Download Quantitative analysis of SMN1 and SMN2 genes based on DHPLC

HUMAN MUTATION 25:460^467 (2005)
METHODS
Quantitative Analysis of SMN1 and SMN2 Genes
Based on DHPLC: A Highly Efficient and Reliable
Carrier-Screening Test
Yi-Ning Su,1 Chia-Cheng Hung,2 Hung Li,3 Chien-Nan Lee,4 Wen-Fang Cheng,4 Po-Nien Tsao,5
Ming-Cheng Chang,4 Chia-Li Yu,1 Wu-Shiun Hsieh,5 Win-Li Lin,2 and Su-Ming Hsu6n
1
Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan; 2Institute of Biomedical Engineering, National Taiwan
University, Taipei, Taiwan; 3Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan; 4Department of Obstetrics and Gynecology, National
Taiwan University Hospital, Taipei, Taiwan; 5Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan; 6Department of
Pathology, National Taiwan University Hospital, Taipei, Taiwan
Communicated by Graham R. Taylor
Autosomal recessive spinal muscular atrophy (SMA) is a common, fatal neuromuscular disease caused by
homozygous absence of the SMN1 gene in approximately 94% of patients. However, a highly homologous
SMN2 gene exists in the same chromosome interval, centromeric to SMN1, and hampers detection of SMN1.
We present a new, rapid, simple, and highly reliable method for detecting the SMN1 deletion/conversion and for
determining the copy numbers of the SMN1 and SMN2 genes by DHPLC. We analyzed SMN1/SMN2 gene
exon 7 deletion/conversion by DHPLC. A total of 25 patients with spinal muscular atrophy lacking the SMN1
gene as well as 309 control individuals from the general population and the family members of patients with
SMA were analyzed. By DHPLC analysis, we could detect the SMA-affected cases efficiently just by recognizing
an SMN2-only peak. Furthermore, after specific primer amplification and adjustment of the oven temperature,
all of the SMA carriers with an SMN1/SMN2 ratio not equal to 1 could be identified unambiguously by this
simple and efficient detection system. To calculate the total SMN1/SMN2 gene dosages further, we developed a
specific multiplex competitive PCR protocol by simultaneously amplifying the CYBB gene (X-linked), the
KRIT1 gene (on chromosome arm 7q), and the SMN1/SMN2 gene ratio by DHPLC. By applying this
technique, we could successfully designate all of the genotypes with different SMN1/SMN2 gene copy numbers,
including equal and unequal amounts of SMN1 and SMN2. We demonstrated that DHPLC is a fast and reliable
tool for detection of carriers of SMA. Hum Mutat 25:460–467, 2005. r 2005 Wiley-Liss, Inc.
KEY WORDS:
DHPLC; SMN1; SMN2; spinal muscular atrophy; SMA; gene dosage
DATABASES:
SMN1 – OMIM: 600354, 253300 (SMA I), 253550 (SMA II), 253400 (SMA III);
GDB: 5215173; GenBank: AH006635, NM_022874; HGMD: SMN1
SMN2 – OMIM: 601627; GDB: 5215175; GenBank: AH006635, NM_022877; HGMD: SMN2
INTRODUCTION
Proximal spinal muscular atrophy (SMA) is an autosomal
recessive disorder with an overall incidence of 1 in 10,000 live
births and a carrier frequency of 1 in 35 to 1 in 50 [Cusin et al.,
2003; Feldkotter et al., 2002; Ogino and Wilson, 2002a; Pearn,
1980]. This severe neuromuscular disease is characterized by
degeneration and loss of alpha motor neurons of the spinal cord
anterior horn cells, which results in progressive symmetric
weakness, atrophy of the proximal voluntary muscles, and death
of infant patients.
On the basis of clinical criteria, patients with SMA are classified
into three types depending on the clinical severity, including age at
onset, achievement of milestones, and life span [Zerres and
Rudnik-Schoneborn, 1995]. Type I SMA (Werdnig-Hoffman
disease; MIM# 253300) is the most severe form, with clinical
onset generally occurring before the age of 6 months and death in
the first 2 years of life. Type II SMA (MIM# 253550) is of
r2005 WILEY-LISS, INC.
intermediate severity, with onset before 18 months. Patients with
type II are able to sit without support, but never gain the ability to
walk; they usually survive beyond 10 years. Type III SMA
(Kugelberg-Welander disease; MIM# 253400) is a mild form of
Received 13 August 2004; accepted revised manuscript 17
November 2004.
n
Correspondence to: Dr. Su-Ming Hsu, Department of Pathology,
National Taiwan University Hospital,Taipei,Taiwan.
E-mail: [email protected]
Yi-Ning Su and Chia-Cheng Hung share the ¢rst authorship.
Grant sponsor: National Science Council of Taiwan; Grant number:
NSC 93 -2314 -B- 002-174; Grant sponsor: National Taiwan University
Hospital; Grant number: NTUH 93A11-1.
The Supplementary Material referred to in this article can be accessed
at http://www.interscience.wiley.com/jpages/1059 -7794/suppmat.
DOI 10.1002/humu.20160
Published online in Wiley InterScience (www.interscience.wiley.com).
DHPLC QUANTITATIVE ANALYSIS OF SMN1 AND SMN2
the disease with onset after the age of 18 months, and with
patients gaining the ability to walk.
A locus for the three clinical types of SMA was mapped to
chromosome 5q13 by linkage analysis [Scheffer et al., 2001; Wirth,
2000] and was refined to the location of the survival motor neuron
(SMN) gene [Lefebvre et al., 1995]. Two almost identical copies of
the SMN gene, telomeric SMN (SMN1; MIM# 600354) and
centromeric SMN (SMN2; MIM# 601627), have been identified.
These two SMN genes are highly homologous and differ in only
two nucleotides in the coding region. These nucleotide differences,
located in exons 7 and 8, allow the SMN1 gene to be distinguished
from the SMN2 gene [Lefebvre et al., 1995]. It has been reported
that approximately 94% of SMA patients were homozygous for
absence of the SMN1 gene [Wirth et al., 1999]. Moreover, small
deletions or point mutations have been found in patients in whom
SMN1 was present [Lefebvre et al., 1995].
Only the SMN1 gene is affected in SMA. The SMN2 gene
cannot compensate for the SMN1 deletion/conversion because,
transiently, a single-nucleotide difference in exon 7 causes exon
skipping [Cartegni and Krainer, 2002]. Therefore, detection of the
absence of SMN1 can be a useful tool for the diagnosis of SMA.
Furthermore, because of the high incidence of SMA, the high
carrier frequency of at least 1 in 50, the severity of the disease in
the patients, and the lack of effective treatment, carrier testing for
SMN1 deletion/conversion is an important step in genetic
counseling. However, a highly homologous SMN2 gene also exists
and hampers the detection of the loss of SMN1, which makes
detection of the gene dosage variations in the SMA carrier test
difficult.
So far, a number of different tools have been available for
diagnosis of SMA. All of the methods that have been used
recognize the difference in nucleotides in exon 7 of the SMN1 and
SMN2 genes and detect the absence of SMN1. SSCP analysis
[Lefebvre et al., 1995) is a simple method, but its sensitivity is low.
Restriction enzyme digestion analysis [van der Steege et al., 1995;
Wirth et al., 1999] is a simple, quick ,and commonly used method
in clinical practice currently. Although these methods can
efficiently detect homozygous SMN1 absence in the majority of
patients, a clear distinction between carriers and noncarriers has
not always been achieved. Thus, several different quantitative
PCR tests for SMN have been developed [Anhuf et al., 2003;
Chen et al., 1999; Feldkotter et al., 2002; Gerard et al., 2000;
McAndrew et al., 1997; Scheffer et al., 2000; Wirth et al., 1999].
These methods directly analyze the SMN1 and SMN2 gene copy
numbers. However, the disadvantages are that fluorescencelabeled probes and a relatively expensive kit are required.
DHPLC is a novel, simple, fast, and high reliable non–gel-based
method that is very sensitive and specific for detection of
variations in DNA including the rapid detection of the homozygous SMN gene absence detection in patients with SMA [Mazzei
et al., 2003; Sutomo et al., 2002]. Here, we present the use of this
method for detecting the SMN1 deletion/conversion and for
further determining the copy numbers of the SMN1 and SMN2
genes.
461
Isolation Kit (Gentra Systems, Minneapolis, MN), according to
the manufacturer’s instructions.
PCR
PCR for the provided DNA fragments was performed in a total
volume of 25 mL containing 100 ng of genomic DNA, 0.12 mM of
each primer, 100 mM dNTPs, 0.5 unit of AmpliTaq GoldTM
enzyme (PE Applied Biosystems, Foster City, CA), and 2.5 mL of
GeneAmp 10 buffer II (10 mM Tris-HCl, pH 8.3, 50 mM KCl),
in 2 mM MgCl2, as provided by the manufacturer. Amplification
was performed in a multiblock system (MBS) thermocycler
(ThermoHybaid, Ashford, UK). PCR amplification was performed
with an initial denaturation step at 951C for 10 minutes, followed
by 35 cycles consisting of denaturation at 941C for 30 seconds,
annealing at 531C for 45 seconds, extension at 721C for 45
seconds, and then a final extension step at 721C for 10 minutes. To
detect the SMN1/SMN2 ratio, we used the intronic primers
spanning intron 6 and 7 (SMN forward 50 -TGTCTTGTGAAACAAAATGCTT-30 , reverse 50 -AAAAGTCTGCTGGTCTGCCTA-30 ).
Multiplex PCR
To amplify the SMN1, SMN2, CYBB (MIN# 306400), and
KRIT1 (MIM# 604214) genes, we used multiplex PCR. The
following primers were used: CYBB forward (50 -CGGGAAATTCACCTACTTGC-30 ) and reverse (50 -AGCATTATTTGAGCATTTGGC-30 ).
KRIT1
forward
(50 -TTCGAA
0
0
TGGCTACTTCTACCTG-3 ) and reverse (5 -AAAACGTCTTTTAAATCAGAGC-30 ). The KRIT1 gene and X-linked CYBB gene
were used as controls for determining the relative gene dosage of
SMN1 and SMN2. The final volume of multiplex PCR was 25 mL
containing 100 ng of genomic DNA, 0.04 mM of each primer of
the CYBB and KRIT1 genes, 0.2 mM of each primer of the SMN
gene, 200 mM dNTPs, 0.5 units of AmpliTaq Gold enzyme (PE
Applied Biosystems), and 2.5 mL of GeneAmp 10 buffer II (10
mM Tris-HCl, pH 8.3, 50 mM KCl), in 2 mM MgCl2 as provided
by the manufacturer. Amplification was performed in an MBS
thermocycler (ThermoHybaid). PCR amplification was performed
with an initial denaturation step at 951C for 10 minutes, followed
by 26 cycles consisting of denaturation at 941C for 30 seconds,
annealing at 531C for 45 seconds, extension at 721C for 45
seconds, and then a final extension step at 721C for 10 minutes.
Cloning and Sequencing of PCR-Generated DNA
Fragments
To generate DNA fragments for use as positive controls in PCR
reactions and to facilitate DNA sequencing, the PCR fragment of
the SMN1 gene and SMN2 gene were subcloned into pGEMs -T
Easy Vector (Promega Corporation, Madison, WI), followed by
digestion according to the manufacturer’s instructions.
For cloning, 5 mL of the PCR fragment were mixed with pGEMT Easy Vector in a final volume of 10 mL and ligated at 41C
overnight. Then 5 mL of the recombinant plasmid was used for
transformation into E. coli, which was then cultured overnight on
selective agar plates containing 20 mL of 50 g/L of ampicillin. The
plates were incubated at 371C overnight. White colonies were
randomly chosen and were routinely cultured at 371C overnight
on Luria-Bertani (LB) broth containing ampicillin. Recombinant
plasmid DNA was extracted and purified by a Mini-Mt Plasmid
DNA Extraction System (Viogene, Sunnyvale, CA).
MATERIALS AND METHODS
Patient Samples
Direct Sequencing
A total of 334 DNA samples were analyzed in this study,
including specimens from 25 patients diagnosed with SMA, 46
obligate carriers from families with SMA patients, and 263
individuals from the general population. Genomic DNA was
collected from peripheral whole blood with a Puregene DNA
Amplicons were purified by solid-phase extraction and bidirectionally sequenced with the PE Biosystems Taq DyeDeoxy
terminator cycle sequencing kit (PE Biosystems) according to the
manufacturer’s instructions. Sequencing reactions were separated
on a PE Biosystems 373A/3100 sequencer.
462
SU ET AL.
DHPLC Analysis
The DHPLC system used in this study is a Transgenomic Wave
Nucleic Acid Fragment Analysis System (Transgenomic, San Jose,
CA). DHPLC was carried out on automated HPLC instrument
quipped with a DNASep column (Transgenomic). The DNASep
column contains proprietary 2-mm nonporous alkylated poly
(styrene divinylbenzene) particles. The DNA molecules eluted
from the column are detected by scanning with a UV detector at
260 nm. DHPLC-grade acetonitrile (9017-03; J.T. Baker, Phillipsburg, NJ) and triethylammonium acetate (TEAA; Transgenomic,
Crewe, UK) constituted the mobile phase. The mobile phases
consisted of 0.1 M TEAA with 500 mL of acetonitrile (eluent A)
and 25% acetonitrile in 0.1 M TEAA (eluent B).
For heteroduplex and multiplex detection, crude PCR products
were subjected to an additional 5minute 951C denaturing step
followed by gradual reannealing from 95 to 251C over a period of
70 minutes. The start and end points of the gradient were adjusted
according to the size of the PCR products by use of an algorithm
provided by WAVEmaker system control software (Transgenomic).
A total of 20 mL of PCR product was injected for analysis in
each run. The samples were run under partially denaturing
conditions according to the nature of each amplicon and provided
by WAVEmaker system control software. The buffer B gradient
increased 2% per minute for 4.5 minutes at a flow rate of 0.9 mL/
minute. Generally, the analysis took about 10 minutes for each
injection.
and known SMN1 or SMN2 only samples, we could distinguish
between the SMN1 and SMN2 peaks in DHPLC (Fig. 1). With
our specific primer design, we could identify the SMA-affected
patients efficiently and sensitively just by recognizing the SMN2only peak in DHPLC.
Under different oven temperatures, the equal (Fig. 2a–e) and
unequal (Fig. 2f–j) SMN1/SMN2 gene dosages could be
differentiated. We proved that the SMN1/SMN2 peak ratio
detected by DHPLC at 52.51C oven temperature was compatible
with gene dosages determined by quantitative real-time PCR
analysis (Fig. 3). To test the validity and reproducibility of our
detection system for gene dosage determination for the SMN1/
SMN2 genes, we analyzed every sample repeatedly at least three
times and all demonstrated the reproducible results.
We further tested the samples with different SMN1/SMN2
ratios in different PCR amplification cycles and demonstrated that
the ratio did not change with increments of the cycle number of
PCR amplification (Supplementary Figure S1, available online
at http://www.interscience.wiley.com/jpages/1059-7794/suppmat).
Quantitative Real-Time PCR of SMN1 and SMN2 Copy
Numbers
For determination of SMN1 and SMN2 gene dosages, TaqMan
technology was used according to the method described by Anhuf
et al. [2003]. Quantification was performed with an ABI Prism
7000 sequence detection system and 96-well MicroAmp optical
plates (Applied Biosystems). The SMN genes were amplified by
use of the forward primer 50 -AATGCTTTTTAACATCCATATAAAGCT-30 and the reverse primer 50 -CCTTAATTTAAGGAATGTGAGCACC-30 . The minor groove binder (MGB) probes
(Applied Biosystems) were designed to distinguish between the
SMN1 and SMN2 genes in exon 7 at position 6. The two specific
hybridization probes were labeled with 50 -FAM as a fluorescent dye
(SMN1-Ex7: 50 -CAGGGTTT CAGACAAA-30 and SMN2-Ex7anti: 50 -TGATTTTGTCTAA AACCC-30 ).
PCR was performed in a total volume of 25 mL containing 50 ng
of genomic DNA, 0.3 mM of each primer, 13 mL Platinums qPCR
Supermix-UDG (Invitrogen, Karlsruhe, Germany), 0.5 mM
6-Carboxy-X-Rhodamine (ROX) as a passive reference (Invitrogen), 2 mM MgCl2, and 100 nmol of each MGB probe. The 96well plate contained 125 ng, 25 ng, and 5 ng standard DNA,
respectively. Each test sample and each amount of standard DNA
were run in duplicate. All reactions of the same run were prepared
from the same master mix.
Reactions for the SMN1 or SMN2 test loci and the Factor VIII
gene reference locus were prepared and run in parallel. The PCR
conditions were one cycle at 501C for 2 minutes, one cycle at 951C
for 10 minutes, followed by 40 cycles of 951C for 15 seconds, 601C
for 1 minute. The analysis was performed with ABI 37000SDS
software (Applied Biosystems).
RESULTS
By DHPLC analysis, we could identify the SMN1/SMN2 peaks
unambiguously at different oven temperatures. To identify the
peaks attributed to SMN1 and SMN2, we constructed the SMN1
and SMN2 gene corresponding to the amplicons which were
analyzed separately to pGEM-T Easy Vector. By comparing the
DHPLC of DNA samples with SMN1 and SMN2 only, PCR
products that were cloned from previously described constructs
Chromatography and sequence analysis of an individual with equal dosage of SMN1/SMN2 genes (a), an individual
with the SMN2 gene only (b), a construct with the SMN2 gene
only (c), an individual with the SMN1 gene only (d), and a construct with the SMN1 gene only (e). [Color ¢gure can be viewed
in the online issue, which is available at www.interscience.wiley.
com.]
FIGURE 1.
DHPLC QUANTITATIVE ANALYSIS OF SMN1 AND SMN2
463
FIGURE 2. E¡ect of DHPLC oven temperature on the power of SMN1/SMN2 discrimination. a^e: Chromatograms of one individual
with SMN1/SMN2 gene ratio of 1; the oven temperature was set at 511C (a), 521C (b), 52.51C (c), 531C (d), and 541C (e). f^j: Chromatograms of one individual with a gene ratio of SMN1:SMN2 = 2:1; the oven temperature was set at 511C (f), 521C (g), 52.51C (h),
531C (i), and 541C (j).
FIGURE 3. DHPLC of an individual with an SMN1/SMN2 gene ratio of1 (a), an individual with a gene ratio of SMN1:SMN2 = 1:2 (b), an
individual with a gene ratio of SMN1:SMN2 = 1:3 (c), an individual with a gene ratio of SMN1:SMN2 = 2:1 (d), an individual with a
gene ratio of SMN1:SMN2 = 3:1 (e), an individual with an SMN2 gene only (f), and an individual with an SMN1 gene only (g).
464
SU ET AL.
Two core families in which affected cases and carriers with
different SMN1/SMN2 ratios could be well distinguished are
presented here (Fig. 4; Supplementary Figure S2).
All of the SMA carriers with SMN1/SMN2 ratios other than
one could be identified by this simple but powerful detection
system simply by injection of the crude PCR products after specific
primer amplification. However, according to the clinical evidence,
a small portion of SMA carriers had an SMN1/SMN2 ratio of 1,
with one copy of SMN1 and one copy of SMN2. By our current
system with DHPLC analysis, it failed to be differentiated from the
most common genotype (SMN1/SMN2 ratio of 1, with two copies
of SMN1 and two copies of SMN2) in the general population.
Therefore, we designed a specific multiplex competitive PCR
protocol, by using primer pairs to amplify the CYBB gene (Xlinked), KRIT1 gene (on chromosome arm 7q), and SMN1/SMN2
genes (as previously described) and also as detected by DHPLC, in
order to: 1) calculate the total gene dosages of SMN1+SMN2 by
adjusting the relative known dosages of the CYBB and KRIT1
genes; and 2) determine the ratio of the SMN1/SMN2 genes by
our previous setting. The results of multiplex PCR in both male/
female and SMN1/SMN2 ratios are shown in Figure 5. The copy
number of the SMN1+SMN2 genes in the unknown samples (U)
in comparison with the control samples (C) was calculated by the
equations below:
Since the CYBB gene is X-linked, K (U) and K (C) represent
the factors for the unknown and control samples: male = 2 and
female = 1.
The putative total copy number of the SMN1+SMN2 genes
calculated from the multiplex competitive PCR protocol is shown
in Figure 6 and was confirmed by quantitative real-time PCR
analysis (data not shown). With our method, we could successfully
designate all of the genotypes with different SMN1/SMN2 gene
copy numbers, including SMN1/SMN2 ratios equal and not equal
to one (Table 1).
DISCUSSION
SMA is the second most frequent autosomal recessive disease
caused by homozygous loss of the SMN1 gene [Lefebvre et al.,
1995]. Homozygous absence of the telomeric SMN1 gene occurs
in approximately 94% of affected individuals with all subtypes of
SMA, and the remaining cases are related to small intragenic
mutations of the SMN1 gene [Ogino and Wilson, 2004; Wirth,
2000]. Because SMA is one of the most common lethal genetic
disorders, with a carrier frequency of 1/35 to 1/50, it is important
to identify carriers of the SMN1 absence for diagnostic purposes
and genetic counseling [McAndrew et al., 1997].
Peak Height of SMN1 þ SMN2 Genes ðUÞ=½Peak Height of CYBB Gene ðUÞKðUÞ
4
Peak Height of SMN1 þ SMN2 Genes ðCÞ=½Peak Height of CYBB Gene ðCÞKðCÞ
Peak Height of SMN1 þ SMN2 Genes ðUÞ=Peak Height of KRIT1 Gene ðUÞ
4
Peak Height of SMN1 þ SMN2 Genes ðCÞ=Peak Height of KRIT1 Gene ðCÞ
Pedigree and DHPLC for one core family. In this family, two sons had the SMN2 gene only and were shown to be patients
with SMA; one daughter had a SMN1/SMN2 ratio of 1 that was classi¢ed as a normal variation; their father, mother, and one daughter
had a gene ratio of SMN1:SMN2 = 1:3 and were considered to be carriers.
FIGURE 4.
DHPLC QUANTITATIVE ANALYSIS OF SMN1 AND SMN2
465
FIGURE 5. DHPLC of multiplex PCR analysis with di¡erent SMN1/SMN2 ratios. I: a^d: Female individuals with various SMN1/SMN2
ratios: 2:2 (a); 2:1 (b);1:2 (c); and 1:1 (d). e^h: Male individuals with various SMN1/SMN2 ratios: 2:2 (e); 2:1 (f);1:2 (g); and 1:1 (h). II:
Female individuals with various copy numbers of SMN1 only: two copies of SMN1 (a), three copies of SMN1 (b), four copies of SMN1
(c). III: Female individuals with various copy numbers of SMN2 only: two copies of SMN2 (a), three copies of SMN2 (b), four copies of
SMN2 (c). ~CYBB gene (X-linked); m KRITI gene; SMN2 gene.
However, the duplication of the SMN locus makes the detection
of SMA carriers difficult. Accurate dosage analysis is necessary to
identify SMA carriers and to distinguish SMA compound
heterozygotes (SMN1 absence in one chromosome 5, and a small
intragenic SMN1 mutation in the other chromosome 5), resulting
in one copy of SMN1 from non-5q SMA-like cases with two copies
of SMN1. Both of these diagnostic applications require a method
that can differentiate between individuals with one and those with
two copies of the SMN1 gene.
In this study, we demonstrate a novel method for detecting
carriers of SMA by DHPLC, which is quite simple to perform, and
is faster than current methods. Recently, applying DHPLC to
detect patients with homozygous SMN gene absence has been
shown to be less laborious when compared with the traditional
approaches [Mazzei et al., 2003; Sutomo et al., 2002]. Because
telomeric (SMN1) and centromeric (SMN2) copies of exon 7 of
the SMN gene differ only by a single base pair, denaturation and
slow renaturation of PCR products of this exon from an individual
with both SMN1 and SMN2 gene copies lead to the formation of
heteroduplexes and homoduplexes. By use of DHPLC, a different
chromatography in normal controls from that in patients carrying
the homozygous deletion/conversion of the SMN1 gene could be
identified. However, this test does not allow the detection of the
hemizygous absence of the SMN1 gene, which characterizes the
compound heterozygous cases (bearing one chromosome with
absence of SMN1 and a subtle mutation in the other) and the
carriers of the disease. In this study, we further extended the
application of DHPLC to detection of the dosage of the SMN1/
SMN2 genes by combining the techniques of heteroduplex analysis
and multiplex competitive PCR.
466
SU ET AL.
With our strategies, first we designed new PCR primers to
amplify four SNPs between the SMN1/SMN2 genes in order to
optimize the resolution, and we adjusted the oven temperature to
normalize the SMN1/SMN2 ratio. Second, to calculate the total
SMN (SMN1+SMN2) gene dosage, we further designed a
multiplex competitive PCR reaction by using two other genes
(one autosomal, one X-linked) as genomic references. DNA
samples from a known absence for SMA patients and family
members were examined retrospectively, and all were identified
unambiguously when compared with a recently developed realtime quantitative assay [Anhuf et al., 2003].
By our current method, one problem is that if hybrid or fused
SMN genes arise through gene conversion that can contain
different base pair differences between SMN1 and SMN2 genes,
these variable DNA content would affect the DHPLC running
conditions. However, up to date, we found that only one case with
Total copy numbers of SMN1+SMN2 genes calculated
by multiplex PCR and DHPLC analysis with di¡erent SMN1/
SMN2 gene ratios. indicates the mean 7 SEM. [Color ¢gure
can be viewed in the online issue, which is available at www.
interscience.wiley.com.]
FIGURE 6.
TABLE 1.
additive single base variation of c.835–50A>G in our tested
samples that could interfere the gene dose analysis (1/334). As we
presented, all of the SMA carriers with SMN1/SMN2 ratios other
than 1 could be identified by first-step heteroduplex analysis.
Therefore, we further designed the multiplex competitive PCR
protocol for calculating the total copy numbers of SMN1+SMN2
gene to identify SMA carriers with SMN1:SMN2 = 1:1 from the
general population with SMN1:SMN2 = 2:2. Indeed, there is a
region of overlap among the various categories concerning the
total copy number especially with the increased total copy number
of the SMN1+SMN2 gene 44. We had evaluated these cases
with estimated total gene copy number 45 by quantitative realtime PCR; no cases have 3:3 copies. However, in our limited cases,
all the SMA carriers with SMN1:SMN2 = 1:1 could all be
identified successfully with the cutoff value of the estimated copy
number o3. Further testing with large number of samples to
estimate the power of this multiplex competitive PCR protocol is
still warranted.
Nevertheless, the current protocols for SMA carrier
testing, including our newly developed method, still have
limitations. The finding of two SMN1 genes on a single
chromosome has serious counseling implications, because a carrier
individual with two SMN1 genes on one chromosome would be
misdiagnosed by SMN1 copy-number analysis [Chen et al., 1999;
Ogino et al., 2002]. Furthermore, the risk of carriers with an
intragenic mutation also should be taken into account. Assuming
that a carrier frequency in the general population is 1 in 50 and
that a conditional probability of a carrier individual having two
copies of SMN1 or point mutation is 5%, then two copies of SMN1
in an individual from the general population would decrease the
carrier risk to B1 in 900 [Ogino and Wilson, 2002a; Ogino and
Wilson, 2002b]. Thus, although the finding of a normal dosage
significantly reduces the risk of being a carrier, our results show
that there is still a small risk of recurrence.
In conclusion, we report here a powerful, rapid quantitative
PCR assay and demonstrate its clinical application for detection
of SMA carriers and possible compound heterozygous patients.
The assay increases the sensitivity of diagnosis of SMA and allows
for direct carrier testing. This assay now can be used for
quantitation of the SMN1 and SMN2 genes in SMA families,
Di¡erent SMN1/SMN2 Gene Copy Numbers Including Equal and Unequal SMN1/SMN2 Ratio
Expected total Measured total
Measured
Genotype
copy number
copy number
(SMN1:SMN2)
(SMN1:SMN2) (SMN1+SMN2)
(mean7SD)
ratio (mean7SD) Interpretation
SMN1 only
SMN2 only
1:1
1:2
2:1
2:2
1:3
3:1
4:1
2:3
3:2
Total
a
2
3
4
2
3
4
2
3
3
4
4
4
5
5
5
1.9670.15
3.0170.21
4.0770.16
1.9670.32
2.9970.29
3.9870.23
2.0570.10
3.0270.21
3.0370.36
4.0270.47
4.0470.20
3.9570.09
5.0670.29
4.9370.35
5.0870.31
No SMN2 peak
No SMN2 peak
No SMN2 peak
No SMN1 peak
No SMN1 peak
No SMN1 peak
1.0870.03
0.5170.02
2.1270.11
1.0570.04
0.3370.02
3.0170.54
4.0670.13
0.6870.02
1.6270.06
Indicates the original number of cases from the general population.
Indicates the number of cases including the patients and their families.
b
N
N
N
0
0
0
1
0.5
2
1
0.33
3
4
0.67
1.5
Number of
subjects
a
b
12 (10 +2 )
5 (4a +1b )
2a
8b
5b
12b
4 (2a +2b )
11 (4 a+7b)
73 (67 a +6b )
176 (160a +16b)
15 (3a +12 b)
2a
2a
2a
5a
334 (263 a + 71b)
Frequency
3.8%
1.5%
0.8%
B
B
B
0.8%
1.5%
25.4%
60.8%
1.1%
0.8%
0.8%
0.8%
1.9%
Status
Normal
Normal
Normal
SMA a¡ected
SMA a¡ected
SMA a¡ected
SMA carrier
SMA carrier
Normal
Normal
SMA carrier
Normal
Normal
Normal
Normal
DHPLC QUANTITATIVE ANALYSIS OF SMN1 AND SMN2
and in the general population, for identifying carriers at risk, and
for providing insight into the frequency and mechanisms of geneconversion events.
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