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• W2040571565 abstract "Prader-Willi syndrome and Angelman syndrome are distinct neurodevelopmental disorders that are associated with the deletion of the chromosomal 15q11-13 region or uniparental disomy of chromosome 15. In this article, we applied SYBR Green I-based real-time PCR and melting curve analysis assay for rapid genotyping of the small nuclear ribonucleoprotein polypeptide N (SNRPN) gene methylation status and for detecting aberrations in copy number in a single tube. A single pair of primers was designed to create a 357 bp fragment containing the cytosine phosphodiester guanine islands in the SNRPN promoter and to amplify both unmethylated and methylated sequences. Genotypes were identified based on the TC value for copy number changes and the characteristic melting temperature of methylated cytosine phosphodiester guanine. Genotyping of SNRPN was performed on blood samples of 20 individuals with Prader-Willi syndrome, 3 individuals with Angelman syndrome, and 20 unaffected individuals. The promoter methylation status and the copy number changes were successfully determined and compared with standard methylation-specific PCR, and were validated by multiplex ligation-dependent probe amplification. This single-tube, SYBR Green I, real-time PCR with melting curve assay is rapid, reliable, sensitive, and easy to perform. It is suitable for high-throughput analysis as an alternative technique for quantitative and qualitative analysis of target genes. Prader-Willi syndrome and Angelman syndrome are distinct neurodevelopmental disorders that are associated with the deletion of the chromosomal 15q11-13 region or uniparental disomy of chromosome 15. In this article, we applied SYBR Green I-based real-time PCR and melting curve analysis assay for rapid genotyping of the small nuclear ribonucleoprotein polypeptide N (SNRPN) gene methylation status and for detecting aberrations in copy number in a single tube. A single pair of primers was designed to create a 357 bp fragment containing the cytosine phosphodiester guanine islands in the SNRPN promoter and to amplify both unmethylated and methylated sequences. Genotypes were identified based on the TC value for copy number changes and the characteristic melting temperature of methylated cytosine phosphodiester guanine. Genotyping of SNRPN was performed on blood samples of 20 individuals with Prader-Willi syndrome, 3 individuals with Angelman syndrome, and 20 unaffected individuals. The promoter methylation status and the copy number changes were successfully determined and compared with standard methylation-specific PCR, and were validated by multiplex ligation-dependent probe amplification. This single-tube, SYBR Green I, real-time PCR with melting curve assay is rapid, reliable, sensitive, and easy to perform. It is suitable for high-throughput analysis as an alternative technique for quantitative and qualitative analysis of target genes. Prader-Willi syndrome (PWS) [OMIM (Online Mendelian Inheritance in Man) No. 176270] is a complex neurodevelopmental disorder. Individuals with PWS exhibit infantile hypotonia, mild-to-moderate mental retardation, hyperphagia with obesity, hypogonadism, mild dysmorphism, and obsessive-compulsive behavior.1Gunay-Aygun M. Schwartz S. Heeger S. O'Riordan M.A. Cassidy S.B. The changing purpose of Prader-Willi syndrome clinical diagnostic criteria and proposed revised criteria.Pediatrics. 2001; 108: E92Crossref PubMed Scopus (379) Google Scholar, 2Holm V.A. Cassidy S.B. Butler M.G. Hanchett J.M. Greenswag L.R. Whitman B.Y. Greenberg F. Prader-Willi syndrome: consensus diagnostic criteria.Pediatrics. 1993; 91: 398-402PubMed Google Scholar Phenotypic features of individuals with Angelman syndrome (AS) (OMIM No. 105830) include severe mental retardation with little or no speech, ataxia, frequent seizures, microbrachycephaly, and inappropriately happy affect.3Williams C.A. Beaudet A.L. Clayton-Smith J. Knoll J.H. Kyllerman M. Laan L.A. Magenis R.E. Moncla A. Schinzel A.A. Summers J.A. Wagstaff J. Angelman syndrome 2005: updated consensus for diagnostic criteria.Am J Med Genet A. 2006; 140: 413-418Crossref PubMed Scopus (428) Google Scholar These two distinct severe neurobehavioral diseases are the most common genomic imprinting genetic disorders and arise from the loss of a cluster of functional genes with either paternal or maternal imprinting at chromosome 15q11-13.4Buiting K. Saitoh S. Gross S. Dittrich B. Schwartz S. Nicholls R.D. Horsthemke B. Inherited microdeletions in the Angelman and Prader-Willi syndromes define an imprinting centre on human chromosome 15.Nat Genet. 1995; 9: 395-400Crossref PubMed Scopus (516) Google Scholar Approximately 70% of PWS cases are associated with a de novo paternally derived deletion in 15q11-13; approximately 25% of PWS cases are derived from maternal uniparental disomy (UPD) of chromosome 15 (UPD15), and the remaining approximate 5% with sequence variants in the imprinting gene.5Fridman C. Varela M.C. Kok F. Setian N. Koiffmann C.P. Prader-Willi syndrome: genetic tests and clinical findings.Genet Test. 2000; 4: 387-392Crossref PubMed Scopus (23) Google Scholar, 6Glenn C.C. Driscoll D.J. Yang T.P. Nicholls R.D. Genomic imprinting: potential function and mechanisms revealed by the Prader-Willi and Angelman syndromes.Mol Hum Reprod. 1997; 3: 321-332Crossref PubMed Scopus (116) Google Scholar Approximately 70% to 80% of AS cases result from deletions of 15q11-13 in the maternal allele, with approximately 11% due to mutation of the ubiquitin protein ligase E3A (UBE3A) gene, approximately 7% caused by paternal UPD15, and the remaining percent having sequence variation in the imprinting gene.6Glenn C.C. Driscoll D.J. Yang T.P. Nicholls R.D. Genomic imprinting: potential function and mechanisms revealed by the Prader-Willi and Angelman syndromes.Mol Hum Reprod. 1997; 3: 321-332Crossref PubMed Scopus (116) Google Scholar, 7Fang P. Lev-Lehman E. Tsai T.F. Matsuura T. Benton C.S. Sutcliffe J.S. Christian S.L. Kubota T. Halley D.J. Meijers-Heijboer H. Langlois S. Graham Jr., J.M. Beuten J. Willems P.J. Ledbetter D.H. Beaudet A.L. The spectrum of mutations in UBE3A causing Angelman syndrome.Hum Mol Genet. 1999; 8: 129-135Crossref PubMed Scopus (132) Google Scholar, 8Lossie A.C. Whitney M.M. Amidon D. Dong H.J. Chen P. Theriaque D. Hutson A. Nicholls R.D. Zori R.T. Williams C.A. Driscoll D.J. Distinct phenotypes distinguish the molecular classes of Angelman syndrome.J Med Genet. 2001; 38: 834-845Crossref PubMed Scopus (277) Google Scholar Nearly all the cytosine phosphodiester guanine dinucleotides around the promoter region of the small nuclear ribonucleoprotein polypeptide N (SNRPN) gene are methylated on the maternal chromosome and completely devoid of methylation on the paternal chromosome.9Dittrich B. Buiting K. Korn B. Rickard S. Buxton J. Saitoh S. Nicholls R.D. Poustka A. Winterpacht A. Zabel B. Horsthemke B. Imprint switching on human chromosome 15 may involve alternative transcripts of the SNRPN gene.Nat Genet. 1996; 14: 163-170Crossref PubMed Scopus (209) Google Scholar The promoter methylation status of SNRPN is recognized as a valid diagnostic characteristic.10Kubota T. Sutcliffe J.S. Aradhya S. Gillessen-Kaesbach G. Christian S.L. Horsthemke B. Beaudet A.L. Ledbetter D.H. Validation studies of SNRPN methylation as a diagnostic test for Prader-Willi syndrome.Am J Med Genet. 1996; 66: 77-80Crossref PubMed Scopus (78) Google Scholar SNRPN, therefore, is a candidate gene for PWS and AS. Based on this important finding, different methods are reported for methylation analyses of PWS and AS, such as a methylation-sensitive enzyme,11Velinov M. Gu H. Genovese M. Duncan C. Brown W.T. Jenkins E. The feasibility of PCR-based diagnosis of Prader-Willi and Angelman syndromes using restriction analysis after bisulfite modification of genomic DNA.Mol Genet Metab. 2000; 69: 81-83Crossref PubMed Scopus (12) Google Scholar methylation-specific PCR,12Kosaki K. McGinniss M.J. Veraksa A.N. McGinnis W.J. Jones K.L. Prader-Willi and Angelman syndromes: diagnosis with a bisulfite-treated methylation-specific PCR method.Am J Med Genet. 1997; 73: 308-313Crossref PubMed Scopus (55) Google Scholar microsatellite analysis,4Buiting K. Saitoh S. Gross S. Dittrich B. Schwartz S. Nicholls R.D. Horsthemke B. Inherited microdeletions in the Angelman and Prader-Willi syndromes define an imprinting centre on human chromosome 15.Nat Genet. 1995; 9: 395-400Crossref PubMed Scopus (516) Google Scholar melting curve analysis,13White H.E. Hall V.J. Cross N.C. Methylation-sensitive high-resolution melting-curve analysis of the SNRPN gene as a diagnostic screen for Prader-Willi and Angelman syndromes.Clin Chem. 2007; 53: 1960-1962Crossref PubMed Scopus (85) Google Scholar denaturing high-performance liquid chromatography,14Baumer A. Wiedemann U. Hergersberg M. Schinzel A. A novel MSP/denaturing high-performance liquid chromatography method for the investigation of the methylation status of imprinted genes enables the molecular detection of low cell mosaicisms.Hum Mutat. 2001; 17: 423-430Crossref PubMed Scopus (60) Google Scholar and pyrosequencing.15White H.E. Durston V.J. Harvey J.F. Cross N.C. Quantitative analysis of SNRPN (correction of SRNPN) gene methylation by pyrosequencing as a diagnostic test for Prader-Willi syndrome and Angelman syndrome.Clin Chem. 2006; 52: 1005-1013Crossref PubMed Scopus (57) Google Scholar Most of these methods, however, are either quantitative or qualitative analyses that determine the presence of a deletion or UPD nondeletion. Melting curve analysis is a high throughput, rapid, and sensitive method that has become common for detection of sequence variants in the specific gene.16Wittwer C.T. High-resolution DNA melting analysis: advancements and limitations.Hum Mutat. 2009; 30: 857-859Crossref PubMed Scopus (366) Google Scholar This method has high sensitivity and cost-effectiveness, which allows as much as 384 samples to be simultaneously screened. The method identifies DNA variant patterns and copy number changes.17Vossen R.H. Aten E. Roos A. den Dunnen J.T. High-resolution melting analysis (HRMA): more than just sequence variant screening.Hum Mutat. 2009; 30: 860-866Crossref PubMed Scopus (385) Google Scholar We developed a SYBR Green I-based real-time PCR method, a fluorescent intercalating double-stranded DNA-specific binding dye for detection of SNRPN gene copy number and combined use of the melting curve analysis that PCR products are detected by their characteristic melting curve profiles. This simple approach concurrently determines copy number and methylation status in a single tube. In this article, we combined SYBR Green I-based real-time PCR and melting curve analysis with a single pair of primers for rapid genotyping of SNRPN methylation status, as well as copy number aberrations. The alternative design improved efficiency of the quantitative and qualitative assays in a single tube for genotyping of PWS and AS patients. All study DNA samples were obtained from the National Taiwan University Hospital and the Mackay Memorial Hospital. A total of 20 individuals with a diagnosis of PWS syndrome, 3 individuals with a diagnosis of AS syndrome, and 20 unaffected controls with the same ethnic background were analyzed. Genotypes were confirmed by methylation specific PCR, fluorescence in situ hybridization, and Methylation-specific multiplex ligation-dependent probe amplification. Genomic DNA was extracted from peripheral whole blood using the Chemagic DNA Blood Kit (Chemagen, Baesweiler, Germany) according to the manufacturer's instructions. This study was approved by the Ethics Committee of National Taiwan University Hospital. The MethylCode Bisulfite Conversion Kit (Invitrogen, Carlsbad, CA) was used to treat 1 μg of genomic DNA with bisulfite according to the manufacturer's instructions. The reaction causes the conversion of unmethylated cytosine to uracil, without changing the status of methylated cytosine. PCR was performed in a final volume of 10 μL containing 50 ng of bisulfite-treated DNA, 0.5 mmol/L of each primer (sense: 5′-GGTTTTAGGGGTTTAGTAGT-3′ and anti-sense: 5′-CCCAAATTCCATTTATTCAA-3′), 2 mmol/L MgCl2 as provided by the manufacturer, 1× FastStart LightCycler DNA master SYBR Green I (Roche Applied Science, Indianapolis, IL), which contained reaction buffer, deoxyribonucleotide triphosphate mix, SYBR Green I binding dye, FastStart TaqDNA polymerase, and 1 mmol/L of MgCl2 (final concentration). Real-time PCR was conducted in a LightCycler 480 instrument (Roche Applied Science). The LightCycler protocol began with an initial denaturation step at 95°C for 10 minutes, followed by 50 cycles with a denaturing temperature of 94°C for 10 seconds, annealing at 57°C for 5 seconds, and extension at 72°C for 20 seconds, with a ramping rate of 4.4°C per second. All primers were designed using LightCycler Probe Design Software 2.0 (Roche Applied Science). After PCR amplification, the PCR products were completely denatured at 95°C, cooled to 65°C at a thermal transition rate of 4.4°C per second, and then heated to 95°C at a thermal transition rate of 2.2°C per second with continuous fluorescence monitoring in the F1 channel. LightCycler 480 software, version 1.3 (Roche Applied Science) was used to analyze the data. The y axis represented the derivative of fluorescence changes and the x axis represented the temperature (°C). In a previous study, we undertook melting curve analysis to detect mutations in the FBN1 and FGFR3 genes by generating 300 to 500 bp PCR products.18Hung C.C. Lin S.Y. Lee C.N. Cheng H.Y. Lin C.Y. Chang C.H. Chiu H.H. Yu C.C. Lin S.P. Cheng W.F. Ho H.N. Niu D.M. Su Y.N. Identification of fibrillin-1 gene mutations in Marfan syndrome by high-resolution melting analysis.Anal Biochem. 2009; 389: 102-106Crossref PubMed Scopus (10) Google Scholar, 19Hung C.C. Lee C.N. Chang C.H. Jong Y.J. Chen C.P. Hsieh W.S. Su Y.N. Lin W.L. Genotyping of the G1138A mutation of the FGFR3 gene in patients with achondroplasia using high-resolution melting analysis.Clin Biochem. 2008; 41 (g): 162-166Crossref PubMed Scopus (28) Google Scholar These amplified lengths are suitable for melting curve analysis with high sensitivity, thus, we planned to amplify similar PCR products in this study. The designed allele-specific primers were tested by regular PCR. After bisulfite treatment, the methylation PCR was performed at 57°C, which is the optimal annealing temperature. All samples were optimized to yield a single, clear band of expected 357 bp using a single set of primers to amplify the unmethylated, as well as methylated alleles (data not shown). To evaluate the SYBR Green I-based real-time PCR assay, a total of 20 patients with PWS, 3 patients with AS, and 20 unaffected individuals with known genotypes (20 wild-type cases, 8 PWS cases with deletion type, 12 PWS cases with UPD type, and 3 AS cases with deletion type) were tested using real-time PCR. All genotypes were previously confirmed by analyses of standard methylation-specific PCR and fluorescence in situ hybridization. There was no case of AS with the UPD type in our study due to the very small number of AS patients, predominantly caused by paternal UPD15. The results of the SYBR green I-based real-time PCR assay on bisulfite-treated DNA for SNRPN genotyping with allele-specific primer sets are shown in Figure 1. In the study, we used only one pair of primers to amplify both alleles of the promoter region of SNRPN. The fluorescent signals increased on the amplification curve in all samples. The threshold cycle (Ct) values correlated with the deleted and nondeleted status of specific alleles in the promoter region of the SNRPN gene. The copy number distributions of the SNRPN gene are shown in Figure 2. The deletion type for the PWS and AS samples resulted in TC values of approximately 29.33 ± 0.13 (eight cases) and 29.43 ± 0.12 (three cases), respectively (mean ± SD). The wild type and PWS with UPD type samples showed TC values of approximately 28.37 ± 0.22 (20 cases) and 28.27 ± 0.08 (12 cases), respectively. There was no AS case with UPD type in our study.Figure 2The scatter plots of the calculated copy numbers by SYBR green-I–based real-time PCR are illustrated for the wild type, Prader-Willi syndrome (PWS) with deletion, PWS with uniparental disomy, and Angelman syndrome (AS) with deletion. UPD, uniparental disomy.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The negative derivative of the fluorescence (F) with respect to temperature (T) versus temperature, which can distinguish the genotype by differential melting temperatures on melting curve analysis, is shown in Figure 3. The melting curve of the wild-type sample showed two marked peaks at Tm = 85°C and Tm = 89°C, respectively. The peak at the lower melting temperature (Tm = 85°C) corresponded to the paternal allele (unmethylated). The maternal allele (methylated) increased the stability of the heteroduplex structure due to the guanine-cytosine rich (GC rich) domain, resulting in the peak at a higher melting temperature (Tm = 89°C). In contrast, the melting curve of the PWS and AS cases showed a single peak at either Tm = 85°C or Tm = 89°C, which belonged to the loss of one of the parental alleles. The Tm of the unmethylated and methylated patterns for SNRPN demonstrated clearer distinction of sequence variation in a derivative plot and in the fluorescence difference plot. By melting curve analysis, all 43 PWS, AS cases, and wild-type samples were successfully identified. PWS and AS are distinct neurodevelopmental disorders and the majority of cases result from chromosome 15q11-q13 deletion or UPD15. The genotype of the deletion in the derived PWS and AS regions or chromosome translocation breaking within the PWS and AS regions can be identified using fluorescence in situ hybridization analysis. Although fluorescence in situ hybridization is the traditional identification method, it cannot be performed in a high throughput manner. Recently, multiplex PCR for the identification of PWS and AS deletions was developed in our laboratory.20Hung C.C. Chen C.P. Lin S.P. Chien S.C. Lee C.N. Cheng W.F. Hsieh W.S. Liu M.S. Su Y.N. Lin W.L. Quantitative assay of deletion or duplication genotype by capillary electrophoresis system: application in Prader-Willi syndrome and Duchenne muscular dystrophy.Clin Chem. 2006; 52: 2203-2210Crossref PubMed Scopus (18) Google Scholar It is easier and faster than the traditional diagnostic approaches. The principle involves three amplifications of a target gene and two reference genes in a single tube. PWS and AS deletions involving large fragments in patients can be identified by multiplex PCR. However, the limitation of multiplex PCR is that only the deletion type of PWS and AS can be identified. To resolve this problem, we designed methylation-specific multiplex PCR to determine copy number changes and methylation status in a single tube.21Hung C.C. Lin S.Y. Lin S.P. Niu D.M. Lee N.C. Cheng W.F. Chen C.P. Lin W.L. Lee C.N. Su Y.N. Identification of cytosine phosphodiester guanine methylation of the SNRPN gene by methylation-specific multiplex PCR.Electrophoresis. 2009; 30: 410-416Crossref PubMed Scopus (5) Google Scholar Recently, the melting curve analysis system was considered a promising tool for detecting sequence variation by the use of a saturating double-stranded DNA dye. Methylation-specific PCR and quantitative melting curve analysis can discriminate between deletional and nondeletional PWS and AS,22Wang W. Law H.Y. Chong S.S. Detection and discrimination between deletional and non-deletional Prader-Willi and Angelman syndromes by methylation-specific PCR and quantitative melting curve analysis.J Mol Diagn. 2009; 11: 446-449Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar and it is based on a single-step procedure. The advantage of our method compared with others is that it uses TC analysis of real-time PCR rather than relative quantitative PCR to detect deletions in SNRPN. Moreover, potential advantages of the assay are that it is relatively easy, fast, and inexpensive; it is capable of being performed in a single closed-tube system; it uses a single primer pair to amplify both maternal and paternal alleles; it is capable of handling a large volume of samples. However, the major limitation of the method is that methylation mosaics are known to represent a significant proportion of AS patients, and it is not clear how such samples would perform in this assay. In this study, we used bisulfite-treated DNA and only one pair of primers to amplify the SNRPN promoter region. We extended the application of real-time PCR and high resolution melting curve analysis for both quantitative and qualitative genotyping of SNRPN easily and accurately. Real-time PCR for the quantitative assay resulted in different TC values for identification of copy number changes. The PWS and AS cases with deletion genotypes had greater TC values. The melting curve for the qualitative assay is based on different melting temperatures for wild type and PWS and AS patients, because the DNA base pair sequences are changed after bisulfite treatment. The wild type had one unmethylated paternal allele, as well as one methylated maternal allele. Because of deletion of the UPD of one parental allele, only one melting peak was observed for PWS and AS patients. Our results were 100% compatible with standard methylation PCR. More importantly, our assay method relies on only one pair of the primers, which enables each sample to be easily genotyped with a single reaction. The method is a closed tube system, using only one PCR reaction without enzyme digestion. We established that the real-time PCR method had the advantage of a single technology sufficient to distinguish the deletion type from the UPD type of PWS. Furthermore, the melting curve analysis protocol distinguished PWS and AS samples from the wild-type samples after real-time PCR. The analysis is performed within a single tube and single machine, and thus it is easier, faster, and avoids potential contamination. Furthermore, the technique is an in-house protocol, and hence we can design primers in any desired target region. The analysis is also flexible and facilitates the study of other genetic disorders by copy number and methylation variants." @default.
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• W2040571565 title "Quantitative and Qualitative Analyses of the SNRPN Gene Using Real-Time PCR with Melting Curve Analysis" @default.
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