MRC-Holland MLPA. Description version 10; 16 March 2017

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1 SALSA MLPA probemix P199-B2 HEXA Lot B As compared to version B1 (lot B1-0410), two reference probes have been replaced and the control fragments have been adjusted (QDX2). Tay-Sachs disease is an autosomal recessive, progressive neurodegenerative disorder which, in the classic infantile form, is usually fatal by the age of 2 or 3. Defects in the HEXA gene encoding the alpha subunit of the hexosaminidase enzyme are the cause of Tay-Sachs disease. Some less severe defects in the HEXA gene cause a late onset form of the disease. Due to some founder mutations, Tay-Sachs disease is frequent among Ashkenazi Jews. This P199 probemix is primarily intended for the detection of copy number changes of (parts of) the HEXA gene but it also contains two probes that are specific for frequent Ashkenazi Jew founder mutations. The HEXA gene comprises 14 exons, spanning more than 33 kb of genomic DNA on chromosome 15q23, about 73 Mb from the p-telomere. This P199-B2 HEXA probemix contains 18 MLPA probes for HEXA sequences. Two of these probes are specific for certain frequent point mutations: 1278insTATC (also known as c.1274_1277duptatc (p.tyr427ilefs) and 1277TATC) and IVS12+1G>C (also known as c g>c). The 1278insTATC mutation is found in 80% of the carriers of Tay-Sachs disease from the Ashkenazi Jew population. A deletion of 7.5 kb, including all of exon 1, is the major mutation found in Tay-Sachs disease carriers from the French-Canadian population. In addition, 11 reference probes are included in this probemix, detecting several different autosomal chromosomal locations. SD030 Sample DNA Please note that the mutation-specific probes have only been tested on control plasmids and not on positive human DNA samples with the point mutation! This SD030 sample DNA is provided with each probemix vial and can be used in data binning in the fragment analysis and as a positive control for the mutation-specific probes (see next page). This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned gene and to detect the presence of the aforementioned mutations 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 these genes are expected to be small (point) mutations, most of 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 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 SALSA P199 HEXA probemix Page 1 of 6

2 Data analysis The P199-B2 HEXA probemix contains 29 MLPA probes with amplification products between 142 and 364 nt. Please note that two of these probes will not generate a signal on the majority of samples as they only detect DNA that contains the 1278insTATC mutation or the IVS12+1G>C mutation. These probes have been tested on artificial test DNA but not on positive human samples! 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 (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. SD030 Sample DNA The SD030 Sample DNA provided with this probemix can be used as Binning DNA sample for binning of HEXA 1278insTATC and IVS12+1G>C mutation-specific probes (HEXA probe L06309 and probe L06312, respectively). Inclusion of one reaction with SD030 DNA in MLPA experiments is recommended as it can aid in data binning of the peak pattern using Coffalyser.Net software and as an artificial positive control for the specific point mutations. Please note that SD030 DNA consists of female DNA mixed with plasmid DNA that contains the target sequences detected by the above mentioned probes + the sequence of the 105 nt chromosome Y specific control fragment. The amount of plasmid used (relative to the genomic DNA) results in a relative probe signal for the 105 nt probe on this female DNA which is similar to the relative probe signal obtained on male DNA samples. As a result, the 100 and 105 nt control fragments indicate the presence of two copies chromosome X and one copy chromosome Y and one copy of the mutation-specific probe (heterozygous mutation). The product description of the SD030 Sample DNA can be found on This product is for research use only. 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 P199 HEXA probemix Page 2 of 6

3 Table 1. SALSA MLPA P199-B2 HEXA probemix Length Chromosomal position SALSA MLPA probe (nt) reference HEXA 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 142 * Reference probe L p HEXA probe L06716 Exon HEXA probe L06304 Exon Reference probe L q HEXA probe L insTATC 172 HEXA probe L06312 IVS12+1G>C 179 Reference probe L q HEXA probe L06715 Exon HEXA probe L06302 Exon Reference probe L p HEXA probe L06998 Exon HEXA probe L06300 Exon Reference probe L q HEXA probe L16923 Exon HEXA probe L06298 Exon Reference probe L q HEXA probe L06306 Exon HEXA probe L06717 Exon * Reference probe L p HEXA probe L06313 Exon Reference probe L q HEXA probe L06307 Exon HEXA probe L06305 Exon Reference probe L p HEXA probe L06310 Exon HEXA probe L16924 Exon Reference probe L p HEXA probe L06315 Exon Reference probe L q35 * New in version B2 (from lot B onwards). Mutation-specific probe. This probe will only generate a signal when the 1278insTATC mutation is Mutation-specific probe. This probe will only generate a signal when the IVS12+1G>C mutation is 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 P199 HEXA probemix Page 3 of 6

4 Table 2. HEXA probes arranged according to chromosomal location Length (nt) SALSA MLPA probe HEXA exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (exon 1) L06998 exon ACCCGAACAACT-TTCAATTCCAGT 0.3 kb L06315 exon nt after exon 1 AGGCACTCCACT-TCCTCCTCGAGC 19.0 kb L06298 exon GAATGTGTTGGT-TGTCTCTGTAGT 1.0 kb L06716 exon 3 3 nt after exon 3 CTCTCCGAGGTA-ACAAATTGGGGC 1.8 kb L06300 exon TAGCCAGCTTGT-TTGGAAATCTGC 0.6 kb L06717 exon CTGCCACTCTCT-AGCATCCTGGAC 1.9 kb L06302 exon GTAGATGATCCT-TCCTTCCCATAT 0.6 kb L16923 exon reverse CAAACTCTGCAA-GCACACGGATAC 0.1 kb L16924 exon 7 24 nt after exon 7 TCTGGGACCAGA-GGGACTCTGCTT 1.4 kb L06304 exon GTCTTCCCAGAT-TTTTATCTTCAT 1.0 kb L06305 exon GTGAGGACTTCA-AGCAGCTGGAGT 0.3 kb L06306 exon TCGTCTCTTCTT-ATGGCAAGGGCT 1.1 kb L06307 exon GATATTCCAGTG-AACTATATGAAG 0.1 kb L06309 exon GAACCGTATATC-TATCCTATGGCC 0.3 kb L06310 exon reverse TTCTCCCCACAT-ACAAGCCTCTCC 0.1 kb L06312 exon 12 1 nt after exon 12 CCCAGGCTCTGC-TAAGGGTTTTCG 0.7 kb L06313 exon AGTTGACATCTG-ACCTGACATTTG 1.7 kb L06715 exon GCCTTTGTGCTG-TTCTGCCTTGCC stop codon (exon 14) Mutation-specific probe. This probe will only generate a signal when the 1278insTATC mutation is Mutation-specific probe. This probe will only generate a signal when the IVS12+1G>C mutation is The NM_ sequence is a reference standard in the NCBI RefSeqGene project. 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 P199 HEXA probemix Page 4 of 6

5 SALSA MLPA probemix P199-B2 HEXA sample pictures Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male DNA analysed with SALSA MLPA probemix P199-B2 HEXA (lot B2-0115). In case the 1278insTATC mutation or IVS12+1G>C mutation is present, a peak will appear at 166 nt or 172 nt, respectively. Figure 2. Capillary electrophoresis pattern of SD030 sample DNA (approximately 50 ng) analysed with SALSA MLPA probemix P199-B2 HEXA (lot B2-0115). The locations of the HEXA 1278insTATC and IVS12+1G>C mutation-specific probes are indicated. SALSA P199 HEXA probemix Page 5 of 6

6 Implemented Changes compared to the previous product description versions. Version March 2017 (55) - The c g>c mutation is renamed to IVS12+1G>C. - Ligation sites in Table 2 are updated according to the new version of the NM_ reference sequence. - Various minor textual and layout changes. Version 09 (54) - Product description adapted to a new product version (version number and lot number changed, small changes in Table 1 and Table 2, new pictures included). - Various minor textual changes. - The mutations described in the text are renamed to 1278insTATC (1277TATC) and c g>c (IVS12+1G>C) Version 08 (53) - Replaced peak area by peak height. - Updated link for Database of Genomic Variants. - SD information added on page 1 and 2. - SD electropherogram added. - Removed the terms old and new MLPA buffer. - Various minor textual changes. Version 07 (48) - Warning added under Table 1, 142 nt probe L Version 06 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 05 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 04 (46) - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Various minor textual changes on page 1. - Various minor layout changes. - Remark on RefSeqGene standard added below Table 2. - Small correction of chromosomal locations in Table 1. Version 04 (46) - 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). - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Ligation sites updated according to new version of the NM_reference sequence. SALSA P199 HEXA probemix Page 6 of 6