MRC-Holland MLPA. Description version 16; 29 May 2017

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1 SALSA MLPA probemix P029-C1 Williams-Beuren Syndrome Lot C As compared to version B1 (lot B1-0315, B1-1211), two target probes have been removed, three reference probes have been replaced and three probe lengths have been adjusted. Williams-Beuren Syndrome (WBS) is an autosomal dominant disorder characterised by cardiovascular disease (supravalvular aortic stenosis (SVAS) and multiple peripheral pulmonary arterial stenosis), distinctive facial features, connective tissue abnormalities, intellectual disability (usually mild), a specific cognitive profile, unique personality characteristics, growth abnormalities and endocrine abnormalities. Williams- Beuren syndrome is estimated to occur at a frequency of approximately 1 in 7500 live births. More information is available on Deletions of the WBS critical region (WBSCR) on 7q11.23, including the ELN gene, are the main cause of WBS. Besides deletions of the WBS region, some duplications have also been described, giving rise to the Williams-Beuren duplication syndrome (OMIM ). The commonly deleted or duplicated chromosomal region has a size of 1.55 Mb (90-95%) or 1.84 Mb (5-10%) and is flanked by highly homologous DNA sequences. However, smaller deletions within the WBSCR, with a variable phenotype, have also been described. The P029-C1 probemix contains 9 probes detecting sequences in the ELN gene, 14 probes detecting sequences outside ELN but within the commonly deleted WBS region, and 5 probes detecting sequences in 7q11.23 but outside the WBS region. In addition, 9 reference probes are included in this probemix, detecting several different autosomal chromosomal locations. Possible copy number changes of this genomic region in healthy individuals can be found in the database of genome variants ( This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned chromosomal 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. Not all deletions and duplications detected by MLPA will be pathogenic; users should always verify the latest scientific literature when interpreting their findings. SALSA 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 test probemixes and reagents includes a limited license to use these products for research purposes. The use of a SALSA 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). More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands Related SALSA probemixes P245 Microdeletion Syndromes-1A: Contains a few probes for the ELN gene. P064 Microdeletion Syndromes-1B: Contains a few probes for the ELN gene. P374 Microdeletion Syndromes 8: Contains probes for the 7q11.23 region. SALSA P029 WBS probemix Page 1 of 7

2 References Dutra RL et al. (2015) Rare genomic rearrangement in a boy with Williams-Beuren syndrome associated to XYY syndrome and intriguing behavior. Am J Med Genet Part A. 167A: Honjo, RS et al. (2015) Williams-Beuren Syndrome: A Clinical Study Of 55 Brazilian Patients And The Diagnostic Use Of MLPA. BioMed Research International Sireteanu, A et al. (2014) Detection Of Chromosomal Imbalances Using Combined MLPA Kits In Patients With Syndromic Intellectual Disability. Romanian Review of Laboratory Medicine. 22: Dutra RL et al. (2012) Copy number variation in Williams-Beuren syndrome: suitable diagnostic strategy for developing countries. BMC Res Notes. 5:13. Beunders C et al. (2010) A triplication of the Williams Beuren syndrome region in a patient with mental retardation, a severe expressive language delay, behavioural problems and dysmorphisms. J Med Genet. 47: Van Hagen JM et al. (2007) Comparing two diagnostic laboratory tests for Williams syndrome: fluorescent in situ hybridization versus multiplex ligation-dependent probe amplification. Genet Test. 11: Depienne C et al. (2007) Autism, language delay and mental retardation in a patient with 7q11 duplication. J Med Genet. 44: Data analysis The P029-C1 WBS probemix contains 37 MLPA probes with amplification products between 132 and 427 nt. In addition, it contains nine control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at nt, three DNA Denaturation control fragments (Dfragments) 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. Data generated by this probemix can first be normalised intra-sample by dividing the peak height of each probe s amplification product by the total peak height of only the reference probes in this probemix (block normalisation). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalised probe ratio in a sample by the average intra-normalised probe ratio of all reference samples. Please note that this type of normalisation assumes no changes occurred in the genomic regions recognised by the reference probes. 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 Many copy number alterations in healthy individuals are described in the database of genomic variants: For example, a duplication of a complete gene might not be pathogenic, while a partial duplication or a deletion may result in disease. For some genes, certain in-frame deletions may result in a very mild, or no disease. Copy number changes of reference probes are unlikely to be the cause of the condition tested for. Users should always verify the latest scientific literature when interpreting their findings. This probemix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P029 WBS probemix Page 2 of 7

3 Table 1. SALSA MLPA P029-C1 WBS probemix Length (nt) SALSA MLPA probe Chromosomal position reference ELN 7q 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 132 Reference probe L q HSPB1 probe L09961 Exon HSPB1 probe L09963 Exon Reference probe L q ELN probe L21523 Exon * Reference probe L q POR probe L21866 Exon «STX1A probe L21869 Exon ELN probe L21525 Exon Reference probe L q HSPB1 probe L09962 Exon LIMK1 probe L21868 Exon LIMK1 probe L29891 Exon TBL2 probe L21872 Exon «FZD9 probe L29979 Exon CLIP2 probe L21530 Exon * Reference probe L q ELN probe L21529 Exon «STX1A probe L21880 Exon «LIMK1 probe L21534 Exon Reference probe L q ELN probe L21881 Exon ELN probe L21883 Exon * Reference probe L q ELN probe L21884 Exon RFC2 probe L21885 Exon ELN probe L21886 Exon «ELN probe L00879 Exon POR probe L13770 Exon Reference probe L p ELN probe L21887 Exon «TBL2 probe L21537 Exon «LIMK1 probe L21889 Exon CLIP2 probe L21888 Exon RFC2 probe L21890 Exon CLIP2 probe L00885 Exon Reference probe L p22 * 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. «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. Notes The LIMK1 exon numbering has changed. From description version 12 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequence (NG_ ) for this gene. This exon numbering used here may differ from literature! The exon numbering used in previous versions of this product description can be found between brackets in Table 2. The identity of the genes detected by the reference probes is available on request: info@mlpa.com. SALSA P029 WBS probemix Page 3 of 7

4 Table 2. P029 probes arranged according to chromosomal location Length (nt) SALSA MLPA probe Gene Exon Ligation site Partial sequence (24 nt adjacent to ligation site) Start of common chromosome 7q11.23 deletion region 240 «21191-L29979 FZD9 exon 1 NM_ ; reverse TTATTCCAGTCA-CAGCTTCCAGAG 382 «17587-L21537 TBL2 exon 7 NM_ ; 13 nt before ex 7 TGAGCTGTGATT-TTCTGTGTTGCA L21872 TBL2 exon 2 NM_ ; 22 nt before ex 2 AGTAACTGGAGA-ACAACATGGCCC 274 «17582-L21880 STX1A exon 7 NM_ ; 5 nt before ex 7 ACCCCACTCGCT-TGCAGCCGGCAG 183 «20987-L21869 STX1A exon 3 NM_ ; reverse GAATCTCCTCCA-CCTGCGGACCAA Distance to next probe kb 3.6 kb kb 4.9 kb kb ELN NM_ start codon (ex 1) L21884 Exon 3 24 nt before ex 3 CCGATGATCTCT-CTTTCTCTTTCT 1.2 kb L21523 Exon reverse CTTACCTGGCTT-AAGAGGTTTGCC 5.0 kb L21886 Exon GGTGGAGTGGCT-GACGCTGCTGCA 1.2 kb L21529 Exon 9 22 nt before ex 9 TGCTGCTAGTAA-CTTTGCTTTCTT 8.0 kb L21525 Exon CAATTCCTGGAA-TTGGAGGCATCG 4.5 kb L21887 Exon TTTCCCGGCTTT-GGTGTCGGAGTC 4.8 kb L21881 Exon nt after ex 26 TGCACCCCACAA-CCACTTGTGGCT 2.0 kb L21883 Exon nt before ex 27 GAGACCCATCGT-TCAGAAATGGAA 5.6 kb 346 «01335-L00879 Exon ACCTCATCAACG-TTGGTGCTACTG 17.1 kb stop codon (ex 33) 281 «17584-L21534 LIMK1 exon reverse CCAGTCCGCGTT-CAGGGCCTGGAG 11.3 kb 390 «01337-L21889 LIMK1 ex 4 (5) TGTGGGACCTTT-ATCGGTGACGGG 10.8 kb L29891 LIMK1 ex 9 (10) TGATGGTGATGA-AGGAGCTGATCC 8.0 kb L21868 LIMK1 ex 13 (14) TGGACGAGAAGA-CTCAGCCTGAGG kb L21885 RFC2 exon 10 NM_ ; reverse TGCCACAAGTGA-GCAAGAATCTAG 14.0 kb L21890 RFC2 exon 3 NM_ ; 48 nt after ex 3 TGTCTCATCCCA-CTGTCCGCTGAA kb L21888 CLIP2 exon 4 NM_ ; 10 nt before ex 4 GAGTCTCCCTGT-GTGCCGACAGGT 22.9 kb L21530 CLIP2 exon 10 NM_ ; AACAGGAGGTCG-AGAGTTTGCGGG 20.4 kb L00885 CLIP2 exon 14 NM_ ; GGAGGCCAATCG-TCACTCCCCAGG kb End of common chromosome 7q11.23 deletion region L21866 POR exon 11 NM_ ; CACCCATTCCCG-TGCCCTACGTCC 0.4 kb L13770 POR exon 12 NM_ ; CCTCATCCTCCA-AGGTGAGGGCCG kb L09961 HSPB1 exon 1 NM_ ; reverse CTGACTCTGCTC-TGGACGTCTGCT 1.2 kb L09962 HSPB1 exon 2 NM_ ; CTTCACGCGGAA-ATACACGTGAGT 0.3 kb L09963 HSPB1 exon 3 NM_ ; reverse GCGGCAGTCTCA-TCGGATTTTGCA Changed in version C1 (from lot C onwards). Small change in length, no change in sequence detected. SALSA P029 WBS probemix Page 4 of 7

5 «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 NM_ sequence for ELN represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Notes The LIMK1 exon numbering has changed. From description version 12 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequence (NG_ ) for this gene. This exon numbering used here may differ from literature! The exon numbering used in previous versions of this product description can be found between brackets in Table 2. Exon numbering used here may differ from literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA P029 WBS probemix Page 5 of 7

6 SALSA MLPA probemix P029-C1 WBS sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P029-C1 WBS (lot C1-0517). SALSA P029 WBS probemix Page 6 of 7

7 Implemented Changes compared to the previous product description version(s). Version May 2017 (55) - 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). - Various minor textual changes. - Related products updated. - Two references added. - Warnings added in Table 1 and 2, 183 nt probe L21869 and 382 nt probe L Version January 2017 (55) - Warning added in Table 1, 274 nt probe L21880 and 346 nt probe L Version December 2015 (55) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - New reference added on page 2. - Figure 2 removed. Version August 2015 (54) - Various minor textual changes. - Figure(s) based on the use of old MLPA buffer (replaced in December 2012) removed. Version May 2015 (54) - Table 2 has been updated with a new ELN NM number and ligation sites, LIMK1 exon numbers, and RFC2 NM number and ligation sites. - Table 1 has been updated with new LIMK1 exon numbers. - Various minor textual changes on page 1. Version 11 (53) - Warning added in Table 1, 346 nt probe L Updated link for Database of genomic variants. - Peak area replaced with peak height. Version 10 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 09 (48) - Product description adapted to a new version (lot number added, changes in Table 1 and Table 2, new picture included). - New references and target probes added on page 1. - Various textual and layout changes. - Remark on RefSeqGene standard added below Table 2. Version 08 (46) - Various minor textual changes. - Reference added on page 1. - Ligation sites of the probes targeting the FKBP6, FZD9, TBL2, STX1A, LIMK1, RFC2 and CLIP2 genes added to table 2. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Warning added in Table 1, 247 nt probe (01164-L00720), 310 nt probe (01333-L00876) and 391 nt probe (01160-L00716). - The CYLN2 gene name changed to CLIP2. - The text Many copy number alterations in healthy individuals are described in... added to page 2. - Additional information added on gene FZD2 on page 2. - Change in disorder name from Williams to WBS. Version 07 (44) - Various minor textual changes on page 1. - Minor changes in the data analysis section on page 2. - Tables have been numbered. - Data analysis paragraph When only a small has been changed. - Added between brackets which standard product description has been used for the update of the product description, e.g. 03 (43). SALSA P029 WBS probemix Page 7 of 7