Detection and Discrimination between Deletional and Non-Deletional Prader-Willi and Angelman Syndromes by Methylation-Specific PCR and Quantitative Melting Curve Analysis
2009; Elsevier BV; Volume: 11; Issue: 5 Linguagem: Inglês
10.2353/jmoldx.2009.090015
ISSN1943-7811
AutoresWen Wang, Hai‐Yang Law, Samuel S. Chong,
Tópico(s)Prenatal Screening and Diagnostics
ResumoPrader-Willi syndrome (PWS) and Angelman syndrome (AS) are clinically distinct neurological disorders caused by a lack of expression of oppositely imprinted genes in chromosomal region 15q11-13. The loss of expression can be due to parent-specific segmental deletions or can arise from non-deletional mechanisms, such as uniparental disomy of chromosome 15 or defects in imprinting. Most current diagnostic methods to distinguish PWS from AS require separate amplification and detection steps, and some methods cannot differentiate between deletional and non-deletional forms of these syndromes. We have developed a single-step, methylation-specific PCR, and quantitative melting curve analysis assay to identify methylation differences and copy number changes in PWS and AS. In this strategy, duplex amplification followed by melting curve analysis was performed to detect the maternally and paternally imprinted SNRPN alleles and LIS1 reference gene. To discriminate between deletional and non-deletional PWS and AS, relative peak height ratios of maternal or paternal SNRPN:LIS1 were determined, respectively. To validate the diagnostic accuracy of the analysis, methylation-specific multiplex ligation-dependent probe amplification was performed on all PWS and AS samples. Complete concordance between the melting curve analysis and methylation-specific multiplex ligation-dependent probe amplification results was observed for all PWS and AS samples. Methylation-specific PCR and quantitative melting curve analysis represents a simple, rapid, and robust alternative to methylation-specific multiplex ligation-dependent probe amplification for the detection of and discrimination between deletional and non-deletional forms of PWS and AS. Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are clinically distinct neurological disorders caused by a lack of expression of oppositely imprinted genes in chromosomal region 15q11-13. The loss of expression can be due to parent-specific segmental deletions or can arise from non-deletional mechanisms, such as uniparental disomy of chromosome 15 or defects in imprinting. Most current diagnostic methods to distinguish PWS from AS require separate amplification and detection steps, and some methods cannot differentiate between deletional and non-deletional forms of these syndromes. We have developed a single-step, methylation-specific PCR, and quantitative melting curve analysis assay to identify methylation differences and copy number changes in PWS and AS. In this strategy, duplex amplification followed by melting curve analysis was performed to detect the maternally and paternally imprinted SNRPN alleles and LIS1 reference gene. To discriminate between deletional and non-deletional PWS and AS, relative peak height ratios of maternal or paternal SNRPN:LIS1 were determined, respectively. To validate the diagnostic accuracy of the analysis, methylation-specific multiplex ligation-dependent probe amplification was performed on all PWS and AS samples. Complete concordance between the melting curve analysis and methylation-specific multiplex ligation-dependent probe amplification results was observed for all PWS and AS samples. Methylation-specific PCR and quantitative melting curve analysis represents a simple, rapid, and robust alternative to methylation-specific multiplex ligation-dependent probe amplification for the detection of and discrimination between deletional and non-deletional forms of PWS and AS. Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two distinct neurological disorders caused by lack of expression of oppositely imprinted genes in chromosomal region 15q11-q13.1Jiang Y Tsai TF Bressler J Beaudet AL Imprinting in Angelman and Prader-Willi syndromes.Curr Opin Genet Dev. 1998; 8: 334-342Crossref PubMed Scopus (103) Google Scholar,2Nicholls RD Saitoh S Horsthemke B Imprinting in Prader-Willi and Angelman syndromes.Trends Genet. 1998; 14: 194-200Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar Absence of a paternally contributed allele leads to PWS whereas absence of a maternally imprinted allele results in AS. Parent-specific segmental deletions, uniparental disomy of chromosome 15 (UPD15), and imprinting defects together constitute the most common causal mechanisms in both PWS and AS. Approximately 70% to 75% of PWS results from a deletion of the paternal chromosome 15q11-q13 region, while another 25% to 29% is caused by maternal UPD15. Imprinting defects and balanced translocations involving breaks in the 15q11-q13 region each account for less than 1% of PWS. Therefore, greater than 99% of PWS is associated with a detectable methylation abnormality in the 15q11-q13 imprinted region (http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=pws; accessed March 24, 2009). In contrast, ∼68% of AS results from a deletion of the maternal chromosome 15q11-q13 region, while paternal UPD15 and imprinting defects account for another ∼7% and ∼3%, respectively. As such, ∼78% of AS is associated with a detectable methylation abnormality in the 15q11-q13 imprinted region (http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=angelman; accessed March 24, 2009). Of the remaining ∼22% of AS patients, whose methylation profiles are normal, approximately half carry a mutation in the UBE3A ubiquitin-ligase gene, while the molecular basis for the remaining patients is undetermined. Small nuclear ribonucleoprotein polypeptide N (SNRPN) is one of several paternally expressed genes in this region, and its parent-of-origin specific methylation has been recognized as a diagnostic indicator of PWS and AS.3Diagnostic testing for Prader-Willi and Angleman syndromes Report of the ASHG/ACMG Test and Technology Transfer Committee.Am J Hum Genet. 1996; 58: 1085-1088PubMed Google Scholar,4Zeschnigk M Schmitz B Dittrich B Buiting K Horsthemke B Doerfler W Imprinted segments in the human genome: different DNA methylation patterns in the Prader-Willi/Angelman syndrome region as determined by the genomic sequencing method.Hum Mol Genet. 1997; 6: 387-395Crossref PubMed Scopus (112) Google Scholar Apart from widely used cytogenetic methods involving high-resolution chromosome studies and fluorescence in situ hybridization for detection of deletional PWS/AS, several molecular diagnostic assays have been developed based on the study of allelic methylation differences at the SNRPN locus, for instance, methylation-specific PCR (MS-PCR),5Buller A Pandya A Jackson-Cook C Bodurtha J Tekin M Wilkinson DS Garrett CT Ferreira-Gonzalez A Validation of a multiplex methylation-sensitive PCR assay for the diagnosis of Prader-Willi and Angelman's syndromes.Mol Diagn. 2000; 5: 239-243Crossref PubMed Google Scholar6Kosaki K McGinniss MJ Veraksa AN McGinnis WJ Jones KL 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 Scholar7Kubota T Das S Christian SL Baylin SB Herman JG Ledbetter DH Methylation-specific PCR simplifies imprinting analysis.Nat Genet. 1997; 16: 16-17Crossref PubMed Scopus (256) Google Scholar methylation-dependent digestion followed by PCR,8Martinez F Leon AM Monfort S Oltra S Rosello M Orellana C Robust, easy, and dose-sensitive methylation test for the diagnosis of Prader-Willi and Angelman syndromes.Genet Test. 2006; 10: 174-177Crossref PubMed Scopus (10) Google Scholar methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA)9Bittel DC Kibiryeva N Butler MG Methylation-specific multiplex ligation-dependent probe amplification analysis of subjects with chromosome 15 abnormalities.Genet Test. 2007; 11: 467-475Crossref PubMed Scopus (52) Google Scholar10Nygren AO Ameziane N Duarte HM Vijzelaar RN Waisfisz Q Hess CJ Schouten JP Errami A Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences.Nucleic Acids Res. 2005; 33: e128Crossref PubMed Scopus (339) Google Scholar11Dikow N Nygren AO Schouten JP Hartmann C Kramer N Janssen B Zschocke J Quantification of the methylation status of the PWS/AS imprinted region: comparison of two approaches based on bisulfite sequencing and methylation-sensitive MLPA.Mol Cell Probes. 2007; 21: 208-215Crossref PubMed Scopus (54) Google Scholar and pyrosequencing.12White HE Durston VJ Harvey JF Cross NC 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 Some of these strategies can discriminate between deletional and non-deletional PWS/AS, but involve separate gene amplification, genotyping and detection steps. Recently, simple and rapid single-step strategies integrating amplification of bisulfite modified DNA and fluorescence melting curve analysis (MCA) have been reported, but they cannot discriminate between deletional and non-deletional PWS/AS.13White HE Hall VJ Cross NC 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 Scholar14Procter M Chou LS Tang W Jama M Mao R Molecular diagnosis of Prader-Willi and Angelman syndromes by methylation-specific melting analysis and methylation-specific multiplex ligation-dependent probe amplification.Clin Chem. 2006; 52: 1276-1283Crossref PubMed Scopus (83) Google Scholar15Worm J Aggerholm A Guldberg P In-tube DNA methylation profiling by fluorescence melting curve analysis.Clin Chem. 2001; 47: 1183-1189PubMed Google Scholar We have developed an improved single-step MS-PCR and MCA assay capable of differentiating between deletional and non-deletional PWS and AS. A total of 143 DNA samples were analyzed in this study, including samples from PWS patients, AS patients and normal controls (Table 1). Genomic DNAs were extracted from peripheral whole blood or cultured lymphoblasts. Each 1 μg of genomic DNA was bisulfite-treated and eluted in 20 μl of elution buffer using the EZ DNA Methylation Kit (Zymo Research). Sodium bisulfite treatment converts unmethylated cytosines to uracil, while methylated cytosines are protected from conversion. Therefore, methylated and unmethylated alleles of a gene fragment can become significantly different in sequence after bisulfite-mediated modification, with resulting differences in amplicon thermal stabilities.Table 1Differentiating between Deletional and Non-Deletional PWS/AS by Analysis of SNRPN:LIS1 Peak Height RatiosSNRPN:LIS1 peak height ratio ± SDGenotypeNo.PaternalMaternalDeletional PWS16—0.67 ± 0.20Non-deletional PWS8—1.60 ± 0.53Deletional AS131.66 ± 0.34—Non-deletional AS24.24 ± 0.86—Paternal duplication (PWS)18.310.62Normal1032.38 ± 0.830.87 ± 0.53 Open table in a new tab All samples were first analyzed by methylation-specific PCR to identify methylation differences at the SNRPN locus.7Kubota T Das S Christian SL Baylin SB Herman JG Ledbetter DH Methylation-specific PCR simplifies imprinting analysis.Nat Genet. 1997; 16: 16-17Crossref PubMed Scopus (256) Google Scholar PWS/AS samples were further analyzed by MS-MLPA (P028 PWS/AS, MRC-Holland) to differentiate between deletional and non-deletional causes.14Procter M Chou LS Tang W Jama M Mao R Molecular diagnosis of Prader-Willi and Angelman syndromes by methylation-specific melting analysis and methylation-specific multiplex ligation-dependent probe amplification.Clin Chem. 2006; 52: 1276-1283Crossref PubMed Scopus (83) Google Scholar In parallel, all samples were analyzed by a single-step MS-PCR and quantitative MCA assay. A 238 bp fragment of the SNRPN promoter was amplified using 0.5 μmol/L each of previously described primers,13White HE Hall VJ Cross NC 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 together with a 107 bp fragment of exon 11 of the LIS1 gene using 0.1 μmol/L each of primers 5′-GTTTTTGGTGTTTTTTAGGGATGTGTAATGTG-3′ (GenBank U72342; 1328→1359) and 5′-CAAACTACTATATTCCTCCCACTCCTTTC-3′ (GenBank U72342; 1434→1406). The LIS1 gene (OMIM # 601545), also known as PAFAH1B1, is located on chromosome 17p13.3. Haploinsufficiency of LIS1 is rare and causes a severe brain malformation and profound mental retardation disorder.16Cardoso C Leventer RJ Ward HL Toyo-Oka K Chung J Gross A Martin CL Allanson J Pilz DT Olney AH Mutchinick OM Hirotsune S Wynshaw-Boris A Dobyns WB Ledbetter DH Refinement of a 400-kb critical region allows genotypic differentiation between isolated lissencephaly, Miller-Dieker syndrome, and other phenotypes secondary to deletions of 17p133.Am J Hum Genet. 2003; 72: 918-930Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar Conversely, LIS1 duplications are even rarer, and are associated with a clinical phenotype distinct from PWS and AS.17Bi W Sapir T Shchelochkov OA Zhang F Withers MA Hunter JV Levy T Shinder V Peiffer DA Gunderson KL Nezarati MM Shotts VA Amato SS Savage SK Harris DJ Day-Salvatore DL Horner M Lu XY Sahoo T Yanagawa Y Beaudet AL Cheung SW Martinez S Lupski JR Reiner O Increased LIS1 expression affects human and mouse brain development.Nat Genet. 2009; 41: 168-177Crossref PubMed Scopus (181) Google Scholar Since loss or gain of LIS1 copy number is phenotypically recognizable from the normal two-copy state, and given the extreme improbability of concurrent LIS1 duplication/deletion and PWS/AS, LIS1 serves as an ideal two-copy reference gene for the PWS/AS quantitative MCA assay. Each duplex amplification reaction was performed in a 20 μl volume containing the pairs of primers, 0.2 mmol/L of each deoxynucleotide triphosphate (Roche Applied Science), 0.5 U HotStarTaq DNA polymerase (Qiagen), 0.5 mmol/L MgCl2, 1× EvaGreen (Biotium), 1× supplied PCR buffer (containing 1.5 mmol/L MgCl2), and 4 μl of the bisulfite-modified DNA. PCR amplification was performed in a LightCycler 480 instrument (Roche Applied Science) under the following conditions: an enzyme activation step at 95°C for 15 minutes followed by 30 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute. The MCA was performed automatically after PCR amplification under the following conditions: fragment denaturation at 95°C for 5 seconds, cooling to 65°C for 1 minute, and slow temperature ramping from 65°C to 95°C, with fluorescence data acquisition rate of 10 readings per degree using the 483–533 nm filter channel. Melting curve fluorescence data were analyzed using the melting temperature (Tm) calling function in the LightCycler 480 software. The Tm calling function converts the melting-curve data into melting-peak plots, determines the Tm for each PCR product as the highest point of the corresponding melting peak, and calculates the peak height for each melting peak. The starting fluorescence signal strength in the raw melting curve plot indicates the reaction quality. A low starting fluorescence intensity in the melting curve plot usually yields a melting peak with high background, leading to inaccuracy in peak height determination. Therefore, we considered the assay to have failed if the starting fluorescence intensity of a melting curve plot falls below the 10 unit threshold.13White HE Hall VJ Cross NC 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 To discriminate between deletional and non-deletional PWS or AS, the peak height data were exported into a Microsoft Excel worksheet to calculate the ratio between the maternal or paternal SNRPN and LIS1 peak heights. This study protocol was conducted under Institutional Review Board approval #EC200711152. The SNRPN amplicon derived from a bisulfite-modified maternal (normally methylated) allele melts at ∼87.7°C, compared with the paternal (normally unmethylated) allele, which melts at ∼83.3°C due to the higher GC content of the former amplicon (Figure 1, A–F). The SNRPN fragment was amplified together with a fragment from LIS1, which functions as the reference two-copy gene for the quantitative MCA analysis. Therefore, DNA samples from normal individuals should produce three melting peaks, including the LIS1 peak with a Tm of ∼76.0°C, and both the paternal and maternal SNRPN peaks. In contrast, the presence of the LIS1 peak and only the maternal or the paternal SNRPN peak is diagnostic of PWS and AS, respectively (Figure 1, A–F). Discrimination between deletional and non-deletional PWS and AS was achieved by determining the ratio between the maternal or paternal SNRPN peak height and the LIS1 peak height. The SNRPN to LIS1 peak height ratio in a deletional PWS/AS patient is expected to be less compared with the SNRPN:LIS1 peak height ratio in a non-deletional PWS/AS patient (Figure 1, Table 1). This is because patients with deletional PWS/AS contain only one copy of SNRPN, while patients with non-deletional PWS/AS, due either to UPD15 or imprinting center defects, contain two copies of SNRPN that are identically imprinted. Comparison of the peak height ratios from deletional and non-deletional PWS and AS patients confirms a higher SNRPN:LIS1 ratio in non-deletional patients compared with patients with deletions (Figure 1, Table 1). In replicate assay runs, we observed variations in the peak height ratio between samples within each genotype group; however, these variations did not result in an overlap in peak height ratios between the deletional and non-deletion groups within each run, thus allowing their unambiguous discrimination (Figure 2). Nevertheless, positive control samples are recommended for each assay run to mitigate the effects of variations between assay runs. Given its quantitative nature, this assay is also capable of detecting rare duplications within the PWS/AS critical region. This is illustrated by the DNA sample from a Coriell Repository cell line GM12135, which was previously shown to carry a paternal segmental duplication of chromosome 15 both cytogenetically and by MS-MLPA.14Procter M Chou LS Tang W Jama M Mao R Molecular diagnosis of Prader-Willi and Angelman syndromes by methylation-specific melting analysis and methylation-specific multiplex ligation-dependent probe amplification.Clin Chem. 2006; 52: 1276-1283Crossref PubMed Scopus (83) Google Scholar In this sample, we observed three peaks corresponding to LIS1, paternal SNRPN, and maternal SNRPN, a normal maternal SNRPN:LISI peak height ratio of 0.62 (range in normal samples 0.87 ± 0.53), but a much higher paternal SNRPN:LISI peak height ratio of 8.31 (range in normal samples 2.38 ± 0.83) (Figure 1F, Table 1). A total of 25 PWS and 15 AS patient samples were analyzed by MS-PCR and quantitative MCA in parallel with MS-MLPA and concordant results were observed for all of the patient samples. Using essentially standard procedures and reagents, we have modified the sodium bisulfite mediated MS-PCR assay for PWS/AS into a quantitative method to enable discrimination between the more common deletional and the rarer non-deletional forms. After sodium bisulfite treatment, PCR and quantitative MCA screening can be performed continuously without further hands-on manipulation and the reaction can be completed within 2 hours. Metaphase fluorescence in situ hybridization has been widely used for the detection of deletional PWS/AS, as well as the rarer translocations, but cannot detect PWS/AS due to UPD15 or imprinting center defects. Furthermore, the procedure requires cell culture, is comparatively more labor-intensive, and requires the use of fluorescent hybridization probes. Recently, MS-MLPA has been developed for the molecular diagnosis of PWS/AS.9Bittel DC Kibiryeva N Butler MG Methylation-specific multiplex ligation-dependent probe amplification analysis of subjects with chromosome 15 abnormalities.Genet Test. 2007; 11: 467-475Crossref PubMed Scopus (52) Google Scholar10Nygren AO Ameziane N Duarte HM Vijzelaar RN Waisfisz Q Hess CJ Schouten JP Errami A Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences.Nucleic Acids Res. 2005; 33: e128Crossref PubMed Scopus (339) Google Scholar11Dikow N Nygren AO Schouten JP Hartmann C Kramer N Janssen B Zschocke J Quantification of the methylation status of the PWS/AS imprinted region: comparison of two approaches based on bisulfite sequencing and methylation-sensitive MLPA.Mol Cell Probes. 2007; 21: 208-215Crossref PubMed Scopus (54) Google Scholar It avoids the need for sodium bisulfite treatment of the DNA sample but instead subjects the DNA to digestion with a methylation sensitive restriction enzyme HhaI. Using multiple specially designed probes specific for gene sequences in and around the PWS/AS critical region as well as reference genes outside this region, MS-MLPA can simultaneously detect methylation pattern differences and copy number changes in patients. However, this method is also comparatively more labor-intensive, makes use of fluorescently labeled PCR primers, and requires post-PCR analysis on a fluorescent fragment separation and analysis instrument. Our MS-PCR and quantitative MCA represents a simple, rapid and robust alternative to MS-MLPA for the detection of, and discrimination between, deletional and non-deletional forms of PWS and AS. As is the case with MS-MLPA, one limitation of quantitative MCA is its inability to differentiate between UPD and imprinting defects, for which multiplex microsatellite marker analysis, single nucleotide polymorphism haplotype analysis, and/or direct PCR and sequencing of the imprinting center is/are required. We thank Lily-lily Chiu and Evelyn S.C. Koay for providing some of the PWS and AS samples.
Referência(s)