MRC-Holland MLPA. Description version 05; 16 March 2018

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1 SALSA MLPA probemix P453-A2 GAA Lot A As compared to previous version A1 (lot A1-0514), one reference probe has been removed and one has been replaced. The GAA gene encodes the enzyme acid alpha-glycosidase (or acid maltase), which is active in lysosomes where the digestive enzyme breaks down glycogen. Mutations in this gene cause Pompe disease, a condition where the activity of acid alpha-glycosidase is significantly reduced. As a results, glycogen builds up to toxic levels in the lysosome. This build-up damages multiple organs and tissue, particularly the muscles, leading to progressive muscle weakness, heart problems, and other features. The GAA gene (20 exons) spans ~18.3 kb of genomic DNA and is located on 17q25.3, 76 Mb from the p- telomere. This P453 probemix contains one probe for each exon of the GAA gene, with the exception of exons 2 and 11. In addition, nine reference probes are included in this probemix, detecting several different autosomal chromosomal locations. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned GAA gene in a DNA sample. Heterozygous deletions of 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. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. Finally, note that most defects in this gene are expected to be small (point) mutations which will not be detected by this SALSA test. 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 Acids 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 References Giugliani, R et al., Current molecular genetics strategies for the diagnosis of lysosomal storage disorders. Expert Rev Mol Diagn 16.1: SALSA P453 GAA probemix Page 1 of 5

2 Data analysis The P453-A2 GAA probemix contains 27 MLPA probes with amplification products between 130 and 400 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 Positive control DNA samples: Inclusion of a positive sample in each experiment is recommended. Coriell Biobank ( and DSMZ ( have a diverse collection of biological resources which may be used as a positive control DNA sample in your MLPA experiments. Sample ID number NA16445 from the Coriell Institute, which has a large heterozygous duplication (6.5 Mb) that includes the complete GAA gene, has been tested at and can be used as a positive control sample. The quality of cell lines can change, therefore samples should be validated before use. 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. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P453 GAA probemix Page 2 of 5

3 Table 1. SALSA MLPA P453-A2 GAA probemix Length (nt) SALSA MLPA probe Chromosomal position reference GAA 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 probe L p Reference probe L q «GAA probe L26719 Exon «GAA probe L26720 Exon GAA probe L26721 Exon GAA probe L27067 Exon «GAA probe L27374 Exon Reference probe L q «GAA probe L27066 Exon GAA probe L26726 Exon «GAA probe L26727 Exon Reference probe L q GAA probe L26728 Exon «GAA probe L26729 Exon «GAA probe L26730 Exon * Reference probe L q «GAA probe L26731 Exon «GAA probe L26732 Exon «GAA probe L26733 Exon Reference probe L q «GAA probe L26734 Exon «GAA probe L26736 Exon Reference probe L q Ж «GAA probe SP0879-L27065 Exon «GAA probe L26738 Exon Reference probe L q Reference probe L q13 * New in version A2 (from lot A onwards). «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. Ж This probe consists of three parts and has two ligation sites. Note: Exon numbering used here may differ from literature! Please notify us of any mistakes. The identity of the genes detected by the reference probes is available on request: info@mlpa.com. SALSA P453 GAA probemix Page 3 of 5

4 Table 2. GAA probes arranged according to chromosomal location Length SALSA MLPA Ligation site Partial sequence (24 nt Distance to Exon (nt) probe NM_ adjacent to ligation site) next probe GAA, at 17q25.3. Ligation sites are indicated according to NM_ , which is a reference standard in the NCBI RefSeqGene project. Exon numbering is according to NG_ start codon (exon 2) 157 «19864-L26720 exon GACAGTGACCTC-GGTGACGCGAAG 4.0 kb No probe exon «19875-L26731 exon TCCCCACTCTAC-AGCGTGGAGTTC 1.8 kb 193 «20050-L27374 exon reverse CTGAGCATCAGG-GGACTGAGGTGC 0.2 kb 221 «19869-L27066 exon TGCTAAACAGCA-ATGCCATGGGTA 0.4 kb 148 «19863-L26719 exon CCCTGCCCTTAG-CTGGAGGTCGAC 0.2 kb 274 «19874-L26730 exon CTGCAGGATACC-CGTTCATGCCGC 0.3 kb 239 «19871-L26727 exon CACGTTCAACAA-GGATGGCTTCCG 1.3 kb 380 «19882-L26738 exon CCGCTGATTGGG-AAGGTAGGGCGA 0.8 kb 310 «19877-L26733 exon CATGGTGGCTGA-GTTCCATGACCA 1.2 kb No probe exon «19878-L26734 exon reverse AGAGGTTGTGCA-GGTTGTAGTGTG 0.7 kb 265 «19873-L26729 exon TCGCCTCCTCCG-TGCCAGGTGAGC 0.3 kb 301 «19876-L26732 exon reverse GTGGTTCCGCAT-GAAGGGGTAGAA 0.4 kb L26728 exon GCCCCTCTTCCT-GGAGTGAGTGAC 3.7 kb L26721 exon GGGAAGGCCGAA-GTGACTGGCTAC 0.6 kb 371 Ж «19881-SP and CCAGTAGAGGCC-26 nt spanning exon 17 L oligo-agctccccgtga 0.7 kb L26726 exon GCGAGGGGCCTA-CACACAGGTCAT 0.4 kb L27067 exon GTGTCCCTGTCT-CCAACTTCACCT 0.8 kb 349 «19880-L26736 exon AATAAGATTGTA-AGGTTTGCCCTC stop codon (exon 20) «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. Ж This probe consists of three parts and has two ligation sites. Note: 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 P453 GAA probemix Page 4 of 5

5 SALSA MLPA probemix P453-A2 GAA sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P453-A2 GAA (lot A2-0617). Implemented Changes compared to the previous product description versions. Version March 2018 (55) - Information added on positive control DNA samples on page 2. - Various minor textual changes. Version July 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). - New reference added on page 1. - Various minor textual changes. Version March 2017 (55) - Minor change: the LPO of the GAA exon 4 probe was erroneously named L This has been updated to L No difference in sequence detected between these two LPOs. Version January 2016 (55) - Various minor textual changes on page 1. Version 01 (47) - Not applicable, new document. SALSA P453 GAA probemix Page 5 of 5