JMD 2001, Vol. 3, No. 4
Copyright © 2001 American Society for Investigative Pathology & Association for Molecular Pathology
Heteroduplex Formation in SMN Gene Dosage Analysis
Shuji Ogino,
Debra G. B. Leonard,
Hanna Rennert,
Sizhen Gao and
Robert B. Wilson
From the Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania
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Abstract
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Most spinal muscular atrophy patients lack both copies of
SMN1 exon 7 and most carriers have only one copy of
SMN1 exon 7. We investigated the effect of
SMN1/SMN2 heteroduplex formation on SMN
gene dosage analysis, which is an assay to determine copy
number of SMN1 exon 7 that utilizes multiplex
quantitative polymerase chain reaction (PCR) with DraI
digestion to differentiate SMN1 from
SMN2. Heteroduplex formation in PCR is a well-described
phenomenon. In addition to demonstrating the presence of heteroduplexes
by sequence analysis of purified SMN1 bands, we
compared the SMN1 signals in various genotype groups
(total n = 260) to those in a group lacking
SMN2 (n = 13), and we
estimated the relative amounts of SMN1/SMN2
heteroduplexes. The SMN1 signal increased as
SMN2 copy number increased despite a constant
SMN1 copy number, although not all pairwise
comparisons showed a statistically significant difference in the
SMN1 signal. In conclusion,
SMN1/SMN2 heteroduplexes form in SMN gene
dosage analysis, falsely increasing the SMN1
signal. External controls for SMN gene dosage analysis
should be chosen carefully with regard to SMN2 copy
number. The effect of heteroduplex formation should be considered when
performing quantitative multiplex PCR.
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Introduction
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Spinal muscular atrophy (SMA) is an autosomal recessive disorder
associated with loss of motor neurons in the anterior horn of the
spinal cord and caused by mutations in the Survival Motor Neuron 1 gene
(SMN1 or SMNt).1
SMN1 and its centromeric homologue, SMN2 (or
SMNc), lie within the telomeric and centromeric halves,
respectively, of a large inverted repeat on chromosome
5q13.1
SMN1 and SMN2 exons differ in
only two bases, one in exon 7 and one in exon 8.2
A C to T
change in a single base of exon 7 of SMN2 affects the
activity of an exonic splice enhancer and changes the splicing pattern
of SMN2 transcripts,3
resulting in a lower
level of full-length SMN protein from SMN2 than from
SMN1.4
Approximately 94% of affected patients lack both copies of
SMN1 exon 7 (reviewed by Wirth5
). To allow
identification of SMA carriers, quantitative polymerase chain reaction
(PCR) methods to determine the copy number of SMN1 exon 7
have been developed.6, 7
These methods, herein referred to
as SMN gene dosage analyses, use PCR amplification of
SMN1 and SMN2 exon 7 segments, followed by
DraI restriction enzyme digestion and quantitation of PCR
products.6, 7
DraI digests PCR products derived
from SMN2 but not those derived from SMN1. A
single copy of SMN1 by gene dosage analysis confirms carrier
status (heterozygous SMN1 deletion). In addition, up to 6%
of affected individuals lack only one copy of SMN1, with a
presumed non-deletion mutation on the other allele. In an individual
with symptoms of SMA, heterozygous SMN1 deletion supports
the diagnosis of SMA. Therefore, SMN gene dosage analysis is
of clinical importance.
Heteroduplex formation during PCR has been described and shown to affect
many genetic tests.8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
We hypothesized that
SMN1/SMN2 heteroduplexes form in our SMN gene
dosage analysis, falsely increasing the SMN1 signal, and
that with a constant SMN1 copy number more
SMN1/SMN2 heteroduplexes form as the SMN2 copy
number increases. The rationale for this hypothesis is as follows. In
the annealing step of the last PCR cycle, most single-strand
SMN1 PCR products anneal with SMN PCR primers, in
which case SMN1/SMN1 homoduplexes form after successful
extension. Some single-strand SMN1 PCR products may anneal
with complementary single-strand SMN1 PCR products as well
as complementary single-strand SMN2 PCR products, forming
SMN1/SMN2 heteroduplexes. As PCR proceeds, the relative
amount of heteroduplex formation may increase, because the
concentrations of SMN1 and SMN2 PCR products
increase and those of SMN primers decrease.
SMN1/SMN2 heteroduplexes would be resistant to restriction
enzyme digestion because of the single nucleotide mismatch present at
the DraI restriction site in the SMN2 PCR
products. After DraI digestion, SMN1 and
SMN2 PCR products, which are distinguished by their fragment
sizes, are denatured and quantitated.6, 7
SMN2
single-strand PCR products denatured from the undigested
SMN1/SMN2 heteroduplexes would be measured as
SMN1 PCR products. In experiments presented here, we tested
the heteroduplex hypothesis. We attempted to detect
SMN1/SMN2 heteroduplexes directly by sequence analysis, and
to quantitate SMN1/SMN2 heteroduplex formation in our
SMN gene dosage analysis.
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Materials and Methods
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Sample Collection and DNA Extraction
Peripheral blood specimens were received by the Molecular
Pathology Laboratory, University of Pennsylvania, for SMN1
copy number determination. A sequential series of samples (total
n = 273) were retrospectively selected and anonymized
for this study. Genomic DNA was extracted from peripheral blood
leukocytes using PUREGENE reagents (Gentra Systems, Inc., Minneapolis,
MN) according to the manufacturer’s protocol.
SMN1 and SMN2 Copy Number Assay (SMN Gene Dosage Analysis)
The method by Chen et al7
was modified for
SMN1 and SMN2 copy number assay, using 23 cycles
of PCR instead of 22 cycles as in the original method. The assay takes
advantage of the single nucleotide difference in exon 7 to distinguish
SMN1 from SMN2 by PCR product size after
DraI digestion. All assays were run in duplicate. Copy
number of SMN1 per cell (or more precisely, per diploid
genome) was determined by quantitation of PCR products and was
calculated using a genomic standard (CFTR exon 4),
internal standards for SMN1 and CFTR
(SMNis and CFTRis, respectively) and five
two-copy-SMN1 controls. These control samples were validated
as two-copy SMN1 controls as described.7
Briefly, during the validation step, we observed small sample-to-sample
and run-to-run coefficient of validations (CVs) of SMN1 gene
dosage (as described in the Appendix) in arbitrarily-selected controls
from individuals without a family history of SMA. We also observed that
a vast majority of samples from individuals without a family history
showed a tightly clustered SMN1 gene dosage, approximating
the dosage of the controls, which was twice the dosage of a vast
majority of samples from known SMA carriers who presumably had only one
copy of SMN1. The internal standards, SMNis and
CFTRis, have similar PCR amplification characteristics
relative to their genomic counterparts. The SMN1 gene dosage
is normalized to five controls with two copies of SMN1. The
normalized SMN1 gene dosage is expressed as
C(SMN1), which stands for calculated SMN1
signal (symbols are defined in Table 1
.). C(SMN1) is
calculated as described in the Appendix. SMN2 copy number
was designated by comparison of the SMN2 signal to the
SMN1 signal, assuming that the SMN2 signal is 70
to 80% of the SMN1 signal for equivalent copy numbers (See
Results, Presence of SMN1/SMN2 Heteroduplexes).
Sequence Analysis of Undigestable 188-bp SMN PCR Products
To test whether heteroduplexes are present after PCR, we amplified
genomic DNA from two samples with one copy of SMN1 and
either three or four copies of SMN2 as well as two samples
with two copies of SMN1 and lacking SMN2 as
negative controls. The PCR method was modified from the SMN
gene dosage analysis PCR as follows: SMNis,
CFTRis, and CFTR primers were excluded from the
PCR master mix. The SMN primers were not fluorescently
labeled. The PCR cycles were increased to 35 and 50 cycles, instead of
23 cycles, to increase the sensitivity of detection. The PCR cycle
number was increased solely for this sequence analysis, but not for
SMN copy number assay or for quantitation of heteroduplexes.
After DraI digestion, PCR samples were electrophoresed on an
ethidium-bromide-impregnated agarose gel [2% UltraPure agarose (Life
Techonologies, Rockville, MD) and 1% NuSieve GTG agarose (FMC
BioProducts, Rockland, ME)]. The undigestable 188 bp SMN
PCR band was visualized under long-wave ultraviolet light and cut from
the gel. The DNA was extracted from the agarose gel slice using the
QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, CA). The DNA
fragments were sequenced with the forward primer SMN7F
(R111)1
using the BigDye Terminator Reaction Kit (Applied
Biosystems, Inc., Foster City, CA) according to the manufacturer’s
instructions. The products obtained after cycle sequencing were
purified through Centri-Sep columns (Princeton Separations, Adelphia,
NJ) and analyzed on an ABI PRISM 310 Genetic Analyzer (Applied
Biosystems, Inc.), according to the manufacturer’s instructions.
Quantitation of SMN1/SMN2 Heteroduplex Formation
SMN1 and SMN2 copy numbers were determined
in samples from 273 individuals. Then C(SMN1) values
were compared among various SMN genotype groups, to test the
hypothesis that the SMN1 signal, C(SMN1),
increasesas SMN2 copy number increases due to SMN1/SMN2
heteroduplex formation, despite a constant SMN1 copy number.
The genotypes tested are shown in Table 2
.A genotype was designated by a code consisting of two integers
separated by a colon: X:Y, where X represents SMN1 copy
number and Y represents SMN2 copy number. Each sample was
tested in duplicate and the average of the two
C(SMN1) values was used. The coefficient of variation
(CV) of the two C(SMN1) values for each sample in the
duplicate testing of the 273 samples ranged from 0% to 12.5% with a
mean of 2.48%, a median of 1.85%, and an 80th percentile point of
4.00%.
The presence of SMN2/SMNis heteroduplexes was negligible
because at least one control sample lacking SMN1 in each run
consistently lacked the 188-bp peak that would have been present due to
undigestable SMN2 if a significant amount of
SMN2/SMNis heteroduplexes formed. Therefore, the presence of
SMN1/SMNis heteroduplexes can also be considered negligible
and virtually all undigested SMN2 PCR products can be
considered as derived from SMN1/SMN2 heteroduplexes.
The extent of heteroduplex formation was calculated as the
percentage of C(SMN1) that was contributed by
SMN2, by comparing the mean C(SMN1) of a
particular genotype to that of the 2:0 group. The 2:0 group lacks
SMN1/SMN2 heteroduplexes due to an absence of
SMN2 and was used as a standard for quantitation of
heteroduplex formation in other genotypes. The mean
C(SMN1) in all samples within each genotype group is
designated as MC(SMN1) followed without space by a
subscript of SMN1 and SMN2 copy number genotype,
designated as X:Y. For example,
MC(SMN1)2:2 represents the mean
C(SMN1) of the 2:2 genotype group. For statistical
analyses of the differences in
MC(SMN1)X:Y between the
genotype groups, an F test was first used to determine whether a
significant difference in variances was present. If there was no
significant difference in variances, then the Student’s
t-test (two-tailed) was used for comparison of
MC(SMN1)X:Y between the two
genotypes. If there was a significant difference in variance,
then the Welch t-test (two-tailed) was used.
MC(SMN1)1:1,
MC(SMN1)1:2 and
MC(SMN1)1:3 were compared to
MC(SMN1)2:0 x 1/2 (which
serves as a standard for the 1:0 genotype). Similarly,
MC(SMN1)3:1 and
MC(SMN1)3:2 were compared to
MC(SMN1)2:0 x 3/2 (which
serves as a standard for the 3:0 genotype). The relative contribution
of SMN2 derived from the SMN1/SMN2 heteroduplexes
among the total SMN1 signal is represented as
%SMN2 and is calculated as described in the Appendix.
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Results
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Presence of SMN1/SMN2 Heteroduplexes
In our experience using the modified method of Chen et
al,7
there seemed to be less SMN2 PCR product
than SMN1 PCR product in most samples. A substantial number
of samples with one copy of SMN1 showed an SMN2
signal intensity approximately 70 to 80% of the SMN1
signal. We presumed that such samples had one copy of SMN2
because the SMN2 signal was approximately equal to the
one-copy SMN1 signal, and because samples with zero copies
of SMN2 showed a complete absence of SMN2 signal.
Other samples with one copy of SMN1 frequently showed an
SMN2 signal approximately 1.5 times stronger than that of
SMN1, implying the presence of two copies of
SMN2. Although a majority of samples with two copies of
SMN1 showed an SMN2 signal intensity
approximately 70 to 80% of that of SMN1 (implying the
presence of two copies of SMN2), more than one third of
samples with two copies of SMN1 showed an SMN2
signal strength close to one third to one fourth of the SMN1
signal (implying the presence of one copy of SMN2). One
hypothesis to explain these discrepancies between copy number and
signal intensity was that the PCR amplification efficiencies of
SMN1 and SMN2 differ. However, this seemed
unlikely because SMN1 and SMN2 sequences in this
PCR differ by only one nucleotide, and this nucleotide difference is
not in the primer sites. Moreover, it could not explain why samples
lacking SMN2 generally exhibited weaker SMN1
signals relative to samples with the same SMN1 copy number
but SMN2 present. An alternative hypothesis was that
incomplete DraI digestion falsely increased SMN1
signal intensity and decreased SMN2 signal intensity. This
also seemed unlikely because of assay controls that lacked
SMN1 in which no undigested SMN2 signal was
detected (see Results, Validation of SMN Gene Dosage
Analysis). We hypothesized that SMN1/SMN2 heteroduplexes,
which formed during PCR and could not be digested by DraI,
falsely increased the SMN1 signal intensity (Figure 1)
. To test this hypothesis, we performed sequence analyses to look for
the presence of the SMN2 sequence in DraI
undigestable PCR products.
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Figure 1. SMN1/SMN2 heteroduplexes contribute to the SMN1 signal
in SMN gene dosage analysis. SMN1
(or SMN2)
homoduplexes form either when complementary SMN1
(or SMN2)
single strand PCR products anneal with each other or when successful
extension occurs after SMN1 (or
SMN2) single strands anneal with
their corresponding PCR primers. SMN1/SMN2
heteroduplexes form when SMN1 single strands anneal with
their near-complementary SMN2 single strands. Since only
SMN2 homoduplexes can be digested by DraI
to form 165-bp DNA fragments, SMN2 strands in
SMN1/SMN2 heteroduplexes cannot be digested and remain
as intact 188-bp PCR products, which are measured as
SMN1.
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The SMN2 sequence derived from SMN1/SMN2
heteroduplexes was identified by sequencing the DraI
undigestable 188 bp band from agarose gels (Figure 2)
. In two samples lacking SMN2 (genotype 2:0), the
SMN2-specific nucleotide T in position 27141 was absent, as
expected (Figure 2A)
. By contrast, in two samples with one
SMN1 copy and three or four SMN2 copies (genotype
1:3 and 1:4), respectively, a significant amount of the
SMN2-specific nucleotide T in position 27141 was present
under the expected C peak of SMN1 (Figure 2B
and 2C)
. The
amount of the SMN2-specific nucleotide T increased as the
PCR cycle number was increased from 35 to 50, as predicted by the
heteroduplex hypothesis.
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Figure 2. Sequence analysis demonstrated the presence of SMN2
sequence derived from SMN1/SMN2 heteroduplexes in the
DraI undigestable 188 bp bands on agarose gels.
Genotypes are described as (SMN1
copy
number):(SMN2
copy number) as in the text. A: The
absence of a nucleotide T peak at position 27141C is shown
(arrow) in one
of the two 2:0 samples after 35 cycles of PCR. The absence of the
nucleotide T peak was observed in both 2:0 samples after both 35 cycles
and 50 cycles of PCR. B: The presence of the nucleotide T
peak at position 27141 is indicated
(arrow) under
the C peak in the 1:4 genotype sample after 35 cycles of PCR. A similar
result was also obtained in the 1:3 genotype sample after 35 cycles of
PCR. C: The increase in the nucleotide T peak relative to
the C peak is shown
(arrow) as PCR
cycle number was increased to 50. A similar result was also obtained in
the 1:3 genotype sample after 50 cycles of PCR.
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Validation of SMN Gene Dosage Analysis
In SMN gene dosage analysis, the calculated
SMN1 signal, C(SMN1), should always be
close to an integer, which is the SMN1 copy number of a
sample, unless there is mosaicism. When individual samples were
classified into SMN1 and SMN2 copy number
genotypes, MC(SMN1)X:Y ± 2 SD
was within the ranges 0.89 to 1.21 for one copy of SMN1 (the
1:1, 1:2, 1:3, and 1:4 genotypes), 1.75 to 2.41 for two copies of
SMN1 (the 2:0, 2:1, 2:2, and 2:3 genotypes) and 2.74 to 3.15
for three copies of SMN1 (the 3:1 and 3:2 genotypes) and
there was no overlap between these ranges (Table 2
and Figure 3
). Incomplete restriction-enzyme digestion could result in a falsely
high signal intensity for SMN1. Therefore, at least one
sample lacking SMN1 but possessing SMN2 served in
every run as a control to verify complete DraI digestion.
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Figure 3. The calculated SMN1 signal,
C(SMN1),
in various SMN genotypes. The x axis
represents SMN genotypes described as
(SMN1 copy
number):(SMN2
copy number) as in the text. The
y axis represents the calculated SMN1
signal,
C(SMN1).
The mean
C(SMN1) is
represented by a column. The solid vertical line across the top of each
column represents ± 1 SD. The mean
C(SMN1)
increased as SMN2 copy number increased despite a
constant SMN1 copy number.
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Quantitation of SMN1/SMN2 Heteroduplex Formation
One sample with a C(SMN1) of 2.218 was initially
included in the 2:0 genotype group but was shown to be an outlier by
the Grubbs-Smirnov test (P < 0.01). Excluding
this sample, the 2:0 genotype group (n = 13)
demonstrated that a distribution of C(SMN1) was close
to a normal distribution with the mean C(SMN1) =
1.902 (95% confidence interval of 1.857 to 1.947), a SD of 0.075 and a
variance of 0.00565. When the outlier sample was included in the 2:0
group, the mean C(SMN1) = 1.924 (95% confidence
interval of 1.860 to 1.989) with an SD of 0.111, reflecting a larger
variance of 0.0124. We used the mean C(SMN1) of the
other 13 samples in the 2:0 genotype as a standard. There are several
possible reasons why the C(SMN1) of 2.218 for the one
outlier sample was greater than 4 SD above the mean
C(SMN1) of the 2:0 genotype excluding this sample.
There might be a polymorphism affecting one of the primer sites on the
CFTR exon 4 genomic reference sequence, causing less
efficient PCR amplification. Other possibilities include mosaicism and
partial DNA degradation in the sample, causing less efficient PCR
amplification of the longer CFTR genomic sequence (239 bp)
compared to the SMN1 sequence (188 bp).
We obtained data for the quantitation of %SMN2 from a total
of 260 samples with various genotypes with SMN2 copies and
the 13 samples with the 2:0 genotype (a standard) (Table 2
and Figure 3
). The C(SMN1) for each genotype exhibited a
distribution that was close to a normal distribution, allowing F-tests,
Student’s t-tests, and Welch t-tests to be
applied to the data. Due to SMN1/SMN2 heteroduplex
formation, the SMN1 signal of the genotypes with two copies
of SMN1 increased as SMN2 copy number increased
despite the constant SMN1 copy number
(P < 0.005). The genotypes 1:Y with at least
one copy of SMN2 also showed significantly higher
C(SMN1) than
[C(SMN1)2:0 x 1/2]
(P < 0.0005) and the genotypes 3:1 and 3:2
showed higher C(SMN1) than
[C(SMN1)2:0 x 3/2]
(P = 0.01 and P = 0.062,
respectively). However, all pairwise comparisons of
C(SMN1) within the genotypes with one copy of
SMN1, and comparison of C(SMN1) between
the 3:1 and 3:2 genotypes, showed only one pair (the 1:1 vs.
1:3 genotypes) with a significant difference (P < 0.032), although there was an overall tendency that
C(SMN1) increased as SMN2 copy number
increased despite a constant SMN1 copy number (Table 2
and
Figure 3
).
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Discussion
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Heteroduplex formation in PCR has been
described.8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
Theoretically, heteroduplexes form
whenever different templates with similar sequences are annealed. In
non-quantitative PCR-Restriction Fragment Length Polymorphism
(RFLP) assays, which are used in many genetic tests,
heteroduplexes usually do not affect the interpretation of results,
because one can determine the presence or absence of a mutant allele
(associated with the presence or absence of a restriction site)
regardless of the presence of a low level of heteroduplex formation.
Heteroduplexes rarely cause diagnostic problems in non-quantitative PCR
analyses, exemplified by PCR analysis for the GAA trinucleotide
expansion in the FRDA gene Friedreich ataxia17
and by PCR-RFLP analysis for the detection of two different mutations
in the steroid 21-hydroxylase gene for congenital adrenal
hyperplasia.14
On the other hand, heteroduplexes may affect the interpretation of
results for quantitative PCR assays, such as in methods for
SMN1 quantitation by McAndrew et al.,6
Chen et
al,7
and Wirth et al.20
Methods that
circumvent problems of heteroduplex formation in quantitative PCR
assays have been reported.21, 22, 23, 24
Most of these methods are
complex and require individual optimization. A relatively simple
method, hot-stop PCR, was recently reported by Uejima et
al,25
but required utilization of radioactivity and an
extra manipulation before the final cycle of PCR. Becker-Andre and
Hahlbrock,11
who first mentioned the possibility of
heteroduplex formation affecting the results of DNA quantitation by
PCR, considered that essentially only homodimeric DNA species formed in
the exponential phase of PCR. Uejima et al25
found that
the quantitated allele ratio in regular 25-cycle PCR was equal to the
theoretical ratio, implying no significant heteroduplex formation after
25 cycles of PCR, whereas there was a significant distortion of the
quantitated allele ratio in regular 35-cycle PCR presumably due to
heteroduplex formation. By contrast, our results indicate that there is
a significant amount of heteroduplex formation in our SMN
gene dosage analysis even using 23 cycles of PCR. Reducing the PCR
cycle number will likely decrease heteroduplex formation. The method of
McAndrew et al6
used 16 cycles of PCR and clearly showed a
smaller effect of heteroduplexes, as well as good accuracy and
precision. Our method has the disadvantage of having greater
heteroduplex formation, but not to the point of affecting test accuracy
or precision, while having the advantage of using a non-radioisotopic
detection system.
This study is the first to evaluate the effect of heteroduplex
formation on the quantitation of SMN1. We demonstrated that
the calculated SMN1 signal for individuals with two
SMN1 copies increased significantly as SMN2 copy
number increased. Although not statistically significant, there was a
consistent tendency for the calculated SMN1 signal to
increase for individuals with one or three SMN1 copies, as
SMN2 copy number increased. We speculate that our assay was
insufficiently accurate to detect statistically significant differences
with these relatively small sample sizes. However, for all practical
purposes, the accuracy and precision of our assay to determine copy
number of SMN1 was quite high. The mean
C(SMN1) ± 2 SD in each SMN1 and
SMN2 genotype group was still within the ranges 0.89 to 1.21
for one copy of SMN1, 1.75 to 2.41 for two copies of
SMN1, and 2.74 to 3.15 for three copies of SMN1,
and there was no overlap between these ranges. It should be noted that
we obtained the results of this study using two-copy SMN1
controls consistently composed of three samples with two copies of
SMN2 (genotype 2:2) and two samples with one copy of
SMN2 (genotype 2:1). Based on our data, two-copy
SMN1 controls for SMN gene dosage analysis should
be chosen carefully with regard to the SMN2 copy number. For
example, if one uses two-copy SMN1 samples lacking
SMN2, as two-copy SMN1 controls, the calculated
SMN1 signal will be overestimated for all samples that have
at least one copy of SMN2. On the other hand, if one uses
two-copy SMN1 samples with three or more copies of
SMN2 as controls, the calculated SMN1 signal will
be underestimated for all samples that have one or two copies of
SMN2, or lack SMN2 entirely. Individuals who lack
SMN2 may have a calculated SMN1 signal between
the one-copy range (consistent with carrier status) and the two-copy
range (more typical of non-carrier status), causing a significant
diagnostic problem. One possible solution to this problem is to modify
the calculated SMN1 signal, C(SMN1), for
each SMN1 and SMN2 genotype. For example, if a
sample lacking SMN2 shows a C(SMN1) of
1.62, this C(SMN1) can be modified to 1.71, which is
closer to two-copy SMN1 samples (by dividing 1.62 by the
factor MC(SMN1)2:0 x 1/2 = 0.95 from Table 2
). For two-copy SMN1 controls for
clinical assays, we recommend using three samples from the 2:2 genotype
and two samples from the 2:1 genotype. This composition of genotypes
roughly reflects the frequencies of SMN genotypes in the
general population (unpublished data). One should be aware of the
effect of SMN1/SMN2 heteroduplexes when interpreting results
of SMN gene dosage analysis.
Intuitively, one might expect a higher %SMN2 in samples
with more total SMN gene copies because more overall PCR
products and fewer primers favor heteroduplex formation rather than
primer binding. However, we estimated nearly identical
%SMN2 (6.6% and 6.4%) in the 1:1 and 2:2 groups. We
speculate that the 23-cycle PCR in our assay is still within the
exponential phase and that the primers are still in relative excess.
One might also predict a higher %SMN2 where there is a
higher fraction of SMN2, i.e., (SMN2 copy
number)/(total SMN copy number), in the specimen. Our
results were generally concordant with this prediction. The observed
values of %SMN2 in each genotype (followed by the fraction
of SMN2 in the specimen and number of samples) from the
lowest to highest are: 2.9% in the 3:1 (0.25, n = 11),
3.6% in the 2:1 (0.33, n = 53), 4.9% in the 3:2
(0.40, n = 6), 6.4% in the 2:2 (0.50,
n = 81), 6.6% in the 1:1 (0.50, n =
27), 8.0% in the 1:2 (0.67, n = 57), 10.5% in the 1:3
(0.75, n = 18), 12.1% in the 1:4 (0.80,
n = 2), and 14.0% in the 2:3 (0.60, n = 4). Therefore, in general, %SMN2 tends to increase as the
fraction of SMN2 in the specimen increases. The only
pairwise comparisons that contradict the prediction involve the 2:3
genotype. This may be due to the small sample size
(n = 4). More samples are necessary to evaluate
this issue.
In conclusion, we have demonstrated that SMN1/SMN2
heteroduplexes form during the final annealing step of PCR in our
SMN gene dosage analysis assay, resulting in an increased
SMN1 signal as SMN2 copy number increases.
Therefore, control samples with two copies of SMN1 for
SMN1 quantitation should be selected judiciously with regard
to SMN2 copy number. The effect of heteroduplex formation
should be considered whenever performing multiplex PCR amplification of
templates with similar sequences.
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Appendix
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1. Calculation of SMN1 Copy Number
To quantitate SMN1 copy number, we defined signal
intensity as the relevant peak area on an ABI PRISM 310 capillary
electropherogram. The signal intensities of the relevant peaks are
defined as follows: A1, SMNis PCR
product; A2, SMN2 PCR product;
A3, SMN1 PCR product;
A4, CFTRis PCR product;
A5, CFTR PCR product.
Because A3 and A1 are
proportional to initial copy numbers of SMN1 and
SMNis, respectively, in a sample tube,
 | (1) |
where a equals the PCR efficiency constant for
SMN. In addition, because the initial copy number of
SMNis equals the PCR master mix volume added into a sample
tube, multiplied by the SMNis concentration in the PCR
master mix,
 | (2) |
where k1 equals the PCR
efficiency difference factor for SMNis compared to
SMN1, c equals the SMNis concentration
in the PCR master mix, and v equals the PCR master mix
volume added into a sample tube.
Similar formulas can be generated for CFTR and
CFTRis, where b equals the PCR efficiency
constant for CFTR, k2
equals the PCR efficiency difference factor for CFTRis
compared to CFTR, d equals the CFTRis
concentration in the PCR master mix, and all cells are assumed to have
2 copies of CFTR exon 4.
 | (3) |
 | (4) |
 | (5) |
 | (6) |
 | (7) |
 | (8) |
 | (9) |
[(A3/A1)/(A5/A4)]
is described as SMN1 gene dosage, which is normalized in the
next step.
[(A3/A1)/(A5/A4)]
is described as SMN1 gene dosage, which is normalized in the
next step.
 | (10) |
 | (11) |
 | (12) |
The formula (12) is designated herein as the calculated
SMN1 signal and described as C(SMN1).
2. Calculation of %SMN2
To quantitate SMN1/SMN2 heteroduplexes, let the mean
C(SMN1) in the 2:0 genotype,
MC(SMN1)2:0 equal to 2
,
where represents the contribution of single-stranded
SMN1 PCR products, all of which were derived from
SMN1/SMN1 homoduplexes in this genotype.
 | (13) |
We assume that the absolute value among the mean
C(SMN1) contributed by single-stranded
SMN1 PCR products to the mean C(SMN1) is
constant ( = 2) regardless of the SMN2 copy number when
the SMN1 copy number remains constant. When we define 2ß
as the contribution of total single-stranded PCR products from
SMN1/SMN2 heteroduplexes to
MC(SMN1)2:Y in a sample from
the 2:Y genotype (where Y is a positive integer; 1, 2, 3, or 4 in this
study), ß represents a contribution of either single-stranded
SMN1 PCR products or single-stranded SMN2 PCR
products, both of which were derived from SMN1/SMN2
heteroduplexes. Then, the contribution of SMN1 strands from
SMN1/SMN1 homoduplexes is equal to 2 - ß, because
total contribution of single-stranded SMN1 PCR products is
constant ( = 2) and some contribution of SMN1 strands,
equivalent to ß, were derived from SMN1/SMN2
heteroduplexes. Therefore, the total contribution of SMN1
and SMN2 single strands to
MC(SMN1)2:Y equals (2 - ß) + 2ß = 2 + ß.
 | (14) |
 | (15) |
From (14) and (15) the fraction of SMN2 contribution to
the total MC(SMN1)2:Y
(represented as %SMN2) equals:
 | (16) |
For genotypes 1:Y and 3:Y,
[MC(SMN1)2:0 x 1/2] and
[MC(SMN1)2:0 x 3/2],
respectively, were used as standards. Therefore, from (16) the fraction
of SMN2 strand contribution to the total
MC(SMN1)X:Y (%SMN2)
equals:
 | (17) |
 | (18) |
|
Acknowledgments
|
|---|
The authors thank Patricia E. McAndrew and Thomas W. Prior for
generously providing us with the plasmids containing SMNis
and CFTRis inserts, Jean B. Patel for assisting us in
sequence analysis, Cynthia Turino and Treasa Smith for performing
SMN gene dosage analysis and their technical support, and
Warren J. Ewens for assisting us in calculating 95% CI for
%SMN2.
|
Footnotes
|
|---|
Address reprint requests to Robert B. Wilson, M.D., Ph.D., Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Stellar-Chance Laboratories Room 509A, 422 Curie Blvd., Philadelphia, PA 19104. E-mail: wilsonr{at}mail.med.upenn.edu
Supported by the Department of Pathology and Laboratory
Medicine, University of Pennsylvania Medical Center, Philadelphia,
Pennsylvania.
Accepted for publication July 2, 2001.
|
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Abstract
Introduction
