MRC-Holland MLPA. Description version 24;
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1 SALSA MS-MLPA probemix ME030-C1 BWS/RSS Lot C As compared to version B2 (lots B2-0309, B & B2-1110), three probes for H19 and two for KCNQ1 have been replaced. One H19 has been removed and one CDKN1C probe added. For the NSD1 gene involved in Sotos syndrome, two probes have been included. Finally, several reference probes, the digestion control probe, and the 88 and 96 nt denaturation control fragments, have been replaced. Beckwith-Wiedemann syndrome (BWS) is a clinically heterogeneous overgrowth syndrome associated with an increased risk for embryonal tumour development. Russell-Silver syndrome (RSS) is a genetically heterogeneous disorder involving both intra-uterine and post-natal growth-retardation. The incidence of both BWS and RSS is estimated to be approximately 1 in 10,000-15,000 and around 85% of the cases are sporadic. These conditions are both caused by a genetic or an epigenetic alteration within two domains of imprinted growth regulatory genes on chromosome 11p15, leading to deregulated expression of the imprinted genes within this region. Approximately 60-70% of the patients have imprinting abnormalities at one of two imprinted domains H19DMR or KvDMR, and these changes are frequently mosaic. Other causes of BWS and RSS are uniparental disomy (UPD), trisomy 11p15 (1%), mutations in the CDKN1C gene (10%), as well as small deletions and translocations. This SALSA MS- BWS/RSS ME030-C1 probemix is capable of rapidly detecting most causes of BWS and RSS, as both copy numbers and methylation status of the 11p15 region can be determined. This MS- MLPA assay for BWS/RSS can also be useful for screening of childhood cancers, in particular Wilms tumour. A strong linkage between methylation of the H19DMR locus, but not KvDMR, has been described in these patients resulting in biallelic expression of the IGF2 gene. Because of similarities between BWS and Sotos syndrome 2 probes for NSD1 have been added. The ME030-C1 probemix contains 26 probes specific for the BWS/RSS 11p15 region. Eleven of these probes contain a HhaI recognition site and provide information about the methylation status of the target sequence. Besides the detection of aberrant methylation, all 26 probes present will give information on copy number changes in the analyzed sample. 2 probes are added in the NSD1 region which is associated with Sotos syndrome, a disease that share a similar phenotype. In addition, 13 reference probes are present which detect genes located outside the BWS/RSS region. Finally, one digestion control probe is included. This is a probe which contains a HhaI recognition site that is unmethylated in most blood derived control DNA samples. Therefore, this probe does not generate a signal after HhaI digestion in normal controls and can be used to confirm complete digestion by the HhaI enzyme. This SALSA MS- probemix can be used to detect aberrant methylation of one or more sequences of the KvDMR and H19DMR domains in the 11p15 BWS/RS region. Methylation levels can be different for different tissues. Please use DNA derived from the same type of tissue and purified by the same method as reference sample. This SALSA probemix can also be used to detect deletions/duplications of one or more sequences in the 11p15 region in a DNA sample. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak area of the amplification product of that probe. Note that a mutation or polymorphism in the sequence detected by a probe can also cause a reduction in relative peak area, even when not located exactly on the ligation site! In addition, some probe signals are more sensitive to sample purity and small changes in experimental conditions. Therefore, deletions and duplications detected by MLPA should always be confirmed by other methods. Not all deletions and duplications detected by MLPA will be pathogenic; users should always verify the latest scientific literature when interpreting their findings. Finally, note that many defects in these genes, such as small (point) mutations, will not be detected by this SALSA test. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. SALSA MS- probemixes and reagents are sold by for research purposes and to demonstrate the possibilities of the MLPA technique. They are not CE/FDA certified for use in diagnostic procedures. Purchase of the SALSA MS- test probemixes and reagents includes a limited license to use these products for research purposes. The use of this SALSA MS- probemix and reagents requires a thermocycler with heated lid and sequence type electrophoresis equipment. Different fluorescent PCR primers are available. The MLPA SALSA probemix ME030-C1 BWS/RSS Page 1 of 8
2 technique has been first described in Nucleic Acid Research 30, e57 (2002). The MS-MLPA method for the detection of both copy numbers and methylation changes was described in Nucleic Acid Research 33, e128 by Nygren et al More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands References of SALSA MS- probemix ME030 Scott RH et al. (2008) Methylation-specific MLPA (MS-MLPA) robustly detects and distinguishes 11p15 abnormalities associated with overgrowth and growth retardation. J Med Genet. 45: Priolo M et al. (2008). MS-MLPA is a specific and sensitive technique for detecting all chromosome imprinting defects of BWS and SRS in a single-tube experiment. Eur J Hum Genet. 16: Zeschnigk M. et al. (2008). IGF2/H19 hypomethylation in Silver-Russell syndrome and isolated hemihypoplasia. Eur J Hum Genet. 16: Figure 1. Scheme of the imprinted gene cluster on chromosome 11p15. Methylation-specific MLPA Please note that each MS-MLPA reaction generates two samples that need analysis by capillary electrophoresis: one undigested sample for copy number detection and one digested sample for methylation detection. A modification of the MLPA technique, MS-MLPA allows the detection of both copy number changes and unusual methylation levels of different sequences in one simple reaction. MLPA probes for methylation quantification are similar to normal MLPA probes, except that the sequence detected by the MS-MLPA probe contains the sequence recognized by the methylation-sensitive restriction enzyme HhaI. Similar to ordinary MLPA reactions, the MS-MLPA protocol starts with sample DNA denaturation and overnight hybridization. The reaction then is split into two tubes. One tube is processed as a standard MLPA reaction. This reaction provides information on copy number changes. The other tube of the MLPA hybridization reaction is incubated with the methylation-sensitive HhaI endonuclease while simultaneously, the hybridized probes are ligated. Hybrids of (unmethylated) probe oligonucleotides and unmethylated sample DNA are digested by the HhaI enzyme. Digested probes will not be exponentially amplified by PCR and hence will not generate a signal when analyzed by capillary electrophoresis. In contrast, if the sample DNA is methylated, the hemimethylated probe-sample DNA hybrids are prevented from being digested by HhaI and the ligated probes will generate a signal. More information about MS-MLPA can be found in the MS-MLPA protocol. Please note that this product cannot be used with an alternative protocol in which the genomic DNA is first digested with Hha1, followed by MLPA reactions on both digested and undigested genomic DNA. Digestion control probes Digestion control probes are unmethylated in most blood derived DNA samples. The signals of the digestion control probes should be gone upon complete digestion by HhaI. SALSA probemix ME030-C1 BWS/RSS Page 2 of 8
3 Data analysis The ME030-C1 BWS/RSS probemix contains 42 MLPA probes with amplification products between 129 and 463 nt. In addition, it contains 9 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at nt, three DNA denaturation control fragments (D-fragments) at nt, one X-fragment at 100 nt and one Y-fragment at 105 nt. More information on how to interpret observations on these control fragments can be found in the MLPA protocol. The analysis of MS-MLPA probemixes consists of two parts: 1) determining copy numbers by comparing different undigested samples (all MLPA probemixes), and 2) determining methylation patterns by comparing each undigested sample to its digested counterpart (MS-MLPA probemixes only). The second part is unique for MS-MLPA probemixes and serves to semi-quantify the percentage of methylation within a given sample. Intra-sample data normalisation (all samples) For analysis of MLPA results, not the absolute fluorescence values but intra-normalized data are used (relative peak areas). The data generated in the digested and undigested sample should first be normalized intra-sample by dividing the signal of each probe by the signal of every reference probe in that sample, thus creating as many ratios per probe as there are reference probes. Subsequently, the median of all these produced ratios per probe should be taken; this is the probe s Normalisation Constant. This Normalization Constant can then be used for sample to reference sample comparison, both for copy number and digestion determination. Copy numbers determination (comparison between undigested samples) The final probe ratio, or ploidy status, of each probe in each sample is calculated by dividing a) the Normalization Constant of each probe obtained on the undigested patient sample by b) the average Normalization Constant of that probe obtained on the undigested reference samples. Methylation analysis (comparing digested and undigested samples) Methylation status of MS-MLPA probes* is calculated by dividing a) the Normalization Constant of each MS- MLPA probe obtained on the digested patient sample by b) the Normalization Constant of each MS-MLPA probe obtained on the corresponding undigested sample. Multiplying this value by 100 gives an estimation of the percentage methylation. Aberrant methylation can then be identified by comparing the methylation status of one or more MS-MLPA probes in the sample in question to that obtained on reference samples. *Note: An MS-MLPA probe targets a single specific HhaI site in a CpG island; an unusual methylation status for a particular CpG-site does not necessarily mean that the whole CpG island has an unusual methylation status! Data used for normalization should be obtained within a single experiment. Only samples purified by the same method should be compared. Confirmation of most exons deletions and amplifications can be done by e.g. Southern blots, long range PCR, FISH. H19 locus and KCNQ1OT1 locus The four MS-MLPA probes targeting the H19 gene and the four MS-MLPA probes targeting the KCNQ1OT1 locus, are located very close to each other within each locus. It is expected that all MS-MLPA probes per locus provide similar results. We recommend using the average, or the median, methylation status of these probes to determine to methylation status of the loci and to disregard aberrant methylation detected by a single MS-MLPA probe. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix ME030-C1 BWS/RSS Page 3 of 8
4 Table 1. SALSA MS-MLPA ME030-C1 BWS/RSS probemix Length (nt) SALSA MLPA probe Hha1 site % methylated in normal blood-derived DNA % expected signal reduction Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA D-fragments: Low signal of 88 or 96 nt fragment indicates incomplete denaturation 100 X-fragment: Specific for the X chromosome 105 Y-fragment: Specific for the Y chromosome Chromosomal position ref. BWS/RSS 129 * Reference probe L q H19 probe L20532 Yes 50% 50% H19DMR/IC1 141 KCNQ1OT1 probe L19191 Yes 50% 50% KvDMR/IC2 148 * Reference probe L q Reference probe L q H19 probe L Region 166 KCNQ1OT1 probe L05782 Yes 50% 50% KvDMR/IC2 171 ± IGF2 probe L20841 Yes 0% 100% 5 DMR0 178 * Reference probe L q H19 probe L08764 Yes 50% 50% H19DMR/IC1 190 * H19 probe L Region 196 ± CDKN1C probe L Exon * Reference probe L q * Reference probe L q H19 probe L Region 221 * KCNQ1 probe L Exon * H19 probe L Exon * H19 probe L16503 Yes 50% 50% H19DMR/IC1 256 Reference probe L p KCNQ1 probe L Exon KCNQ1OT1 probe L19204 Yes 50% 50% KvDMR/IC2 284 ± IGF2 probe L Exon * Reference probe L q H19 probe L05772 Yes 50% 50% H19DMR/IC1 310 Reference probe L q * NSD1 probe L (Exon 24) 328 KCNQ1 probe L Exon * Reference probe L q *± CDKN1C probe L18042 Yes 10% 90% Exon *# Digestion Control probe L09311 Yes 0% 100% 8p KCNQ1 probe L Exon * KCNQ1 probe L Exon * Reference probe L q KCNQ1OT1 probe L06781 Yes 50% 50% KvDMR/IC2 400 KCNQ1 probe L Exon ± KCNQ1 probe L Exon * NSD1 probe L (Exon 22) 427 * Reference probe L q ± KCNQ1 probe L Exon ± CDKN1C probe L Exon ± H19 probe L Region 463 * Reference probe L p25 * New in version C1 (from lot C onwards). Changed in version C1 (from lot C onwards). Small change in length, no change in sequence detected. ± These probes are located within, or close to, a very strong CpG island. A low signal of these probes can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. SALSA probemix ME030-C1 BWS/RSS Page 4 of 8
5 # Digestion control: warns for insufficient digestion. Upon digestion, this probe should not give a signal. More variable. Note: Exon numbering might be different as compared to literature! Please notify us of any mistakes: info@mlpa.com. The identity of the genes detected by the reference probes is available on request: info@mlpa.com. Table 2. ME030 probes arranged according to chromosomal location Length (nt) SALSA MLPA probe Gene / exon Hha1 site 418 * L02071 NSD1-319 * L02529 NSD1 - GenBank Ligation site NM_ ; NM_ ; Chromosomal position Distance to next probe kb * L19241 H L01713 H L05772 H19 Yes 238 * L16503 H19 Yes L08764 H19 Yes L20532 H19 Yes L11143 H L11141 H * L19242 H L05778 IGF L05775 IGF2 Yes L02903 KCNQ1-221 * L16502 KCNQ L04802 KCNQ L19240 KCNQ L20510 KCNQ L19204 KCNQ1OT1 Yes L05782 KCNQ1OT1 Yes L06781 KCNQ1OT1 Yes L19191 KCNQ1OT1 Yes L18343 KCNQ1-373 * L16504 KCNQ1 - NR_ ; NR_ ; 178nt NR_ ; 342nt NR_ ; 486nt NR_ ; 657nt NR_ ; 961nt NR_ ; 3282 nt NR_ ; 3798 nt NR_ ; 6778 nt NM_ ; NM_ ; 318nt after exon 4; NM_ ; nt before exon NR_ ; NR_ ; NR_ ; NR_ ; 190 nt kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb SALSA probemix ME030-C1 BWS/RSS Page 5 of 8
6 Length (nt) SALSA MLPA probe Gene / exon Hha1 site L21092 KCNQ1 - GenBank Ligation site Chromosomal position Distance to next probe kb L20842 CDKN1C - NM_ ; kb L05768 CDKN1C - NM_ ; kb 346 * L18042 CDKN1C Yes NM_ ; * New in version C1 (from lot C onwards). Changed in version C1 (from lot C onwards). Small change in length, no change in sequence detected. The NM_ , NR_ , NM_ , NM_ , and NM_ sequences are reference standards in the NCBI RefSeqGene project. Table 3. Sequences detected by the ME030-C1 probes Length SALSA MLPA (nt) probe Partial sequence with HhaI site L20532 CAGGCCCTCTGGGATGTGGAAGGGCTGGC-CGCGCCTTCGGCAAACCCTCTGTTCCCA L19191 GATCGGTTTTGATGCCACCCGGGCTCAGAT-TGGCCCAGCGGGTCCAGCGCCGCATGAG L01713 GCGGGGAGGCCGTCTTTGGAGAA-TTCCAGGATGGGTGCTGGGTGAGAGAGACGTGGTA L05782 GACCGTGTTCAAACCCTCCCAGAGAGA-TGGGGAGGGCCGCGCTGAGGAGAGTCTG L05775 TCAAGCCACCTGCATCTGCACTCA-GACGGGGCGCACCCGCAGTGCAGCCTCC L08764 GTAGAGTGCGCCCGCGAGCCGTA-AGCACAGCCCGGCAACATGCGGTCTTCAGAGT L19242 GGAGCAGGCAAACACCTCGGGCTATTTGCTTT-CTAATGGGGCATTTGCATGTGTATAGG L05768 GTCCCTCCGCAGCACATCCACGAT-GGAGCGTCTTGTCGCCCGTGGGACCTTC L11141 GAATCGATAAAGGATGGGGATCAATGG-TGTTTGTGCACTGTGCGGTCTGTGCCCAATT L16502 GACCTACCTCTAGTGTGTGCTTACCAGCTA-ATGGATGACTGGGTTTTCGAAGCACTGTC L19241 GACCACCAGCCACCACATCATCCCA-GAGCTGAGCTCCTCCAGCGGGATGACGCCGTCCC L16503 CTGAGGGGCAGAGGGAAGTGCCGCAA-ACCCCCTGGTGGGCGCGGTGCCAGCCCCCCA L18343 ACTCTCCACTGCAGGCTGCGGGAAC-ACCATCGGGCCACCATTAAGGTCATTCGACGCA L19204 GCGGGGCACACAGCTCACCTCAGCAA-CGCCAGTGATCACCCGTCCCGCGCCGTCCGC L05778 GCCAGGTCACAGCTGCGGAAACA-GCACTCCTCAACGATGCCACGGCTGCGAC L05772 CGGCCCCCAGCCATGTGCAAAGTA-TGTGCAGGGCGCTGGCAGGCAGGGAGCA L02529 CCAGAAGGAGCGGGCAGCTTCACCTCA-TCAGGTCACACCACAGGCTGATGAGAAGATGC L04802 CTACATCGGCTTCCTGGGCCTCATCT-TCTCCTCGTACTTTGTGTACCTGGCTGAGAAG L18042 CCTCTCCTTTCCCCTTCTTCTCGCT-GTCCTCTCCTCTCTCGCTGCCCGCGTTTGCGCAG L09311 CCTCCTAGCCTGGCGCGCGATT-ATTTGAAGACGCTCACGGAGCGGCTGGCTAGGCTGA L19240 CCTGCAGGTCACAGTCACCACCATCGGCT-ATGGGGACAAGGTGCCCCAGACGTGGGTC L16504 CTTCTCTCCAGGCTGGACCAGTCCAT-TGGGAAGCCCTCACTGTTCATCTCCGTCTCAG L06781 CGTCCTCCTCGGTGCGTCAGTCAT-CGTGGTTCTCCCCGGCGCGCCCCTCGGC L20510 CCTTCTTGGCTCGGGGTTTGCCCTGAAGGT-GCAGCAGAAGCAGAGGCAGAAGCACTTC L21092 CCTCCAGCAGCCAGCCAAACACACAG-AAGGGGACTGCCACCTCCCCTTGCCAGCT L02071 CTAGAATGTCTTGGGAATGGAAAGACTGTT-TGCAAATGTGGAGCCCCGAACTGCAGTG L02903 GCTCTCGGGAATTTGAGGCCTGT-GGCTGCTGTGGACCCTGGGAAAGAGCCTGTGC L20842 CCTCGCTGGGCCTCGGCTGGGACCGTTCATGT-AGCAGCAACCGGCGGCGGCTGCCGCA L11143 CCTGCCTGCAGAAACATCCCGGGTCAA-CAGGCCAGGCACCGCATTGGTTCGCGAG The Hha1 sites are marked with grey. Ligation sites are marked with Note: Exon numbering might be different as compared to literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA probemix ME030-C1 BWS/RSS Page 6 of 8
7 SALSA MS-MLPA probemix ME030-C1 BWS/RSS sample pictures Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng undigested human male control DNA analyzed with SALSA MLPA probemix ME030-C1 BWS/RSS (lot C1-0711) for the quantification of copy numbers. Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng digested human male control DNA analyzed with SALSA MLPA probemix ME030-C1 BWS/RSS (lot C1-0711) to determine the methylation status. SALSA probemix ME030-C1 BWS/RSS Page 7 of 8
8 Implemented Changes the following has been altered compared to the previous product description version(s). Version 24 (06) - Methylation percentage errors corrected in Table 1. - Reference sequence information added below Table 2. Version 23 (05) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new pictures included). Version 22 (05) - Product description adapted to a new lot (lot number added, new picture included). - Warning added about a SNP for probe L04803 below table 1. - Warning added for CDKN1C probes under table 2d. on page 6. Version 21 (05) - Various minor textual changes on page 1. - Mapview locations updated in table 1. - Various minor layout changes. Version 20 - Product description adapted to a new lot (lot number added, new picture included). - Small textual and layout modifications. Version 19 - Exon numbering of the KCNQ1 gene adjusted to the exon numbering of the NCBI. - More details added about SNP in 142 nt probe L Minor changes to text of Figures 2 & 3. SALSA probemix ME030-C1 BWS/RSS Page 8 of 8
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