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Determination of SMN1/SMN2 gene dosage by a quantitative genotyping platform combining capillary electrophoresis and MALDI-TOF mass spectrometry.

Spinal muscular atrophy (SMA) [6] one of the most common autosomal recessive disorders, has an estimated incidence of at least 1 in 10 000 live births and a carrier frequency of 1 in 35 to 1 in 50 (1, 2). The disease is caused by a loss of a-motor neurons in the spinal cord anterior horn cells and results from mutations in the survival motor neuron (SMN) genes. Patients with SMA are classified into 3 groups based on the clinical severity of the disease: severe, intermediate, and mild (SMA type I, II, and III, respectively) (3). 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 intermediate severity and causes some patients to be unable to walk. Type III SMA (Kugelberg-Welander disease; MIM# 253400) is a mild form of the disease with onset after the age of 18 months (4), and with patients gaining the ability to walk.

The SMA-determining gene group, SMN, is located in a complex region of chromosome 5813 and includes 2 highly homologous genes, telomeric SMN1 (MlM# 600354) and centromeric SMN2 (MlM# 601627). The 2 closely related genes differ at only 5 by within their 3'-terminal regions. Among the differences in exons 7 and 8, substitution of a single nucleotide (c.840C>T) decreases the activity of an exonic splicing enhancer. As a consequence, lower amounts of full-length transcript are generated from SMN2 than from SMN1, leading to a deficiency of normal, stable SMN protein (5, 6).

Deletions of the functional SMN1 appear to be directly involved in SMA; approximately 94% of clinically typical SMA-affected patients lack both copies of SMN1 exon 7 (7). Moreover, small deletions or point mutation that cause gene conversion from SMN1 to SMN2 have been found in patients in whom SMN1 was present (8). The clinical phenotypes of SMA depend on the mutation mechanism. Although absence of the SMN2 allele cannot cause SMA, the SMN2 copy number correlates inversely with disease severity and length of survival (9,10). Potential therapies for SMA include approaches to increase expression of full-length SMN2 transcripts (11); however, SMN2 cannot compensate for SMN1 deletion/conversion. Hence, identification of SMN1 deletions and determination of SMN1 and SMN2 copy numbers are of clinical importance in genetic counseling and are of therapeutic significance.

The high-performance DNA analysis (HDA) system, a 12-channel capillary electrophoresis instrument equipped with a disposable cartridge for rapid DNA separation and detection, was developed recently (12). The HDA system uses superbright light-emitting diodes (LEDs) based on InGaN material technology, which allows transmission of 524 run excitation light from the LEDs to gel-filled multicapillaries. In contrast to all previous capillary electrophoresis instruments, the microoptical collector in the HDA system can improve detection sensitivity. Therefore, HDA provides a non-fluorescence-based approach with sensitive, inexpensive, and high-throughput feature.

For single-nucleotide polymorphism (SNP) analysis, primer extension (minisequencing) is a direct method for multiplexed high-throughput genotyping using allele-specific DNA products. Minisequencing is the most widely used strategy for SNP genotyping because it provides high specificity and sensitivity with use of various detection techniques (13,14). Compared with other SNP analysis methods, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) offers direct and accurate differentiation of the allele-specific extension products (15). Furthermore, an additional advantage of high-throughput MALDI-TOF MS analysis is that simultaneous multiplexed genotyping can be achieved in a single experiment (16). Therefore, MALDI-TOF MS in combination with a minisequencing strategy has become one of the most promising tools for mutation analysis to allow study of polymorphisms in disease-causing genes (17,18).

The capability of the MALDI-TOF MS-based genotyping approach has been reported for quantitative SNP analysis using standard mixtures of pooled PCR products at several test loci (19). In this study, single-base primer extension by the addition of a dideoxynucleotide (PinPoint assay) yields allele-specific products that differ in relative molecular mass (Mr) by 9 to 40 units; this small mass difference is difficult to differentiate by routine low-resolution MALDI MS in the presence of trace cation (N[H.sub.4.sup.+], [Na.sup.+], and [K.sup.+]) adducts. Therefore, in that study, a very short extension strategy using alternating deoxynucleoside triphosphates (dNTPs) and dideoxynucleoside triphosphates was adopted so that the products differed in mass by [M.sub.r] 300 to 400 to resolve the 50/50 heterozygote for direct quantification from mass spectra. In addition, a calibration curve had to be constructed to correct the mass spectra response. We previously reported a sequential desalting method (20) to decrease cation adducts and consequently increase the required resolution for the multiplexed Pinpoint assay (21) in mass analysis. Among all minisequencing methods, the PinPoint assay produces the smallest extension products, which are least susceptible to variations in extension efficiency (22). The Pinpoint assay is therefore presumed to provide accurate relative quantification of gene dosage based on the smallest difference in primer extension products.

Here we report a quantitative genotyping platform that combines capillary electrophoresis for determining absolute gene dosage and the Pinpoint assay and MALDI-TOF MS for quantitative SNP analysis. With respect to SMA diagnostics, we evaluate the method using the critical C-to-T substitution at position +6 of exon 7 (c.840C>T) in SMA for differentiation between SMN1 and SMN2. The copy numbers and relative ratio of the SMN1 and SMN2 genes obtained with this platform can be determined to diagnose the status of SMA carriers in the general population. We confirmed the reliability of the platform in a study of 9 SMA-affected patients, 33 SMA carriers, and 478 healthy individuals. Our results demonstrate the feasibility of rapid and accurate quantitative genotyping for high-throughput, unambiguous diagnosis of SMA.

Materials and Methods

PATIENT SAMPLES

A total of 520 DNA samples were analyzed in this study, including samples from patients diagnosed with SMA, from carriers (i.e., families of SMA patients), and from apparently healthy individuals from the general population. The donors of all samples gave informed consent. Genomic DNA was collected from peripheral whole blood with a Puregene DNA Isolation Kit (Gentra Systems, Inc.), according to the manufacturer's instructions.

PRIMER DESIGN AND PCR

The PCR primers used to amplify the SMN1/SMN2 genes are shown in Table 1 of the Supplemental Data that accompanies the online version of this article at http: // www.clinchem.org/content/vol52/issue3/. Genomic DNA template (100 ng) was amplified in a total volume of 25 [micro]L containing 0.12 [micro]M each of the primers, 100 [micro]M each dNTP, 0.5 U of AmpliTaq Gold enzyme (PE Applied Biosystems), and 2.5 [micro]L of GeneAmp 10x buffer II (10 mM Tris-HCI, pH 8.3; 50 mM KCl) in 2 mM Mg[Cl.sub.2], as supplied by the manufacturer. PCR amplification was performed with an initial denaturation step at 95[degrees]C for 10 min, followed by 35 cycles of 94[degrees]C for 30 s, 53[degrees]C for 45 s, and 72[degrees]C for 45 s, with a final extension step of 72[degrees]C for 10 min. PCR amplification was performed in a multiblock system (MBS) thermocycler (ThermoHybaid).

MULTIPLEX COMPETITIVE PCR

We used multiplex competitive PCR to amplify the SMN1, SMN2, CYBB, and KRIT1 genes with the primers listed in Table 1 of the online Data Supplement. KRIT1 and X-linked CYBB were used as internal controls for determining the absolute gene dosages of SMN1 and SMN2. Multiplex competitive PCR was carried out in final volume of 50 [micro]L containing 100 ng of genomic DNA, 0.02 [micro]M each of the CYBB primers, 0.04 [micro]M each of the KRIT1 primers, 0.2 [micro]M each of the SMN primes, 200 [micro]M each of the dNTPs, 0.5 U of AmpliTaq Gold enzyme (PE Applied Biosystems), and 2.5 [micro]L of GeneAmp 10x buffer II (10 mM Tris-HCI, pH 8.3; 50 mM KCl) in 2 mM Mg[Cl.sub.2], as provided by the manufacturer. Amplification was performed in an MBS thermocycler with an initial denaturation step at 95[degrees]C for 10 min, followed by 26 cycles of 94[degrees]C for 30 s, 53[degrees]C for 45 s, and 72[degrees]C for 45 s, with a final extension step at 72[degrees]C for 10 min.

CAPILLARY ELECTROPHORESIS

The HDA system with a GCK-5000 Cartridge Kit provided by eGene was used to analyze multiplex competitive PCR products to determine the gene dosage. PCR products were diluted 20-fold with deionized water and placed in the instrument sample tray. The DNA samples were automatically injected into the capillary channel and subjected to electrophoresis according to the eGene operation protocol. BioCalculator software (eGene) labeled the integrated peak area automatically.

PURIFICATION OF PCR PRODUCTS

For the primer extension reaction, the Microcon YM-100 system (Millipore) was used to remove excess primers and unincorporated dNTPs to purify the PCR products. The PCR products were quantified by use of a GeneQuant pro spectrophotometer (Amersham Pharmacia Biotech), and the samples were diluted to a final concentration of 10 ng/[micro]L for further experiments.

PinPoint ASSAY

The primer used to detect the critical C-to-T substitution by primer extension is listed in Table 1 of the online Data Supplement. Primer extension reactions were carried out in a 20-micro]L volume containing 50 ng of input PCR product and 0.75 [micro]M primer, 25 [micro]M each of the dideoxynucleoside triphosphates (Amersham Pharmacia Biotech), and 0.5 U of ThermoSequenase DNA polymerase (Amersham Biosciences) in 1 x supplied reaction buffer. The reaction was carried out in an MBS thermocycler. Each reaction was subjected to initial denaturation step at 96[degrees]C for 60 s, followed by 50 cycles of denaturation at 96[degrees]C for 15 s, primer annealing at 43[degrees]C for 15 s, and extension at 60[degrees]C for 100 s.

SAMPLE PURIFICATION FOR MALDI-TOF MS

The primer-extension products were dried under reduced pressure and then desalted by use of sequential POROS and cation-exchange chromatography. The DNA was purified by use of POROS 50-R2 (PerSeptive Biosystems) reversed-phase chromatography medium (23). Pipette tips were filled with 20 [micro]L of POROS (80 g/L in 300 mL/L ethanol), the ethanol solution was drained out, and then 20 [micro]L of 100 mmol/L triethylammonium acetate (TEAR, pH 6.5) was added to the tip. The DNA sample was extracted in the presence of 100 mmol/L TEAA (pH 6.5), rinsed sequentially with 100 mmol/L and 10 mmol/L TEAR (pH 6.5), and eluted with 200 mL/L acetonitrile. Solvent was removed by applying compressed air to the tip after all steps (24). Finally, the sample was dried in an SPD111V SpeedVac (Savant).

The sample was redissolved in 3-5 [micro]L of water and desalted by cation-exchange chromatography with 5WX8-200 Dowex cation-exchange resin (Supelco Park) according to the protocol of Harksen et al. (25). The cation-exchange beads were first exchanged with 1 mol/L ammonium acetate and placed on a piece of Parafilm. DNA sample (5-10 [micro]L) and an equal amount of matrix (see the section on MS Analysis) were added to 0.1 mg of beads and mixed up and down in the pipette tip several times. The supernatant was loaded on the sample plate for MALDI analysis.

MS ANALYSIS

Before MALDI-TOF MS analysis, the sample was mixed with matrix solution (50 g/L 3-hydropicolinic acid in a 4:5:1 mixture of water-acetonitrile-50 g/L diammonium citrate) and spotted on a 96 x 2-well Teflon sample plate (PerSeptive Biosystems). MALDI-TOF mass spectra were acquired by a reflectron time-of-flight mass spectrometer (Voyager-DE PRO; Applied Biosystems). The instrument was equipped with a 337-nm nitrogen laser source at 3-20 Hz. Measurements were taken in linear, negative-ion mode at a 20-kV acceleration voltage and 380 ns delayed ion extraction. The excess unextended oligonucleotide primers were used as internal standards for mass calibration. A typical mass spectrum was obtained by averaging 100 laser shots followed by noise reduction and gaussian smoothing using Data Explorer software (Applied Biosystems).

Results

ANALYTICAL SCHEME

As shown in Fig. 1, the analytical scheme combines 2 complementary assays: (a) determination of the total copy numbers of SMN genes by multiplex competitive PCR and the capillary electrophoresis-based HDA system; and (b) determination of the relative ratio of SMN1 and SMN2 by Pinpoint minisequencing and MALDI-TOF MS. We first determined the absolute gene dosages (sum of SMN1 and SMN2), using CYBB and KRIT1 as internal quantification references (Fig. 1A). After multiplex competitive PCR amplification, the PCR products were directly analyzed by the HDA system without further purification. The copy numbers of the SMN genes in the unknown samples (U) in comparison with the control samples (C) were calculated by use of Eqs. 1 and 2 below:

Copy number = [Peak area of SMN (U)/[Peak area of CYBB (U)] x K (U)]]/[Peak area of SMN (C)/[Peak area of CYBB (C) x K (C)] x 4 (1)

Copy number = [Peak area of SMN (U)/Peak area of KR1T1 (U)]]/[[Peak area of SMN (C)/Peak area of KR1T1 (C)] (2)

[FIGURE 1 OMITTED]

Because CYBB is X-linked, K (U) and K (C) represent the following factors: male = 2 and female = 1. Both CYBB and KRIT1 were used for calculations, and the results were averaged.

To further determine the copy numbers of SMN1 and SMN2, we determined the relative ratio of SMN1 and SMN2 by Pinpoint minisequencing and MALDI-TOF MS analysis (Fig. 1B). After sequential desalting with POROS and cation-exchange chromatography, the minisequencing products of the SMN1 and SMN2 alleles were subjected to MALDI-TOF MS analysis. The intensity ratio resulting from the C/T target-specific minisequencing products reflects the relative quantities of SMN1 and SMN2. From a quantitative analysis standpoint, the different gene products in the Pinpoint assay have similar masses to circumvent problems derived from distinct ionization efficiency. The relative ratio of SMN1 to SMN2 can be determined by Eq. 3:

Ratio of copy number = Intensity of SMN1/Intensity of SMN2 (3)

The combination of the 2 methods enables unambiguous determination of the individual copy numbers of SMN1 and SMN2 for the diagnosis of SMA.

DETERMINATION OF SMN COPY NUMBER BY CAPILLARY ELECTROPHORESIS

To quantify SMN copy number, we used a specific multiplex competitive PCR using primer pairs to amplify CYBB (X-linked), KRIT1 (on chromosome arm 7q), and SMN/SMN2 (as described above). CYBB and KRIT1, with known dosages, were used as genomic references to calculate the absolute gene dosages of SMN1 + SMN2, collectively. Multiplex competitive PCR is based on the concept that the studied gene and its genomic references have equivalent PCR amplification efficiencies. Typical electrophoresis spectra of multiplex competitive PCR followed by detection with capillary electrophoresis are shown in Fig. 2. The spectra clearly show the different patterns of copy numbers 2, 3, and 4 for 3 male individuals (confirmed by quantitative real-time PCR). The fragments are well separated from each other; thus, the area of each peak can be used to calculate the total copy number of SMN1 + SMN2 in an unknown sample by use of Eqs. 1 and 2.

[FIGURE 2 OMITTED]

GENOTYPING AND QUANTIFICATION BY MALDI-TOF MS

In the second stage of our platform, the of SMN1 / SMN2 ratio was determined by minisequencing and MALDITOF MS. Typical mass spectra obtained from homozygotes and heterozygotes (with different SMN1 /SMN2 ratios) are shown in Fig. 3. Mass differences of 15 were observed between primer extension products derived from the wild-type (i.e., SMN1) or mutant allele (i.e., SMN2), which unambiguously identified the sequence difference, C or T, at the polymorphic site. The measured gene ratios (SMN1 / SMN2) were consistent with previous diagnoses made by quantitative real-time PCR analysis (data not shown). Quantification was performed by determining the ratio of the corrected peak height in Eq. 3, after baseline subtraction and gaussian smoothing, between minisequencing products of SMN1 and SMN2 in each MALDI mass spectrum.

[FIGURE 3 OMITTED]

For MALDI-TOF MS quantification of the SMN1/ SMN2 ratio, the between-run CV was 15% (n = 6). To further assess the accuracy of the quantitative method, we also evaluated the fluctuation between different positions on the MALDI sample plate. To minimize the spectral noise and spot-to-spot fluctuations in MALDI-TOF MS, we used the mean of 5 replicate spectra to calculate the peak-area ratio. The CV from individual MALDI spots was <2% (n = 5). We evaluated the contribution of interference attributable to partial resolution in mass spectra to the quantification error based on peak intensity by deconvolution of the SMN1/SMN2 genotype peaks, using gaussian peak fitting in the Origin 7.0 prognm (see Fig. 1 of the online Data Supplement). The difference was <5% of the measured peak height ratio. Experimental bias from instrument or sample handling therefore did not influence determination of the SMN1/SMN2 ratio.

GENOTYPING OF SMA CARRIERS AND SMA-AFFECTED PATIENTS

Finally, to validate the suitability of our quantitative genotyping platform based on capillary electrophoresis and MALDI-TOF MS for high-throughput screening, we analyzed 520 DNA specimens, including 478 samples from apparently healthy individuals from the general population, 9 from SMA-affected patients, and 33 from SMA carriers. In the first stage, the copy number of each genotype was determined by the HDA system (see Table 1 and Fig. 2 of the online Data Supplement). Calculation of the ratios of the peak areas SMN/CYBB or SMN/KRIT1 gave mean (SD) SMN copy numbers for each genotype of 3.90 (0.14) for 4 copies, 2.87 (0.19) for 3 copies, and 2.09 (0.02) for 2 copies. In the second stage, we used all 520 DNA specimens from SMA patients, carriers from families of affected patients, and individuals from the general population to further evaluate the ability of the SMN1/ SMN2 ratio to identify SMA-affected individuals. Representative mass spectra for different SMN1/SMN2 ratios are shown in Fig. 4. The statistical data indicate that the CV from individuals is between 4% and 10%, demonstrating that use of the Pinpoint assay combined with MALDI MS can be a suitable method for quantification of the SMN1/SMN2 ratio. In combination with the total copy number determined by the HDA system, the method unambiguously determined the genotypes of the 520 persons studied (Table 2). Consequently, the diagnostic accuracy of our method for detecting SMA is 100%.

[FIGURE 4 OMITTED]

The genotype distributions in the group of apparently healthy individuals, carriers, and SMA-affected patients derived from the MALDI-TOF MS and capillary electrophoresis-based HDA analyses are shown in Fig. 5. Among the healthy individuals, 437 of 478 had 2 SMN1 copies (SMN1/SMN2 ratios of 2:2, 2:1, or 2:0), 31 of 478 carried 3 SMN1 copies (SMN1/SMN2 ratios of 3:1 or 3:0), and the remaining 10 carried 4 SMN1 copies (SMN1 / SMN2 ratio of 4:0). Among the SMA patients, 5 of 9 had 4 SMN2 copies (SMN1/SMN2 ratio of 0:4), 2 of 9 had 3 SMN2 copies (SMN1/SMN2 ratio of 0:3), and 2 of 9 had 2 SMN2 copies (SMN1/SMN2 ratio of 0:2). To confirm the SMN gene dosages, we performed quantitative real-time PCR, using TagMan technology with minor groove binder probes (26), and a specific multiplex competitive PCR protocol coupled with denaturing HPLC (data not shown) (27). The excellent agreement demonstrated that our approach is capable of making an unambiguous distinction between carriers and noncarriers.

Discussion

SMA is one of the most frequent severe autosomal recessive muscular diseases, with a birth prevalence of 1 in 10 000. The absence of both copies of the SMN1 gene is the most common mutation type of this disease and accounts for 94% of all SMA alleles in this population. Because of the high carrier frequency of 1 in 35 to 1 in 50 in the worldwide population (1, 2), prenatal diagnosis and carrier screening of SMA are currently the best solutions to minimize the incidence of SMA. Various assays have been used for the screening and diagnosis of SMA. Several different quantitative PCR tests for SMN analysis have been developed (3,10, 26, 28, 29). Although these methods efficiently determine SMN1 and SMN2 gene copy numbers, most of the methods are based on fluorescent labels or hybridization assays, which are often susceptible to complications attributable to nucleic acid secondary structure (30, 31). Heteroduplex formation may affect quantitative PCR-restriction fragment length polymorphism analyses (32). Heteroduplexes between SMN1 and SMN2 PCR products cannot be digested by DraI, an enzyme that is commonly used in the restriction fragment length polymorphism analyses, which increases the SMN1 signal while decreasing the SMN2 signal (33).

Our parallel assays for screening SMA carriers and patients determine both the SMN1/SMN2 ratio and the absolute SMN gene dosage by 2 independent methods. Our results demonstrate that the complementary assays together create a rapid and reliable method for quantifying SMN1 /SMN2 genes. In an analysis of 520 genomic DNA samples of known genotype from healthy individuals, carriers, and SMA patients with homozygous deletions or conversions, the quantification platform had 100% assay specificity and sensitivity. In all cases, the carriers had only 1 SMN1 gene; because of the small size of our screening sample, no carriers with 1 SMN1 and 1 SMN2 gene or 1 SMN1 and no SMN2 gene were observed. In the case of SMN2, the SMN2 copy number of affected patients was related to the phenotype (data not shown). It should be noted that, like all other previously described assays, our assay does not detect the remaining mutations. Therefore, patients who are compound heterozygotes for a common point mutation will not be identified by the current method. For affected patients who are genotyped as having 1 SMN1 gene, it might be prudent to genotype the SMN1 gene to determine the possibility of compound heterozygosity with a point mutation allele.

Current capillary electrophoresis instruments use lasers as the light source for DNA analysis, which is bulky and expensive. By combining HDA with multiplex competitive PCR, we can identify SMN gene dosage based on the peak area compared with known genes. There was no loss of resolution with the LED light source for detecting multiplex competitive PCR products. Additionally, capillary electrophoresis was optimized for rapid screening of multiplex PCR products. In our study, automated capillary electrophoresis of multiplex competitive PCR products can be completed with the HDA system within 10 min. For high-throughput analyses, up to 96 DNA samples can be analyzed within 1.5 h.

Primer extension combined with MALDI MS is a direct method for detecting SNPs. Mass can be measured accurately to determine the exact nucleotide at the mutation site without the need for additional confirmation. Detection specificity is therefore better than can be achieved with fluorescence-based genotyping. Another advantage of this combination of techniques is its high-throughput capacity to screen large populations. In our improved MALDI-TOF MS analysis platform, sequential desalting can be completed within a few minutes and is amenable to automation, thereby allowing adaptation for robust high-throughput SNP genotyping.

The development of new molecular tools will favor a viable and cost-effective detection method. We calculated the costs of this assay at approximately $5.00 (US) per sample. Excluding the instruments, this cost includes chemicals, reagents, and primers for PCR amplification, and reagents and supplies for purification, primer extension, capillary electrophoresis-based HDA, and MALDITOF MS. The assay may be most cost-effective in clinical laboratories with available equipment. Furthermore, the automated HDA system and MALDI-TOF MS can provide a rapid assay for molecular diagnostic. The strategy described here can be adapted to characterize individual genotypes with respect to other disease-causing mutations; as such, it may be widely applicable in the clinical setting for diagnostic genotyping.

We gratefully acknowledge financial support from Academia Sinica, the National Science Council, and National Taiwan University Hospital.

References

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HUNG-YI KAO, [1,2] ([dagger]) YI-NING SU, [3] ([dagger]) HSIN-KAI LIAO, [1] MING S. LIU, [4] and YU-JU CHEN [1,5] *

[1] Institute of Chemistry, and Genomics Research Center, Academia Sinica, Taipei, Taiwan, ROC.

[2] Department of Bioinformatics and Structure Biology, National Tsing Hua University, Hsinchu, Taiwan, ROC.

[3] Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan, ROC.

[4] Institute eGene, Inc., Irvine, CA.

[5] Institute of Molecular Biology, National Chung-Hsing University, Taichun, Taiwan.

[6] Nonstandard abbreviations: SMA, spinal muscular atrophy; SMN, survival motor neuron; HDA, high-performance DNA analysis; LED, lightemitting diode; SNP, single-nucleotide polymorphism; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; dNTP, deoxynucleoside triphosphate; MBS, multiblock system; and TEAA, triethylammonium acetate.

([dagger]) Hung-Yi Kao and Yi-Ning Su contributed equally to this study.

* Address correspondence to this author at: Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan, ROC. Fax 886-2-2783-1237; e-mail yjchen@chem.sinica.edu.tw.

Received October 2, 2005; accepted December 29, 2005.

Previously published online at DOI: 10.1373/clinchem.2005.061192
Table 1. Statistical evaluation of the measured SMN gene
copy numbers determined by capillary electrophoresis.

SMN absolute Mean (SD) measured No. of
copy number absolute copy number individuals Frequency, %

 4 3.90 (0.14) 320 (a) + 9 (b) 64
 3 2.87 (0.19) 170 (a) + 9 (b) 34
 2 2.09 (0.02) 10 (a) + 2 (b) 2

(a) Original number of cases from the general population.

(b) Number of cases, including the patients and their families.

Table 2. Summary of SMN1/SMN2 genotype in 520
individuals, as determined by capillary electrophoresis
and MALDI-TOF MS.

 SMN1/SMN2 result

SMN2 CV,
genotype Interpretation Mean (SD) %

1/2 0.5 0.49 (0.02) 4.1
1/3 0.33 0.32 (0.03) 9.0
2/1 2 2.00 (0.13) 6.5
2/2 1 0.99 (0.06) 6.1
3/1 3 3.19 (0.30) 9.4
SMN1 only [infinity] No SMN2 peak
SMN2 only 0 No SMN1 peak
Total

SMN2 No. of Predicted
genotype individuals status

1/2 23 SMA carrier
1/3 10 SMA carrier
2/1 144 Normal
2/2 283 Normal
3/1 21 Normal
SMN1 only 30 Normal
SMN2 only 9 SMA patient
Total 520

Fig. 5 Distribution of copy numbers among apparently healthy
individuals, SMA carriers, and SMA-affected patients.

Number of individuals

 Four copies Three copies Two copies

Patient 5 2 2
 0:4 (n = 5) 0:3 (n = 2) 0:2 (n = 2)

Carrier 10 23
 1:3 (n =10) 1:2 (n =23)

Healthy 314 154 10
 4:0 (n = 10) 3:0 (n = 10) 2:0 (n = 10)
 3:1 (n = 21) 2:1 (n = 144)
 2:2 (n = 283)

Note: Table made from bar graph.
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Title Annotation:Molecular Diagnostics and Genetics
Author:Kao, Hung-Yi; Su, Yi-Ning; Liao, Hsin-Kai; Liu, Ming S.; Chen, Yu-Ju
Publication:Clinical Chemistry
Date:Mar 1, 2006
Words:5410
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