MRC-Holland MLPA. Description version 08; 30 March 2015

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1 SALSA MLPA probemix P351-C1 / P352-D1 PKD1-PKD2 P351-C1 lot C1-0914: as compared to the previous version B2 lot B one target probe has been removed and three reference probes have been replaced. P352-D1 lot D1-0215: as compared to the previous version C1 lot C one target probe has been removed and two reference probes have been replaced. Adult polycystic kidney disease (PKD) is an autosomal disorder with the cardinal manifestations of renal cysts, liver cysts, and intracranial aneurysm. Genetic heterogeneity is recognized, with one locus (PKD1), responsible for the most common form, being tightly linked to markers on the extreme distal portion of 16p in the region of the alpha-haemoglobin locus. There is evidence that PKD may arise from mutations in any of several human disease loci, e.g. PKD1 on chromosome 16 and PKD2 on chromosome 4. The PKD1 gene (46 exons), spans ~47 kb of genomic DNA and is located at chromosome 16p13.3, only 2.1 Mb from the p-telomere. DNA analysis of the PKD1 gene is complicated due to the presence of several PKD1 pseudogenes on 16p13.11 at 16 Mb from the p-telomere. The PKD2 gene (15 exons), spans ~70 kb of genomic DNA and is located at chromosome 4q22.1, 88.9 Mb from the p-telomere. This P351-C1/P352-D1 PKD1-PKD2 probemixes contain PKD1 probes for 36 of the 46 exons and 17 PKD2 probes covering all PKD2 exons with the exception of exon 13. Two probes are present for PKD2 exons 1, 2 and 6. In addition, 3 probes are added to the P351 probemix for the TSC2 gene located just downstream of PKD1 and 11 reference probes are included in each probemix, detecting several different autosomal chromosomal locations. Please note that these P351 and P352 probemixes are different from the probemixes used by Consugar, M.B. et al. (2008, Kidney Int 74: ) and Kozlowski, P. et al. (2008, Genomics 91: ). According to Consugar et al., a considerable portion of PKD1 deletions are mosaic cases. These are challenging cases to detect as the MLPA probe signals will show only a small reduction in mosaic samples. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned genes 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 Acid Research 30, e57 (2002). Related SALSA probemixes P046 TSC2: Contains probes for TSC2 gene P337 TSC2: Contains probes for TSC2 gene, used as confirmation of P046 results P341-P342 PKHD1: Contains probes for PKHD1 gene, responsible for the recessive form of PKD 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 probemix P351/P352 PKD1-PKD2 Page 1 of 8

2 Data analysis The P351-C1 PKD1 probemix contains 41 MLPA probes with amplification products between 135 and 459 nt. The P352-D1 PKD1-PKD2 probemix contains 41 MLPA probes with amplification products between 136 and 471 nt. In addition, both probemixes contain 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. 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 probemix P351/P352 PKD1-PKD2 Page 2 of 8

3 Table 1. SALSA MLPA P351-C1 PKD1 probemix Length Chromosomal position SALSA MLPA probe (nt) reference PKD 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 135 Reference probe L q ± PKD1 probe L24677 Exon ± PKD1 probe L16105 Exon ± PKD1 probe L24689 Exon ± PKD1 probe L24698 Exon Reference probe L p ± PKD1 probe L24681 Exon ± PKD1 probe L24696 Exon ± PKD1 probe L24700 Exon Reference probe L p ± PKD1 probe L27456 Exon ± PKD1 probe L24699 Exon Reference probe L p ± PKD1 probe L24690 Exon ± PKD1 probe L24702 Exon ± PKD1 probe L24687 Exon Reference probe L q ± PKD1 probe L24686 Exon ± PKD1 probe L24694 Exon ± PKD1 probe L27362 Exon ± PKD1 probe L24692 Exon Reference probe L q ± PKD1 probe L24688 Exon ± PKD1 probe L24678 Exon ± PKD1 probe L24682 Exon * Reference probe L q ± PKD1 probe L24693 Exon ± PKD1 probe L27363 Exon ± PKD1 probe L24685 Exon Reference probe L p ± PKD1 probe L24683 Exon ± TSC2 probe L24630 TSC2 Exon ± PKD1 probe L24697 Exon Reference probe L p ± PKD1 probe L24679 Exon ± PKD1 probe L24691 Exon ± TSC2 probe L24628 TSC2 Exon * Reference probe L q ± TSC2 probe L24629 TSC2 Exon ± PKD1 probe L24695 Exon * Reference probe L q14 * New in version C1 (from lot C onwards). ± These probes are 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: - Nearly all probes have a small change in length, no change in sequence detected. - 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 probemix P351/P352 PKD1-PKD2 Page 3 of 8

4 Table 2. SALSA MLPA P352-D1 PKD1-PKD2 probemix Length Chromosomal position SALSA MLPA probe (nt) reference PKD1 PKD 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 136 Reference probe L q ± PKD2 probe L24648 Exon PKD2 probe L24659 Exon ± PKD1 probe L24666 Exon ± PKD1 probe L24674 Exon PKD2 probe L24658 Exon Reference probe L q PKD2 probe L24661 Exon ± PKD1 probe L11651 Exon ± PKD1 probe L27419 Exon PKD2 probe L24654 Exon * Reference probe L q PKD2 probe L24652 Exon PKD2 probe L24653 Exon Reference probe L q ± PKD1 probe L24667 Exon ± PKD1 probe L24664 Upstream 263 PKD2 probe L20398 Exon ± PKD1 probe L24669 Exon Reference probe L q ± PKD1 probe L24668 Exon ± PKD2 probe L24647 Exon * Reference probe L q ± PKD1 probe L24671 Exon PKD2 probe L24656 Exon Reference probe L q PKD2 probe L24651 Exon PKD2 probe L24657 Exon Reference probe L p PKD2 probe L24650 Exon PKD2 probe L24655 Exon ± PKD2 probe L24646 Exon Reference probe L q ± PKD1 probe L24675 Exon ± PKD1 probe L24673 Exon PKD2 probe L24662 Exon Reference probe L q ± PKD1 probe L24663 Upstream 452 ± PKD1 probe L24670 Exon PKD2 probe L24649 Exon Reference probe L q26 * New in version D1 (from lot D onwards). ± These probes are 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: - Nearly all probes have a small change in length, no change in sequence detected. - 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 probemix P351/P352 PKD1-PKD2 Page 4 of 8

5 Table 3. P351/P352 probes arranged according to chromosomal location Table 3a. PKD1 gene Length (nt) SALSA MLPA PKD1 Ligation site Partial sequence (24 nt Distance to P351/P352 probe exon NM_ adjacent to ligation site) next probe start codon (ex 1) L24663 Upstream 2026 nt before exon 1 TCTTCCCTGAAC-CAAACAAGATCT 0.5 kb L24664 Upstream 1736 nt before exon 1 CATTGAGACTTG-GTTAATCTGTTT 18.2 kb L24677 Exon 3 10 nt before exon 3 CCGAATCCCCCG-TCTCTTCCAGGG 1.1 kb L24678 Exon GAGACGGCTCCG-CCGAGGTGGATG 0.5 kb L11651 Exon GGAGCAGTGTCA-GGCCTGGGCCGG 0.7 kb L24666 Exon CTGCGAGCTGCA-GCCCGGAGGTGT 0.7 kb L24679 Exon 9 64 nt before exon 9 GAAGTTCGGGTA-GGGGGAGTCTGG 0.9 kb L27362 Exon nt after exon 10 GGGTCTGTGCAC-CAGACACACCCA 1.0 kb L24681 Exon TGCCGTGGCTCA-GTGAGGGGGAGC 1.0 kb L24682 Exon nt before exon 12 GGCTGACACCAT-TCCCCCCGCAGA 0.4 kb L24683 Exon AACTACAACGTA-ACCGTGGAGCGG 0.4 kb L27456 Exon nt before exon 14 TCACTCACTGCG-TCCCACCGCCCC 1.0 kb L24667 Exon GAGCTCCGCGGA-CTCAGCGTGGAC 1.3 kb L24685 Exon reverse GTGCCATCCCTA-ACCACGGCCTGC 1.5 kb L24686 Exon ACAGCGCAACTA-CTTGGAGGCCCA 0.9 kb L24687 Exon nt after exon 16 GTGGAGGCCGCA-CGCTCTCCCCTC 1.2 kb L24688 Exon ACCTTCACGCTC-ACGGTGCTGGGC 0.3 kb L24668 Exon GCAGCCTCTCCA-GCTACGGAGCCG 0.4 kb L24689 Exon nt after exon 20 TGGGAGGGGACG-TCACATCTGCTG 0.3 kb L24669 Exon nt before exon 21 CGGCCTCCTGCG-CTGCTGACAGCT 0.9 kb L24670 Exon GGCCATGATGCT-CATCCTGCAGGC 0.7 kb L24690 Exon GGCACCACAGCC-CTCAGAGCTGGG 1.3 kb L24691 Exon GCGAGGAGGACA-TGGTGTGGCGGA 0.5 kb L24671 Exon nt after exon 26 GTGAGGGGCGCA-GCGGGGTGGCAG 1.4 kb L24692 Exon nt before exon 27 CTTGAGTGCGCA-CAGGCCAAAGCT 0.6 kb L24693 Exon GACGCAGCCCTT-TTGCGCTTCCGG 0.6 kb L24694 Exon nt after exon 30 GAAGCCAGAATG-GTGAAAGAACGA 1.6 kb L27419 Exon 31 7 nt after exon 31 CTGAGGTGAGGA-CTCTACTGGGGG 0.5 kb L24673 Exon GCCAAATCCTTC-TCAGCATCAGGT 3.1 kb L24695 Exon nt before exon 35 CTGCCTCCTGGA-GGCCGGGATGAA 0.3 kb L24674 Exon AGGGTCTGCGGA-AGCGCCTGCTGC 0.4 kb L24675 Exon TGCACTCTTCCT-GGCCAAGGAAGA 0.6 kb L24696 Exon CAAAGCGCCATC-AAGCAGGAGCTG 0.4 kb L24697 Exon TCTGGCCATGGA-TGGCCCACGTGC 0.5 kb L16105 Exon AGCACCAGCGAT-TACGACGTTGGC 0.3 kb L24698 Exon GAGCTGGGCCTG-AGCCTGGAGGAG 0.3 kb L24699 Exon CTGTGTTCCTGG-AGCTCACGCGCT 0.4 kb L24700 Exon TCGCCGTGCACT-TCGCCGTGGCCG 0.4 kb L27363 Exon CCAGTGGTCCGT-CTTTGGCAAGAC 1.3 kb L24702 Exon ACGGTTTCTAGC-CTCTGAGATGCT 1.2 kb stop codon (ex 46) L24630 TSC2 exon 41 GCTCCGCCACAT-CAAGCGGCTCCG 1.5 kb L24629 TSC2 exon 37 GGCAACGACTTT-GTGTCCATTGTC 1.5 kb L24628 TSC2 exon 35 AGATCCCATCAT-ACGACACCCACA Note: The complete PKD1 gene is difficult to denature but the region between PKD1 exon 31 and TSC2 exon 40 is even more difficult to denature due to an extremely high % GC. This may cause false positive results in samples containing salt (for more information: -> MLPA procedure -> Sample preparation). The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. SALSA probemix P351/P352 PKD1-PKD2 Page 5 of 8

6 No probes are present for exons 1, 2, 4, 8, 17, 24, 28, 32, 34 and 45. In many cases this is due to the existence of a PKD1 pseudogene with very high similarity to the real gene. Because of the high similarity between this pseudogene and some of the probe target sequences, apparent deletions of single probes may often be false positives. Feedback on the performance of this product is very much appreciated. Table 3b. PKD2 gene Length (nt) SALSA MLPA probe PKD2 exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 1) 388 ± L24646 Exon GTGAACTCCAGT-CGCGTGCAGCCT 0.2 kb 297 ± L24647 Exon GGAGATCGAGAT-GCAGCGCATCCG 11.6 kb 142 ± L24648 Exon AGCACTAACCGA-GAGAAATACCTT 0.1 kb L24649 Exon 2 2 nt after exon 2 TGTGCATCTGTA-AGTAGAATATTT 16.2 kb L24650 Exon nt before exon 3 TATTGCAGGGAG-AGTAGGCTGGTG 2.5 kb L24651 Exon GGCTGTACTGGA-AGATGCAGCCCA 5.0 kb L24652 Exon TGGATTTGTCAA-GAACAAGAGAGG 3.4 kb L20398 Exon TGTCACAACTTT-TGATTTCTTCCT 0.1 kb L24653 Exon CGCATTCACAAA-CTACACTATTTC 5.2 kb L24654 Exon AGTTTCTGGAAG-ATCAAAATACTT 4.1 kb L24655 Exon GCCAGCTCTCGA-CAACCATGTCTC 1.9 kb L24656 Exon GGAAGCTAATCG-AGTTTTGGGACC 3.9 kb L24657 Exon CTGACTTGGCAC-AGCAGAAAGCTG 3.5 kb L24658 Exon AGGAGGAGGCAA-GTTAAACTTTGA 0.4 kb L24659 Exon GATGGAGACCAA-GAACTGACCGAA 9.0 kb No probe Exon L24661 Exon CATAGTGTCCAA-GATTGACGCCGT 0.7 kb L24662 Exon CAGCTTCCCAGA-TCAGTCATGGTT stop codon (ex 15) ± These probes are 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 is a reference standard in the NCBI RefSeqGene project. Notes: - Nearly all probes, both PKD1 and PKD2, have a small change in length, no change in sequence detected. - 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 probemix P351/P352 PKD1-PKD2 Page 6 of 8

7 SALSA MLPA probemix P351-C1/P352-D1 PKD1-PKD2 sample pictures Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA probemix P351-C1 PKD1 (lot C1-0914). Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA probemix P352-D1 PKD1-PKD2 (lot D1-0215). SALSA probemix P351/P352 PKD1-PKD2 Page 7 of 8

8 Implemented Changes compared to the previous product description version(s). Version March 2015 (54) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 2 and Table 3, new picture included). Version 07 (48) - Warning added in Table 1, 427 nt probe L Version 06 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 05 (48) - Remark on RefSeqGene standard and transcript variant added below Table 2. - Various minor textual changes on pages 1. Version 04 (46) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 2 and Table 3, new picture included). - Various minor textual changes and Tables 3a and 3b have been numbered. SALSA probemix P351/P352 PKD1-PKD2 Page 8 of 8