18, 2001.
|
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Abstract
Introduction
