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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. REFERENCES Anhuf D, Eggermann T, Rudnik-Schoneborn S, Zerres K. 2003. Determination of SMN1 and SMN2 copy number using TaqMan technology. Hum Mutat 22:74–78. Cartegni L, Krainer AR. 2002. Disruption of an SF2/ASFdependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1. 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