MRC-Holland MLPA. Description version 52; 22 July 2015

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1 SALSA MS-MLPA probemix ME028-B2 Prader-Willi/Angelman Lot B2-0413, lot B As compared to version B1 (lot B1-0609), the control fragments have been replaced (QDX2). PRADER-WILLI SYNDROME (PWS) and ANGELMAN SYNDROME (AS) are distinct neurogenetic disorders, both usually caused by chromosomal deletions on chromosome 15q11 or by uniparental disomy. The chromosomal alterations result in an aberrant expression profile of gene loci that are subject to imprinting. Absence of a paternal allele of chromosome 15q11, due to chromosomal deletion or uniparental disomy, results in PWS. The absence of the maternal copy of the same region causes AS. This SALSA MS- probemix ME028 can be used to detect copy number changes, as well as to analyse CpG island methylation of the 15q11 region in a semi-quantitative manner. This SALSA ME028-B2 PWS/AS probemix contains 32 probes specific for sequences in or near the PWS/AS critical region of chromosome 15q11, which can be used to detect copy number changes in this region. Five of these probes are specific for an imprinted sequence and contain a recognition site for the methylation sensitive HhaI enzyme. These five probes can be used for the presence of aberrant methylation patterns in the 15q11 locus, either caused by uniparental disomy or by imprinting defects. For the analysis of results, 14 reference probes for genes located outside the PWS/AS region are present. In addition, two probes are present that will indicate complete digestion by the HhaI enzyme in the methylation quantification reaction. This SALSA MS- probemix can be used to detect aberrant methylation of one or more sequences of the Prader-Willi/Angelman 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 MS- probemix can also be used to detect deletions and duplications of one or more sequences of the 15q11 region in a DNA sample. Heterozygous deletions of probe recognition sequences should give a 35-50% reduced relative peak height 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 height, 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. Please note that several copy number changes affecting only a part of the 15q11 PWS/AS region have been described in healthy persons. Analysis of parental DNA samples can therefore in some cases be required for correct interpretation of results. 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 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 Related SALSA probemixes ME030 BWS: Contains probes for Beckwith-Wiedemann syndrome. P325 OCA2: Contains probes for each exon of OCA2 with the exception of exon 8. P336 UBE3A: Contains probes for each UBE3A exon. P343 Autism-1: Contains additional probes for UBE3A and the 15q13 region. More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands SALSA MS- probemix ME028 Prader-Willi/Angelman Page 1 of 11

2 References for SALSA probemix ME028 Ramsden SC et al. (2010). Practice guidelines for the molecular analysis of Prader-Willi and Angelman syndromes. BMC Med Genet. 11:70. Bittel DC et al. (2007). Methylation-specific multiplex ligation-dependent probe amplification: analysis of subjects with chromosome 15 abnormalities. Genet Test. 11(4): Dikow N et al. (2007). 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. 21(3): Procter M et al. (2006). Molecular Diagnosis of Prader-Willi and Angelman Syndromes by Methylation- Specific Melting Analysis and Methylation-Specific Multiplex Ligation-Dependent Probe Amplification. Clin Chem. 52(7): Reference DNA samples Please note that the NIBSC provides an excellent panel of WHO certified genomic DNA samples for Prader Willi and Angelman syndrome: 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 analysed 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 can not be used with an alternative protocol in which the genomic DNA is first digested with HhaI, followed by MLPA reactions on both digested and undigested genomic DNA. Digestion control probes The target sequences of the 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. Besides the two digestion control probes at 346 and 463 nt, the sequence detected by the 184 nt UBE3A MS-MLPA probe is also unmethylated in most blood-derived DNA samples. A total absence of these three probes in the digested sample therefore indicates a complete digestion by HhaI. The 346 nt probe is the most sensitive to incomplete HhaI digestion; HhaI-digestion can be considered sufficient when less than 10% of the 346 nt digestion control probe signal remains in the digested reaction compared to the undigested reaction. Data analysis The ME028-B2 Prader-Willi/Angelman probemix contains 48 MLPA probes with amplification products between 130 and 480 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. SALSA MS- probemix ME028 Prader-Willi/Angelman Page 2 of 11

3 Please note that the great majority of Prader-Willi and Angelman samples will show a deletion of probes, AND/OR a methylation change of four SNRPN and one NDN MS-MLPA probes. Abnormal copy numbers or methylation changes detected by only one or two probes should be treated with caution. 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. 1) Copy number analysis Intra-sample data normalisation For analysis of MLPA results, not the absolute fluorescence values but intra-normalised data are used (relative peak heights). The data generated in the undigested sample should first be normalised intrasample 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 Normalisation Constant can then be used for sample to reference sample comparison, both for copy number and digestion determination. Inter-sample normalisation (comparison with reference samples) The final probe ratio, or ploidy status, of each probe in each sample is calculated by dividing a) the Normalisation Constant of each probe obtained on the undigested test sample by b) the average Normalisation Constant of that probe obtained on the undigested reference samples. 2) Methylation analysis - Intra-sample data normalisation For analysis of MLPA results, not the absolute fluorescence values but intra-normalised data are used (relative peak height). The data generated in the digested sample should first be normalised 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 Normalisation Constant can then be used for sample to reference sample comparison. Methylation analysis (comparison with reference samples) The methylation status of each MS-MLPA probe* in each sample is calculated by dividing a) the Normalisation Constant of each probe obtained on the digested test sample by b) the Normalisation Constant of each MS-MLPA probe obtained on the corresponding undigested sample. Multiplying this value by 100 gives an estimation of the percentage of 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. *Notes: An MS-MLPA probe targets a single specific HhaI site in a CpG island; if methylation is absent for a particular CpG-site, this does not necessarily mean that the whole CpG island is unmethylated! With this probemix no discrimination between uniparental disomy and imprinting defects can be made. For this, it is necessary to perform microsatellite analysis in the patient and parents. Aberrant methylation of the 15q11 locus In addition to the large de novo deletions of the 15q11 on the paternal or on the maternal chromosome, PWS or AS can be caused by aberrant methylation of the 15q11 locus. This can be caused either by uniparental disomy or by imprinting defects. Aberrant methylation of the 15q11 locus can be detected by use of the 5 methylation specific MLPA probes detecting sequences in the SNRPN gene and the NDN gene, when compared to results obtained on DNA samples from healthy individuals. Table 5 and Figure 3 contain an overview of the expected copy number changes and methylation profiles in PWS/AS patients with deletions or aberrant methylation. SALSA MS- probemix ME028 Prader-Willi/Angelman Page 3 of 11

4 SNRPN locus The four probes targeting the SNRPN gene are located very close to each other and it is expected that all four probes provide similar results. We recommend using the average, or the median, methylation status of these SNRPN probes to determine to methylation status of the SNRPN locus and to disregard aberrant methylation detected by a single SNRPN MS-MLPA probe. NDN locus Please note that the 419 nt NDN probe has a tendency to overdigest and may seem to be only 30% methylated in DNA samples from healthy persons. We noticed that the percentage of methylation found using this and two alternative NDN probes that we tested varies between individuals. Methylation changes of the NDN probe only may not have any clinical significance. We have been informed that the NDN locus is significantly hypomethylated in chorionic villi (Karin Buiting & Jasmin Beygo, Essen, unpublished results). We therefore recommend interpreting the SNRPN locus methylation only in case of prenatal diagnosis. Data normalisation should be performed within one 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 blotting, long range PCR, qpcr, FISH. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website This probemix was developed at in collaboration with several European laboratories, represented by K. Buiting (Essen) and S. Ramsden (Manchester). Info/remarks/suggestions for improvement: info@mlpa.com. SALSA MS- probemix ME028 Prader-Willi/Angelman Page 4 of 11

5 Table 1. SALSA MS-MLPA ME028-B2 PWS/AS probemix Length Chromosomal region HhaI Methylated SALSA MLPA probe (nt) Ref. UBE3A SNRPN Other 15q site in blood (%) 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 130 Reference L q Reference L q SNRPN L02951 CpG island (PWS-SRO) + 50% imprinted 148 Reference L q ٨ TUBGCP L00865 BP1-BP2 region 160 UBE3A L00863 Exon Reference L q MKRN L q SNRPN L13905 CpG island (PWS-SRO) + 50% imprinted 184 UBE3A L04293 Exon 1 + 0% 190 SNRPN L04294 CpG island (PWS-SRO) + 50% imprinted 197 UBE3A L11550 Exon APBA L q Reference L q SNRPN L13907 SNORD116 snorna cluster 220 GABRB L q ATP10A L q ± Reference L q ٨ SNRPN L13090 exon u1b* 244 Reference L p SNRPN L13997 CpG island (PWS-SRO) + 50% imprinted 256 SNRPN L12881 exon u5 (AS-SRO) 264 Reference L q SNRPN L13094 intron u2 278 SNRPN L13383 intron u2 287 ٨ SNRPN L11862 exon u1b 294 SNRPN L13088 SNURF-SNRPN exon UBE3A L13474 Exon Reference L q Reference L q SNRPN L13798 SNORD116 snorna cluster + (100%) 337 Reference L q ๑ Digestion L q26 Digestion Control Probe. Signal <10% upon digestion. + 0% 355 UBE3A L12925 Exon ATP10A L q UBE3A L11548 Exon GABRB L q SNRPN L13519 exon U5 (AS-SRO) 400 SNRPN L11861 SNURF-SNRPN exon ± MAGEL L q ± NDN L q % imprinted 427 Reference L p ٨ NIPA L05755 BP1-BP2 region 445 NDN L q Reference L p # ± Digestion L p22 Digestion Control Probe. No signal upon digestion. + 0% 472 SNRPN L13796 SNORD116 snorna cluster 480 Reference L q23 ٨ BP1-BP2 region and u1b-u1b* region: copy number variation in these regions has been described in healthy individuals. NDN: (1) both NDN probes, especially 445 nt, are affected by denaturation problems. Check the 96 nt D-fragment: if this and one or both NDN probes are lower than the 92 nt fragment, denaturation was incomplete. (2) the 419 nt probe has a tendency to overdigest. The SNORD116 snorna gene cluster was previously named HBII nt: target site contains 2 SNPs which may affect probe signal. In case of a reduced probe signal, sequence the target region! ๑ 346 nt: Digestion control, warns for insufficient digestion. HhaI-digestion can be considered sufficient when <10% of the signal remains in the digested reaction compared to the undigested reaction. SALSA MS- probemix ME028 Prader-Willi/Angelman Page 5 of 11

6 #463 nt: Digestion control, warns for insufficient digestion. Upon digestion, this probe should not give a signal. The target sequence of the 328 nt SNORD116 probe appears to be fully methylated in blood-derived DNA but hypomethylated in fetal DNA (K. Buiting & J. Beygo, pers. communication). We have received various reports of different methylation ratios for this probe also in blood-derived DNA and strongly recommend to use this probe only for copy number analysis, not for methylation analysis. ± This probe is located within, or close to, a very strong CpG island. A low signal of this probe can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. The 202 nt APBA2 probe is outside the common PWS/AS region. Size of the region showing aberrant copy number will differ between different PWS/AS patients. More 15q13 probes are available in SALSA MLPA probemix P297 Microdeletion- 2. Table 2. 15q probes arranged according to chromosomal location Length (nt) SALSA MLPA probe Gene HhaI site Distance to p- telomere Chromosomal position Distance to next probe 154 ٨ L00865 TUBGCP5 BP1-BP2 region kb 434 ٨ L05755 NIPA1 BP1-BP2 region kb End of copy number variable region L12922 MKRN Exon kb 409 ± L11839 MAGEL Exon kb L11833 NDN Exon kb 419 ± L13937 NDN Exon kb 287 ٨ L11862 SNRPN Exon u1b 6.2 kb 238 ٨ L13090 SNRPN Exon u1b* 64.9 kb L13383 SNRPN Intron u kb L13094 SNRPN Intron u kb L12881 SNRPN Exon u5 (AS-SRO) 0.6 kb L13519 SNRPN Exon u5 (AS-SRO) 33.8 kb L13997 SNRPN SNRPN CpG island (PWS-SRO) 0.1 kb L13905 SNRPN SNRPN CpG island (PWS-SRO) 0.3 kb L04294 SNRPN SNRPN CpG island (PWS-SRO) 0.3 kb L02951 SNRPN SNRPN CpG island (PWS-SRO) 12.5 kb L13088 SNRPN SNURF-SNRPN exon kb L11861 SNRPN SNURF-SNRPN exon kb L13907 SNRPN SNORD116 snorna cluster 24.4 kb L13796 SNRPN SNORD116 snorna cluster 16.0 kb L13798 SNRPN SNORD116 snorna cluster kb L12925 UBE3A Exon kb L13474 UBE3A Exon kb L00863 UBE3A Exon kb L11550 UBE3A Exon kb L11548 UBE3A Exon kb L04293 UBE3A Exon kb L11846 ATP10A Exon kb L11843 ATP10A Exon kb L09339 GABRB Exon kb L11544 GABRB Exon kb L00867 APBA Exon 14; Outside common kb PWS-AS region 346 # L12924 BLM q26 # Digestion control, warns for insufficient digestion. HhaI-digestion can be considered sufficient when <10% of the signal remains in the digested reaction compared to the undigested reaction. 202 nt APBA2 probe is outside the common PWS/AS region. Size of the region showing aberrant copy number will differ between different PWS/AS patients. More 15q13 probes are available in SALSA MLPA probemix P297 Microdeletion-2. ٨ BP1-BP2 region and u1b-u1b* region: The copy numbers of the TUBGCP5 and NIPA1 genes show variation in some healthy individuals! According to Stefansson, H. et al (2008; Nature 455, ), a deletion of this BP1-BP2 region is present in 0.19% of normal individuals and in 0.55% of schizophrenia patients. More probes for this BP1-BP2 region are in the P211 HSP probemix. The chromosomal order of these probes in the NCBI and BLAT databases is different. We used the order that is mentioned by Jiang et al. (2008) BMC Genomics 9:50. Copy number changes of the exon u1b/u1b* regions have been described in some healthy individuals: SALSA MS- probemix ME028 Prader-Willi/Angelman Page 6 of 11

7 ± This probe is located within, or close to, a very strong CpG island. A low signal of this probe can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. 366 nt: target site contains 2 SNPs which may affect probe signal. In case of a reduced probe signal, sequence the target region! The new name for the HBII-85 snorna gene cluster is SNORD116. Deletions within the SNORD116 cluster have been described as cause of Prader-Willi phenotype (Sahoo,T. et al (2008) Nature Genetics 40: ). Additional probes in this region are present in the P343 Autism-1 and P336 UBE3A probemixes. Small deletions in this region might be a rare cause of PWS and will only be of interest when they are not present in parental samples. The target sequence of the 328 nt SNORD116 probe appears to be fully methylated in blood-derived DNA but hypomethylated in fetal DNA (K. Buiting & J. Beygo, pers. communication). We have received various reports of different methylation ratios for this probe also in bloodderived DNA and strongly recommend to use this 328 nt probe only for copy number analysis, not for methylation analysis. The AS-SRO is part of the PWS-AS Imprinting Centre (located upstream of the SNURF-SNRPN gene) and is required for a chromosome to have the maternal pattern of epigenetic modification and gene expression only if the chromosome has an intact PWS-SRO. The PWS-SRO is also part of the PWS-AS IC and is a 4.1kb region that includes the SNRPN promoter. The PWS-SRO is unconditionally required for a chromosome to have the paternal pattern of epigenetic modification and gene expression. Note: Exon numbering used here may differ from literature! Please notify us of any mistakes: info@mlpa.com. Table 3. Location of the SNRPN/UBE3A probes in Genbank Length SALSA MLPA Distance to Location in Genbank Distance to Gene (nt) probe p-telomere NG_ next probe 287 SNRPN L Reverse 6.2 kb 238 SNRPN L kb 278 SNRPN L kb 270 SNRPN L kb 256 SNRPN L kb 391 SNRPN L kb 250 SNRPN L kb 178 SNRPN L kb 190 SNRPN L kb 142 SNRPN L kb 328 SNRPN L kb 294 SNRPN L Reverse 8.3 kb 400 SNRPN L kb 214 SNRPN (SNORD116) L Reverse 24.4 kb 472 SNRPN (SNORD116) L kb 328 SNRPN (SNORD116) L kb 355 UBE3A L Reverse 20.3 kb 301 UBE3A L Reverse 11.1 kb 160 UBE3A L Reverse 4.2 kb 197 UBE3A L Reverse 29.8 kb 373 UBE3A L Reverse 33.5 kb 184 UBE3A L Reverse kb Notes: More UBE3A and 15q13 probes are available in SALSA MLPA probemix P336 UBE3A and in SALSA MLPA probemix P343 Autism-1. Please contact us with suggestions or questions about this probemix: info@mlpa.com. SALSA MS- probemix ME028 Prader-Willi/Angelman Page 7 of 11

8 Table 4. Sequences detected by the ME028-B2 probes Length (nt) Gene Sequences detected by the ME028-B2 probes, with HhaI site 130 CTTN GGCTTCCTTCAAGGCAGAGCTGA-GCTACAGAGGCCCTGTGAGTGGGACGGAGCCG 136 DPYSL4 TGGGCTCTGACGCTGACCTGGTCATA-TGGAACCCCAAGGCCACCAAGATCATCTCTGCCAA 142 SNRPN AGGGGGTGTTGAGCGCAGGT-AGGTGTATAATAGTGACCACTGCGTGGTGGAGCAGGGTAC 148 AATF TAGGTTTCATGTCCTTAGCAAGCTACTGAG-TTTCATGGCACCTATTGACCATACTACAATGAATGATGATGCC 154 TUBGCP5 GGACCAACACTTGTACAGCAGTGATCCATT-GTATGTTCCAGATGACAGGGTTTTGGTTACTGAGACTCAGGTT 160 UBE3A GAAGAAGACTCAGAAGCATCTTCCTCAAGG-ATAGGTGATAGCTCACAGGGAGACAACAATTTGCAAAAATTAG 166 CHEK1 GTCACAGGAGAGAAGGCAATATCCAATATT-TATTTCTGGAGTACTGTAGTGGAGGAGAGCTTTTT 172 MKRN3 CGACATGTGTGGGCTGCAGACCT-TGCACCCCATGGATGCTGCCCAGAGGGAAGAACATAT 178 SNRPN GCCGCTGCTGCAGCGAGTCTGGCGCAGAGT-GGAGCGGCCGCCGGAGATGCCTGACGCATCTGTCTGAG 184 UBE3A GAGATCCGTGTGTCTCCCAAGA-TGGTGGCGCTGGGCTCGGGGTGACTACAGGA 190 SNRPN CCGATGGTATCCTGTCCGCTCGCAT-TGGGGCGCGTCCCCCATCCGCCCCCAACTGTGGT 197 UBE3A GCATCTAATAGAACGCTACTACCACCAGTT-AACTGAGGGCTGTGGAAATGAAGCCTGCACGAATGAG 202 APBA2 CAAAGGGTGTGCCCTCACCACCCACTT-GATTTTTTTCATTTTGCCAAAAAGGGGTATGTCTTTATCAAAG 208 COL2A1 GGACCTTCTGTTCCTGTCTCTTCTGGAACA-TTCTTCTCTGAGCCTGAGACCTCTCTCCTGACAGGGT 214 SNRPN CCTGGTGACCAATGCTGCAGGGTA-GGCTGAAGAGGCTTTCACTGTTTGCCCCGACACC 220 GABRB3 AGGCGGCATTGGCGATACCAGGAA-TTCAGCAATATCCTTTGACAACTCAGGAATCCAGTACAGGAAA 226 ATP10A CATGGATCCTTTGGATTTTGATTCCAG-TTGATCCCTGGAGTAAGGTCCTAACCGGGGT 232 VAT1 GCTGATGCCATGAAACAGATGCAGGAGAA-GAAGAATGTGGGCAAGGTCCTCCTGGTTCCAG 238 SNRPN GTGGCCAGCTCACCACCACCTGA-TGAAAGATACACCACAGGGTGAGAGCATCCTAACAGCAAAC 244 DNAH5 GTTTCAAAGGAAAGTCAGTGCTGTGAAAAT-TGACCTGGAAAAAAGCTGTACCATGCCCTCCTGGCAG 250 SNRPN GAGGGAGCTGGGACCCCTGCA-CTGCGGCAAACAAGCACGCCTGCGCGGCCGC 256 SNRPN CCTTGGTTTGCGGCAGAAGAATCTGCA-TTTCGAACAAGTGCCAGGACTGGTCTGAGGAACACACGTT 264 CYP3A5 GAATTCCAGGGCCCACACCTCTGCCTTTG-TTGGGAAATGTTTTGTCCTATCGTCAGGTGAGTTGCTTGAGCTT 270 SNRPN CAGGGATTTGAGAAGAGGGGATTCAGA-TGACAGAGAACATCCCAAGGGAGAAGCAGAATCCAATATAGAG 278 SNRPN CACAGAGATCTGGGAGAGCTGACTTTGGTGAAA-ATTCTCACCTTGCACAGGCTTGTGTCTGTTTTCCAGAATC 287 SNRPN GACCTTGCCCCCGCAAACCA-CACAGAAGGCTCTGAGCTCAGCTACAGTGGCTGC 294 SNRPN CCTGGTTTTTGCTTGGAATCAGATTCCTCGCTA-CTCCAATATGGCTTTAACCACCTCTTGGTGTCTCAGCTAA 301 UBE3A GAAAATCCTGCAGACTTGAAGAAGCAGTTG-TATGTGGAATTTGAAGGAGAACAAGGAGTTGATGAGGGAGGT 309 NF2 GGCAGATCAGCTGAAGCAGGA-CCTGCAGGAAGCACGCGAGGCGGAGCGAAGAGCCAAGCAG 319 CHEK1 CACGACAAAATGGTCAGGTTTAGAATAGTTGT-TCGTGGTTGAAAGACTTGGATAAATGTGGATGAGATAGAA 328 SNRPN GGTGATGGATACTCCAGGTGGGAATGAGAA-AGCGCCATACATGTGTTGACTTGGTACCCTAAGA 337 DLEU1 AGGAGGTTGTTTGCTGTACTCTCCCTTGT-ACAGTTAGCTGTCTCTAGTGCCTGAATGCACTAATTGTCCTTT 346 # BLM GCGGGGTCGCCGTACAGCGC-CGGGAGGGACGCGTATCTCCAAAGCCCAATCAGAGTC 355 UBE3A GGAGTTCTGGGAAATCGTTCATTCATTTAC-AGATGAACAGAAAAGACTCTTCTTGCAGTTTACAACGGGCACA 366 ATP10A GAGAGGCAGATGAACTGCGACGT-GCTCTGGTGTGTCCTGCTCCTTGTTTGCATGTCT 373 UBE3A CAGGAGAACCTCAGTCTGACGACATTG-AAGCTAGCCGAATGTAAGTGTAACTTGGTTGAGACTGTGG 382 GABRB3 CTGAGCTTTCGGTTGAAGAGGAACATTGGA-TACTTCATTCTTCAGACTTATATGCCCTCTATACTGATAACGA 391 SNRPN GAAGTGGCATATATCCAAGGACAGCCATAA-TATTCCACTTGGCCAAGGCTCTTCTGACAGGCTCATG 400 SNRPN GCGAAGCAACCAGAGCGTGAAGAA-AAGCGGGTTTTGGGTCTGGTGTTGCTGCGTG 409 MAGEL2 GCATTCCTGGAAACCAGCAAGATGCTT-GTCCTGAGGTTTTTGGCCAAGCTCCATAAGAAAGATCCAC 419 NDN CTTGCCAGACGGCGCAGACAT-GTCAGAACAAAGTAAGGATCTGAGCGACCCTAACTTTGCAGCC 427 DPYD CAATATGGAGCTTCCGTTTCTGCCAA-GCCTGAACTACCCCTCTTTTACACTCCTATTGATCTGGTGG 434 NIPA1 CAGTGGCTGTTGGCCAGATTGGAA-ACTTCCTGGCTTACACGGCGGTCCCCACGGTCCTGG 445 NDN GAAGGGTGGGGGTGGGTCATTAT-AGTATTCAGGATTTACAGTGCAGTATTCACGTGTAACTTT 454 SLC6A5 CTGGGGCCCATTCTTAGCTCA-ACACCGCGGGGAGCGTTACAAGAACATGATCGAC 463 # MLH1 CTGCTGAGGTGATCTGGCGCAGA-GCGGAGGAGGTGCTTGGCGCTTCTCAGGCTCCTCCTCT 472 SNRPN TCACCTGCATGCTTCACATCCAGGCCAG-AGTGCCTCTGTTGTACCCTCTGTGTTATGAAGACATA 480 BRIP1 CAACACCTGAGCTCGGGTCATCAGAGAATA-GTGCCTCTAGTCCTCCCCGTTTCAAAACAGAGAAGATGGAAA # Digestion control, warns for insufficient digestion. HhaI-digestion can be considered sufficient when <10% of the signal remains in the digested reaction compared to the undigested reaction. The HhaI sites are marked with grey. Ligation sites are marked with Note: Please notify us of any mistakes: info@mlpa.com. SALSA MS- probemix ME028 Prader-Willi/Angelman Page 8 of 11

9 SALSA MLPA probemix ME028-B2 Prader-Willi/Angelman sample pictures * D y e S i g n a l Size (nt) Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng undigested human male control DNA analysed with SALSA MLPA probemix ME028-B2 Prader-Willi/Angelman (lot B2-0413) for the quantification of copy numbers. Arrows indicate the location of the two digestion control probes (open arrows) and the five probes detecting the imprinted 15q11 sequences (closed arrows). * The 184 nt probe indicated is unmethylated in most blood-derived DNA samples D y e S i g n a l * Size (nt) Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng digested human male control DNA analysed with SALSA MLPA probemix ME028-B2 Prader-Willi/Angelman (lot B2-0413) to determine the methylation status. Arrows indicate the location of the two digestion control probes (open arrows) and the five probes detecting the imprinted 15q11 sequences (closed arrows). * The 184 nt probe indicated is unmethylated in most blood-derived DNA samples SALSA MS- probemix ME028 Prader-Willi/Angelman Page 9 of 11

10 Table 5. Interpretation of copy number and methylation ratio results PWS Deletion PWS Disomy Reference AS Disomy AS Deletion Genomic situation of the 15q11 region * M_ MM PM PP P_ Copy number Copy number ratio % imprinted 100% 100% 50% 0% 0% Methylation ratio * In this row, the paternal and maternal copies of the 15q11 region are indicated with a P or M, respectively. Next to uniparental disomy, PWS/AS can also be caused by aberrant methylation due to imprinting defects. With the ME028 probemix no discrimination between uniparental disomy and imprinting defects can be made. Figure 3. Simplified schematic representation of results that may be obtained with PWS/AS samples. A cartoon of both copies of chromosome 15 in normal individuals (centre), PWS (left) and AS (right) is included at the top. A white box with a red cross indicates a deletion of the 15q11 region; indicates methylation; and and indicate the maternal and paternal copy (except in PWS/AS UPD samples), respectively. The top row of electropherograms schematically shows undigested samples, which are used for copy number analysis. The example shows three reference probes (,, ), three probes for the 15q11 region without a HhaI site (1, 2, 8), one probe for the 15q11 region with a HhaI site ( ), and one digestion control probe with a HhaI site ( ). Peaks with a reduced height relative to the reference (normal) sample are indicated with blue arrows ( ). A reduced copy number ratio is only present in PWS/AS deletion samples. The bottom row of electropherograms schematically shows the accompanying samples that have been digested with HhaI. Compared to the undigested samples, only probes with a HhaI site are affected (, ). The digestion control ( ) is unmethylated, and disappears from the electropherograms (indicated with *). The peak height of the 15q11 probe with a HhaI site ( ) is reduced relative to the undigested samples in the reference sample and in AS samples, as indicated with a red arrow ( ). In PWS samples, the peak height of this probe is not reduced relative to the undigested samples, as indicated with a red square ( ). SALSA MS- probemix ME028 Prader-Willi/Angelman Page 10 of 11

11 Implemented Changes the following has been altered compared to the previous product description version(s). Version July 2015 (13) - Section about the interpretation of results expanded and clarified, including a new table and figure. - Various minor textual changes. - Various minor layout changes. Version 51 (12) - Extra information about genetic region of certain probes (PWS-SRO) added to Table 1 and 2. Version 50 (12) - Error corrected in description of digestion control probes. - Small textual changes. Version 49 (08) - This product description has been changed to incorporate a new lot (lot number added, new picture included). Version 48 (07) - Warning added in Table 1, 232 nt probe L08267, 409 nt probe L11839, 419 nt probe L13937, and 463 nt probe L Version 47 (05) - Information added about the digestion control probes on page 2 and table 1 and 2. - Information added to the figures about the 184 nt probe. Version 46 (05) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 45 (05) - p2: Remark on the availability of reference DNA samples - p2: Remark on partial digestion of the 346 nt DNA digestion control probe as an indication for incomplete HhaI digestion. Version 44 (05) - Added on page 6: More probes are available in SALSA MLPA probemix P343 Autism. Version 43 (05) - Warning on the 328 nt probe below tables 1 and 2 updated. This probe should only be used for copy number analysis. Version 42 (05) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new picture included). - Warning about variability updated under table 1 for NDN probes at 419 nt (04027-L13937) and 445 nt (11149-L11833). Version 41 (05) - Interpretation of methylation specific MLPA probes with regard to distinguishing uniparental disomy from imprinting defects has been changed. - Reference of Ramsden et al added. - Warning about SNPs added under table 1 for ATP10A probe at 366 nt. - Warning about variability added under table 1 for NDN probe at 455 nt. - Various minor textual and layout changes. Version 40 (05) - Remark added under table 2 about the role of the AS-SRO in AS. - Name of the HBII-85 snorna gene cluster adapted to the new nomenclature (SNORD116). - Small changes of probe lengths in Table 1, 2 and 3 in order to better reflect the true lengths of the amplification products. Version 39 (04) - Remark added in tables 1 and 2 that exon u5 is part of the AS-SRO. Version 38 (04) - This product description has been changed to incorporate a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). Various minor textual changes on page 1, data analysis has been modified, tables have been numbered and standard symbols have been added under Table 1. SALSA MS- probemix ME028 Prader-Willi/Angelman Page 11 of 11