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Design and Validation of a Conformation Sensitive Capillary Electrophoresis-Based Mutation Scanning System and Automated Data Analysis of the More than 15 kbp-Spanning Coding Sequence of the SACS Gene

2009; Elsevier BV; Volume: 11; Issue: 6 Linguagem: Inglês

10.2353/jmoldx.2009.090059

1943-7811

Sascha Vermeer, Rowdy Meijer, Tom Hofste, Daniëlle Bodmer, Ermanno Bosgoed, Frans P.M. Cremers, B. Kremer, Nine V.A.M. Knoers, Hans Scheffer,

DNA Repair Mechanisms

In this study, we developed and analytically validated a fully automated, robust confirmation sensitive capillary electrophoresis (CSCE) method to perform mutation scanning of the large SACS gene. This method facilitates a rapid and cost-effective molecular diagnosis of autosomal recessive spastic ataxia of Charlevoix-Saguenay. Critical issues addressed during the development of the CSCE system included the position of a DNA variant relative to the primers and the CG-content of the amplicons. The validation was performed in two phases; a retrospective analysis of 32 samples containing 41 different known DNA variants and a prospective analysis of 20 samples of patients clinically suspected of having autosomal recessive spastic ataxia of Charlevoix-Saguenay. These 20 samples appeared to contain 73 DNA variants. In total, in 32 out of the 45 amplicons, a DNA variant was present, which allowed verification of the detection capacity during the validation process. After optimization of the original design, the overall analytical sensitivity of CSCE for the SACS gene was 100%, and the analytical specificity of CSCE was 99.8%. In conclusion, CSCE is a robust technique with a high analytical sensitivity and specificity, and it can readily be used for mutation scanning of the large SACS gene. Furthermore this technique is less time-consuming and less expensive, as compared with standard automated sequencing. In this study, we developed and analytically validated a fully automated, robust confirmation sensitive capillary electrophoresis (CSCE) method to perform mutation scanning of the large SACS gene. This method facilitates a rapid and cost-effective molecular diagnosis of autosomal recessive spastic ataxia of Charlevoix-Saguenay. Critical issues addressed during the development of the CSCE system included the position of a DNA variant relative to the primers and the CG-content of the amplicons. The validation was performed in two phases; a retrospective analysis of 32 samples containing 41 different known DNA variants and a prospective analysis of 20 samples of patients clinically suspected of having autosomal recessive spastic ataxia of Charlevoix-Saguenay. These 20 samples appeared to contain 73 DNA variants. In total, in 32 out of the 45 amplicons, a DNA variant was present, which allowed verification of the detection capacity during the validation process. After optimization of the original design, the overall analytical sensitivity of CSCE for the SACS gene was 100%, and the analytical specificity of CSCE was 99.8%. In conclusion, CSCE is a robust technique with a high analytical sensitivity and specificity, and it can readily be used for mutation scanning of the large SACS gene. Furthermore this technique is less time-consuming and less expensive, as compared with standard automated sequencing. Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) belongs to a heterogeneous group of neurodegenerative disorders characterized by ataxia, mostly due to progressive degeneration of the cerebellum and associated structures. The clinical phenotype of these disorders is broad and quite variable. Clinically it can sometimes be hard to distinguish the different types of cerebellar ataxia. Molecular diagnosis can be extremely helpful to differentiate between the different forms of ataxia. Using DNA analysis to confirm the clinical diagnosis of ARSACS (MIM 270550) has been hampered in the majority of patients worldwide due to the lack of an efficient and cost-effective mutation scanning system for the SACS gene. The large size of the total coding sequence of this gene has precluded sequencing of the total coding sequence of the SACS gene as a routine diagnostic service. Since automated sequencing of this large gene is labor-intensive, costly, and time-consuming, we developed mutation screening by designing a heteroduplex analysis system using conformation-sensitive capillary electrophoresis (CSCE), which is a simple, rapid, reliable, and sensitive technique enabling a high sample throughput1Davies H Dicks E Stephens P Cox C Teague J Greenman C Bignell G O'meara S Edkins S Parker A Stevens C Menzies A Blow M Bottomley B Dronsfield M Futreal PA Stratton MR Wooster R High throughput DNA sequence variant detection by conformation sensitive capillary electrophoresis and automated peak comparison.Genomics. 2006; 87: 427-432Crossref PubMed Scopus (34) Google Scholar and automated DNA variant identification. We have chosen this method because it can be used for the analysis of larger fragment sizes (up to 500 bp), it has a higher sensitivity, and it can easily be implemented in existing automated sequencer infrastructure. DNA analysis therefore becomes more reliable, more robust, less expensive, and has a higher throughput, in comparison with established techniques such as single-strand confirmation polymorphism, denaturing gradient gel electrophoresis, and denaturing high performance liquid chromatography.2Hestekin CN Barron AE The potential of electrophoretic mobility shift assays for clinical mutation detection.Electrophoresis. 2006; 27: 3805-3815Crossref PubMed Scopus (43) Google Scholar The SACS gene involved in ARSACS is located on chromosome 13q12.12 and encodes the large protein sacsin.3Engert JC Berube P Mercier J Dore C Lepage P Ge B Bouchard JP Mathieu J Melancon SB Schalling M Lander ES Morgan K Hudson TJ Richter A ARSACS, a spastic ataxia common in northeastern Quebec, is caused by mutations in a new gene encoding an 11.5-kb ORF.Nat Genet. 2000; 24: 120-125Crossref PubMed Scopus (339) Google Scholar The gene consists of at least 11 exons, of which 9 contain coding sequences, spanning 13,737 bp and 4579 amino acids (NM_014363.4). To date, apart from the two mutations originally identified in the ARSACS patients from Québec, 30 different additional mutations, including nonsense, missense, small deletions and insertions, have been identified in ARSACS patients from different countries. These mutations occur throughout the entire gene. Not all types of mutations will be detected by sequencing or alternative subtle mutation scanning techniques such as CSCE. For instance, recently a Belgian ARSACS patient has been described with a 1.5-Mb deletion on chromosome 13q12.12 encompassing the SACS gene and a missense mutation on the other allele.4Breckpot J Takiyama Y Thienpont B Van VS Vermeesch JR Ortibus E Devriendt K A novel genomic disorder: a deletion of the SACS gene leading to spastic ataxia of Charlevoix-Saguenay.Eur J Hum Genet. 2008; 16: 1050-1054Crossref PubMed Scopus (42) Google Scholar This underlines that additional mutation detection techniques will still be needed. Here, we report the development of a fully automated, robust CSCE method to perform mutation scanning of the large SACS gene, facilitating a rapid and cost-effective molecular diagnosis of ARSACS patients. Since the cohort of Dutch ARSACS patients had been thoroughly analyzed for SACS mutations by automated direct sequencing, a good starting point for the analytical validation of the CSCE mutation scanning including automated heteroduplex pattern analysis had already been created. A technical validation of the CSCE technique in general has been performed as an interlaboratory collaborative project previously5Grieco GS Malandrini A Comanducci G Leuzzi V Valoppi M Tessa A Palmeri S Benedetti L Pierallini A Gambelli S Federico A Pierelli F Bertini E Casali C Santorelli FM Novel SACS mutations in autosomal recessive spastic ataxia of Charlevoix-Saguenay type.Neurology. 2004; 62: 103-106Crossref PubMed Scopus (63) Google Scholar; details will be published elsewhere. The analytical validation of CSCE specifically for the SACS gene presented here has been performed in two phases: a retrospective analysis of samples containing known DNA variants in the SACS gene and a prospective analysis of samples of patients for whom SACS mutation analysis was requested by a physician. Issues addressed during the development of a CSCE system for mutation scanning are discussed and illustrated with examples. These include the position of a DNA variant relative to the primers, and the CG content of the amplicons. Finally an overview of all novel SACS mutations and single nucleotide polymorphisms (SNPs) identified in a cohort of 76 patients for whom SACS mutation analysis was requested is presented (Tables 15Grieco GS Malandrini A Comanducci G Leuzzi V Valoppi M Tessa A Palmeri S Benedetti L Pierallini A Gambelli S Federico A Pierelli F Bertini E Casali C Santorelli FM Novel SACS mutations in autosomal recessive spastic ataxia of Charlevoix-Saguenay type.Neurology. 2004; 62: 103-106Crossref PubMed Scopus (63) Google Scholar and 2).Table 1Known and Novel Mutations Detected by CSCE and SequencingExonAmpliconVariantPredicted protein changePredicted consequenceMutation typePositionNumber of occurrences66c. 502G>Tp. Asp168TyrAmino acid substitutionMissense174/390278c. 961C>Tp.Arg321XPremature termination codonNonsense226/46629//10c. 1475G>Ap.Trp492XPremature termination codonNonsense448/484//84/4491812c. 2094-2A>GAberrant splicingSplice site92/2 991c. 2182C>Tp.Arg728XPremature termination codonNonsense182/2992c. 2185+1delAberrant splicingSplice site186/2992918*Known Italian mutations (Grieco et al5).c. 4108C>Tp.Gln1370XPremature termination codonNonsense365/46421c. 4957G>Tp.Glu1653XPremature termination codonNonsense100/4842c. 5125C>Tp.Gln1709XPremature termination codonNonsense268/4841c. 5143A>Tp.Lys1715XPremature termination codonNonsense286/484123/24c. 6000_6004delp.Arg2002fsPremature termination codonDeletion468–472/492//100–104/466126*Known Italian mutations (Grieco et al5).c. 6835dupAp.Glu2280fsPremature termination codonInsertion241–242/46327*Known Italian mutations (Grieco et al5).c. 7250_7254delp.Thr2417fsPremature termination codonDeletion318–322/48630c. 8401-8403delp.Gln2801delSingle amino acid deletionIn frame deletion319–321/489234/35c. 9911_9912delp.Leu3304fsPremature termination codonDeletion413–414/481//49–50/474136c. 10442T>Cp.Leu3481ProAmino acid substitutionMissense260/496137c. 10906C>Tp.Arg3636XPremature termination codonNonsense393/464141c. 12160C>Tp.Gln4054XPremature termination codonNonsense195/4851143c. 12992G>Ap.Arg4331GlnAmino acid substitutionMissense351/4861* Known Italian mutations (Grieco et al5Grieco GS Malandrini A Comanducci G Leuzzi V Valoppi M Tessa A Palmeri S Benedetti L Pierallini A Gambelli S Federico A Pierelli F Bertini E Casali C Santorelli FM Novel SACS mutations in autosomal recessive spastic ataxia of Charlevoix-Saguenay type.Neurology. 2004; 62: 103-106Crossref PubMed Scopus (63) Google Scholar). Open table in a new tab Table 2Polymorphisms Detected by CSCE and SequencingExonAmpliconVariantPredicted protein changePredicted consequenceMutation typePositionNumber of occurrences11c.1-219 A>G145/58510c.1-13A>G351/5851822c.171+6C>T3c.171+24G>A133c.172-129A>G1/3431c.259+28A>G245/343155c.346-143G>A79/391477c.696T>Ap.Asn23 2LysAmino acid substitutionMissense189/327138c.909A>Gp.Ala3 03AlaAmino acid substitutionMissense174/4669c.973G>Ap.Gly325ArgAmino acid substitutionMissense238/466110c.1656A>Gp.Leu552LeuAmino acid substitutionMissense265/4491411c.1846G>Cp.Ala6 16ProAmino acid substitutionMissense385/4881c.2080G>Ap.Ala6 94ThrAmino acid substitutionMissense151/4887913/14c.2472G>Ap.Ser 824SerAmino acid substitutionMissense374/459//76/475115c.2983G>Tp.Val995PheAmino acid substitutionMissense252/4941c.2988A>Gp.Leu996LeuNo changeMissense257/494219c.4188C>Tp.His1396HisNo changeMissense86/4982c.4466A>Gp.Asn14 89SerAmino acid substitutionMissense364/498324c.6195T>Cp.Ile 2065IleNo changeMissense294/46640c.6267G>Ap.Ser 2089SerNo changeMissense366/466226c.6781C>Ap.Leu2261IleAmino acid substitutionMissense187/4631c.6946A>G*SNP not validated.p.Thr 2316AlaAmino acid substitutionMissense127c.7140T>Ap.Asn24 80LysAmino acid substitutionMissense208/4861c.7149C>Tp.Arg2383ArgNo changeMissense217/486128c.7539C>Tp.Val2513ValNo changeMissense243/498131/32c.8853T>Cp.Val2951ValNo changeMissense404/491//52/4944434c.9801A>G*SNP not validated.p.Thr 3267ThrNo changeMissense1c.9846A>Gp.Pro3282ProNo changeMissense348/481135c.9981T>Cp.Ala3327AlaNo changeMissense119/47387c.10106T>Cp.Val3369AlaAmino acid substitutionMissense244/4734336c.10338G>Ap.Gln344 6GlnNo changeMissense156/494438c.11032C>Gp.Pro36 78AlaAmino acid substitutionMissense153/499541c.12304T>Cp.Leu4102LeuNo changeMissense339/4852743c.12813T>Cp.Pro42 71ProNo changeMissense121/486145c.13522A>Cp.Lys4508GlnAmino acid substitutionMissense183/4831c.13717A>Cp.Asn45 73HisAmino acid substitutionMissense377/4831* SNP not validated. Open table in a new tab Peripheral blood was collected using EDTA as an anticoagulant from index patients with recessive cerebellar ataxia. Automated genomic DNA extraction was performed using a Tecan Freedom EVO 150 workstation combined with a Chemagic Magnetic Separation Module 1 from Chemagen. The concentration of DNA samples were standardized at 100 ng/μl. For the validation, in total 52 DNA samples of patients were used who either have ARSACS confirmed at the DNA level or known variants in the SACS gene (retrospective phase), or who were suspected of having ARSACS (prospective phase). The remaining 24 patients for whom mutation data were available have not been included in the validation study to avoid redundancy, ie, no additional fragments would have been validated by their inclusion. To facilitate the detection of homozygous mutations, before CSCE, the PCR product of a patient was mixed with the PCR product of a mutation-negative reference sample to allow the formation of heteroduplex bands. For this purpose, we used two different reference samples (V3 and V4). These samples were sequenced in advance to identify the DNA variants present in the different amplicons of the SACS gene. Informed consent was obtained from all patients included in this study. We designed 45 overlapping PCR primer pairs for the nine coding exons (Table 3), including the gigantic exon 9 described previously3Engert JC Berube P Mercier J Dore C Lepage P Ge B Bouchard JP Mathieu J Melancon SB Schalling M Lander ES Morgan K Hudson TJ Richter A ARSACS, a spastic ataxia common in northeastern Quebec, is caused by mutations in a new gene encoding an 11.5-kb ORF.Nat Genet. 2000; 24: 120-125Crossref PubMed Scopus (339) Google Scholar and the flanking intronic sequences of the SACS gene (GenBank reference sequence accession number NM_014363.4, NCBI). All forward primers (FPs) and reverse primers (RPs) contain next to the specific primer an universal primer tag (M13), (FP) 5′-TGTAAAACGACGGCCAGT-3′ and (RP) 5′-CAGGAAACAGCTATGACC-3′. The primer sequences, positions and size of each amplicon are listed in Table 3. The DNA-amplification mix included (per amplicon per sample) 1.0 μl genomic DNA, 0.36 μl (8.33 mmol/L) dNTPs, 1.44 μl MgCl2 (50 mmol/L, AB), 1.80 μl 10× PCR Gold Buffer (AB), 0.14 TaqDNA polymerase Gold (5 U/μl), 6.26 μl distilled H2O (mQ), and 7 μl FP and RP (10 pmol/μl) from a mix of 0.16 μl FP and RP per 1 μl mQ. The reaction volume per sample was 18 μl. The samples were PCR amplified using the following PCR program on a Perkin-Elmer (ABI) Geneamp 9700: an initial denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and elongation at 72°C for 1 minute, with a final step at 72°C for 7 minutes, after which the samples are cooled down to 10°C. Before CSCE, 5 μl of each PCR-product with 7 μl Loading Orange G was run on a 1% agarose gel with a 100-bp marker for comparison. Next, all PCR-products (13 μl) were purified using a Multiscreen PCRμ96 filter plate (Millipore, Tullagreen, Carrigtwohill, County Cork, UK).Table 3PCR Primers and Amplicon SizeExonAmpliconPrimer sequenceAmplicon size (bp)GC%11F: 5′-ACGTTTTCCCCAAGAACCTT-3′R: 5′-TTTAACCCTTGGCTTGAAAAA-3′585382*Amplicon that has not been validated.2F: 5′-GGCTTCCTCTAGCGTTTCCTCCT-3′R: 5′-ACGGAAAAGGCAAGTGATGA-3′5777433F: 5′-TCCGTGGAATATTCACTTCTCC-3′R: 5′-AGGGCGAGACTCAGTTTCAA-3′3433344F: 5′-TTGAATCTGCTTTACTTTCTGCTG-3′R: 5′-GCCCCAGAAAACCTCACATA-3′4753655F: 5′-TGTTGCAAATAGTGGGTTTCC-3′R: 5′-CCCACCAAAAGCAGAGAAAA-3′3913066F: 5′-TTCAGTCACTCTATTCACTCCTCTC-3′R: 5′-GAATTTGGGTAAGATGGCTTTT-3′3913977F: 5′-TTTAATGGCTCATGCTTTTTCA-3′R: 5′-TTGTAGGCGAAGAGGGAAAC-3′328368F: 5′-CAGTTTGCACCATTTGTTGG-3′R: 5′-GCCCACCCACACTGTTACAC-3′466419F: 5′-ATGAGCGGCCGAATTCTAT-3′R: 5′-GCTTTGGGGACAACATTCAT-3′4844410F: 5′-CAGTGGGTTCTTTGGCCTTA-3′R: 5′-CTGAACAGCAGCATCCACAT-3′4494711F: 5′-AGCTGTGACTGGGTCAGGTT-3′R: 5′-CCATTGAATTTCACAACAAAGG-3′48850812F: 5′-TCGGCTTAACTGACTTGAAAA-3′R: 5′-GCTTGAGCCATAAGAAATTGTTG-3′29932913F: 5′-CCTTCCAGTACTGTGTTATTTGTGA-3′R: 5′-AAGGACAAACCCTCCAAGTTT-3′4593714F: 5′-GATGCCACTTATCCCCAGAA-3′R: 5′-TGGGAGTTTGGCAGTATGGT-3′4753715F: 5′-CCGATAGCAGTGAGAAAGAGAAA-3′R: 5′-GGGTGGGAAATAGGTTCCTT-3′4943616F: 5′-TCCAAATGTGCTTGAGTGGT-3′R: 5′-ATTGCATGGCCTACATCACA-3′4984017F: 5′-GAAAGGCCTCCAAATTATCCA-3′R: 5′-CTGGGCAAGTGGACAAAACT-3′3843918F: 5′-CCAGCATATTTTGCTTGAGATTT-3′R: 5′-TAATGTCACAATAACAGCATTC-3′4653719F: 5′-CAGTGAACAAGAAAGCAAACAAA-3′R: 5′-TCCCTGGGTCTAGGAGATTC-3′4983820F: 5′-TTCTGGAAGAATACCCTTCAGTG-3′R: 5′-CTGTTGAGTTCTAAAGGACAGTCG-3′5003621F: 5′-CCATTTATAGATGTATTTGGCTGTCA-3′R: 5′-GTGGCGACTGTAAATCAGCA-3′4843822F: 5′-AGGCTGCTAAGCTCATGAAGA-3′R: 5′-TAACAGCAAAGCACCCATTG-3′4774523F: 5′-CATGTGGGGCAGTAGGAGTT-3′R: 5′-AGGCTGCTGAACCAACATCT-3′4924124F: 5′-GCTCATGGAAAAGGGAAAGA-3′R: 5′-ACTTTGCAACTCGTCCTTCG-3′4663825F: 5′-GGAGTTCTTCGTGTTACTCC-3′R: 5′-GTCAGTTGCTGCAAACATGG-3′4663926F: 5′-AAGGGATCCTAGAGCAAAGGA-3′R: 5′-TGCATTCTCAACTAGAATGAAGC-3′4633727F: 5′-CCAGGAGAATATCACCAATGC-3′R: 5′-CAAGGGCAATCATTGTAGCA-3′4863528F: 5′-TGGAGTCTCATTAGAGAAAAGAAAC-3′R: 5′-TTCTGTAAATGGCTGGTTGTTG-3′4983929F: 5′-TTCTGTAAATGGCTGGTTGTTGA-3′R: 5′-GCGCAGTTTGTCCAAAAGAT-3′4964230F: 5′-TGCACAATGTTCAGATTTCCTC-3′R: 5′-AATGCAGGCAGCTACTCCAC-3′4893831F: 5′-CGTGGCTAATTTGTAATAGATCAGG-3′R: 5′-TGCTTTCACTAGACAATATAAATCTGG-3′4914032F: 5′-CCCTGGTTCTGATCCAACAT-3′R: 5′-TGATATCAGCAGGGGTCACA-3′4943633F: 5′-CACGCAAAACAGTAGCAGAGA-3′R: 5′-TGGAAATGTCAAACACTTTTGC-3′4983434F: 5′-GATGCAAAACGACCCAAGTT-3′R: 5′-GCTTTCATTAGAGCATGAAAAAC-3′4813635F: 5′-CTGTTTCAGCCAACCAGCTT-3′R: 5′-TGTCCATTTCTCCACTTCAGC-3′4733936F: 5′-TTGAATCATTTGATGTCCCAAG-3′R: 5′-GGAATAAACAATTTTTCAGGAAGC-3′4943437F: 5′-CAAGTGCTGAGGAATTATCAGAGA-3′R: 5′-GGCCCGCTCAGGACATAA-3′4643338F: 5′-TCCTTCTGCATCATATATTCCAA-3′R: 5′-CCTCGCAACTGAAAACGAA-3′4993839F: 5′-ATGCAACATAACGACGTTGG-3′R: 5′-AAGCGCAAGGTCTCGTACAT-3′4573840F: 5′-TGAGGGCAAACAATTAGATCC-3′R: 5′-TGGCCAGAAAAGCATTATCA-3′4974041F: 5′-TTTGTCAGTTTGGAGCGTTG-3′R: 5′-TTTTGATGGCTCCGAAGAGT-3′4853642F: 5′-GGCAATGACTCTTAAATCAGCA-3′R: 5′-GGCTGGTTGGTGTAGAAGGA-3′4853843F: 5′-TGCTGACAATTCTAGTTTTCTAGG-3′R: 5′-GAGGCCCTGTCTGCATTTT-3′4864044F: 5′-TGAAATGGCATCCTGACAAA-3′R: 5′-TCAGACTTTCCCCTCACAGC-3′4904245F: 5′-CACGCAGATGGCTAAGACAA-3′R: 5′-TGGCAATGAAGCTTAATGAAGTA-3′48339* Amplicon that has not been validated. Open table in a new tab For fragment 2 (exon 2) a different PCR program and amplification mix is used; 0.5 μl (8.33 mmol/L) dNTPs, 3.0 μl MgCl2, 3.0 μl PCR Gold Buffer, 7.5 μl Betaine, 0.2 μl Tag DNA polymerase Gold, 13.8 mQ, 1 μl FP and RP (10 pmol/μl) with 1.0 μl genomic DNA in a total reaction volume of 30 μl per sample. The PCR program is as follows: initial denaturation at 94°C for 5 minutes, then 38 cycles of denaturation at 98°C for 5 seconds, annealing at 68°C for 30 seconds, and elongation at 72°C for 50 seconds, with the final step at 72°C for 7 minutes, after which the samples are cooled down to 10°C. During CSCE, the target gene is PCR-amplified and PCR products are denatured and re-annealed slowly, so that four different double stranded (ds) DNA molecules, ie, two homoduplexes and two heteroduplexes, can be formed if a heterozygous mutation is present. To identify conformational changes resulting in a mobility shift, the re-annealed PCR products can be analyzed using any capillary DNA sequencer, and by using non-denaturing conditions, ie, an ABI 3730 sequencer with conformation analysis polymer-containing capillaries. The re-annealed PCR products can be detected by using a four-dye filter set, and fluorescently labeled primers. The presence of many SNPs in the gene of interest will lead to the identification of these as DNA variants in several samples, depending on their allele frequency. These DNA variants will all have to be subsequently analyzed by automated sequencing. CSCE therefore is most suited for genes containing just a limited number of (common) SNPs. The peak patterns are analyzed automatically by specially designed software (BioNumerics, Applied Maths, St-Martens-Latem, Belgium). Before CSCE, 5 μl purified PCR-product was mixed with 2 μl PCR-product of a mutation-negative reference sample (V3). For the amplicons 7 and 10 of the SACS gene a different reference sample (V4) was used since the V3 sample was heterozygous for common SNPs in these amplicons. The labeling-PCR mix contained 0.2 μl 6-carboxyfluorescein-labeled FP, 0.2 μl reverse M13 primer, 0.5 μl dNTPs (8.33 mmol/L), 1.5 μl MgCl2 (50 mmol/L, AB), 2.5 μl 10× PCR Gold Buffer (Applied Biosystems, Inc, Foster City, CA), 0.2 μl TaqDNA polymerase Gold (5 U/μl), and 17.9 μl mQ. Of this mix 18 μl was used per sample for the labeling-PCR. The following PCR program was used for the labeling-PCR: an initial denaturation at 94°C for 5 minutes, followed by 10 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and elongation for 1 minute at 72°C, with a final step at 72°C for 7 minutes after which the samples are cooled down to 10°C. After the labeling PCR, the products (25 μl) are again purified as described before. Per sample 1 μl pooled PCR product, was mixed with 9 μl standard LIZ-500 (Applied Biosystems)/mQ-mix (in proportion 0.2 μl Liz to 8.8 μl mQ), before running the actual CSCE. The analysis was performed using an ABI 3730 automated sequencer (Applied Biosystems) using capillaries containing conformation analysis polymer. Results were analyzed using BioNumerics 5.0 (Applied Maths NV, Sint-Martens-Latem, Belgium) software. We choose to run four patient samples in a single CSCE run. To automatically analyze the CSCE electropherograms of the different amplicons of the SACS gene by the data analysis software program Bionumerics an optimized set of parameters (quantified characteristics of the shapes of the electropherogram) has been used. The parameters include (1) the relative position of the peak within the electropherogram, and (2) the signal-width as a percentage of the run length, and (3) the left and right control range, ie, the area flanking the main peak signal in which the algorithm will search for additional peaks. To further characterize the shape of the peaks (curves), the algorithm calculates five different pattern-matching parameters, ie, SRMS, MAX, SECPK, DFH3, and DFH4. SRMS stands for single-sided root mean square value, and is calculated from the highest 50% of square differences between the curves compared. MAX determines the highest difference between any corresponding values on the two curves. SECPK calculates the proportion of a secondary peak relative to the primary peak of the curve. DFH3 is a parameter for the difference in H3 parameters between the signal and the reference (H3 is a measure of the deviation from symmetry, compared with a Gaussian curve derived from reference samples without DNA variants), whereas DFH4 determines the deviation from a theoretical Gaussian curve. The parameters SRMS and MAX take into account any difference between the curves, the parameters SECPK, DFH3, and DFH4 each apply to a specific component of the curve. For a correct match interpretation a peak height has to be above 1000 and below 32000 relative fluorescence units. Peak heights outside this range will be regarded as failures by the BioNumerics program. In this validation study, all samples have been analyzed in parallel by CSCE and by automated sequencing. Sequence reactions were run on an ABI 3730 automated sequencer and analyzed with a combined software program: Phred, Phrap, and Polyphred (University of Washington). In affected siblings or relatives of patients with one homozygous or two compound heterozygous mutations in the SACS gene, the presence of the mutations was identified or excluded using direct sequencing. Due to the occurrence of two relatively frequent polymorphisms in amplicon 35, which are heterozygous in both reference samples V3 and V4, direct sequencing is preferred over CSCE for this amplicon, since aberrant heteroduplex patterns will occur in the majority of cases. As the PCR-mix and program conditions for exon 2 of the SACS gene differ substantially from standard conditions, CSCE conditions for this specific exon have not been developed. Primers were designed (a) to allow for an identical annealing temperature for all 45 amplicons, except for amplicon 2 (exon 2) for which a different annealing temperature is used because of the high CG-content of this fragment, (b) to obtain amplicon sizes ranging from 299 to 585 bp in length, (c) to allow for a distance of at least 30 bp between the 5′-end of a particular primer and the nearest exon-intron boundary within the amplicon, and (d) to have at least 100 bp overlapping sequence for neighboring amplicons of the same exon. An overlap will ensure the identification of a heterozygous deletion encompassing a single or multiple amplicons that could otherwise escape detection. Furthermore a minimum of 30 bp between the beginning of a primer and an exon-intron boundary are present in each amplicon, where applicable. To enable a rapid confirmation by automated sequencing of sequence variants identified by CSCE, the amplicons to be analyzed by CSCE were identical to the amplicons to be analyzed by sequencing. This approach also reduces the consumable costs, since identical primer pairs for both CSCE as well as sequencing can be used. This method is feasible, since there is no need to split amplicons into smaller fragments to comply with the presence of multiple melting domains, like in denaturing gradient gel electrophoresis- or denaturing high performance liquid chromatography-based approaches. To automatically analyze the CSCE electropherograms of the different amplicons of the SACS gene by the data analysis software program Bionumerics an optimized set of parameters (quantified characteristics of the shapes of the electropherogram) has been used that was developed during a separate technical validation of CSCE. Essentially, the position and shape of the peak of a normal (no DNA variant-containing) reference electropherogram is compared with the peak positions and shapes of the electropherogram to be analyzed. The parameters used are described in the Materials and Methods. A generic technical validation of the experimental conditions of CSCE and the optimal general settings of the software program BioNumerics has been performed previously. In this collaborative study (Interlaboratory Diagnostic Validation of Confirmation Sensitive Capillary Electrophoresis, to be published in detail elsewhere) parameters including (1) CG-content of fragments, (2) position of the DNA-variant relative to the primers, (3) fragment length, and (4) temperature during electrophoresis have been analyzed. The main results are that (1) CG-content of 30% to 60% gives optimal results, whereas for amplicons with a CG-content up to 80% different conditions may be necessary (2), SNPs close to a primer may escape detection, an issue that can be circumvented by the design of the analytical strategy, eg, by taking sufficient overlap of amplicons into account, (3) length of amplicons up to 500 bp can still be analyzed reliably, and (4) cooling of the oven temperature below 15°C, which improves separation of heteroduplex bands.6Mattocks C, Watkins G, Janssens T, Matthijs G, Bosgoed E, Donck van der K, Scheffer H, Pot B, Theelen J, Aspholm T, Cross N: Inter-laboratory diagnostic validation of confirmation sensitive capillary electrophoresis. Poster presented at the Annual Meeting of the European Society of Human Genetics, 2008 Jun 3, Barcelona, SpainGoogle Scholar As a result of the design and analytical validation of the CSCE system for mutation scanning of the SACS gene presented in this paper, the five pattern matching parameters were set at specific lower and upper threshold values (Table 4). Patterns with parameter values above one or more of these thresholds are considered to be divergent (indicated by red) and patterns with one or more parameter values below a certain threshold are considered to be normal (indicated by green). Patterns with one or more parameter values between these two thresholds are slightly divergent (indicated by orange). These patterns always need to be visually inspected.Table 4Peak Matching ParametersThresholdParameterGreen (II)Green (I)OrangeRedSRMS<3.50 20.00MAX<3.50 20.00SECPK<200.00 400.00DFH3<1.00 1.70DFH4<0.70 1.20The parameters and settings are described in the Materials and Methods. Peaks indicated in green should be considered as normal and do not require subsequent sequencing. Green II indicates the adapted parameters. Peaks indicated in orange need further visual inspection and peaks indicated in red always require sequencing. Open table in a new tab The parameters and settings are described in the Materials and Methods. Peaks indicated in green should be considered as normal and do not require subsequent sequencing. Green II indicates the adapted parameters. Peaks indicated in orange need further visual inspection and peaks indicated in red always require sequencing. For analyt