SALSA MLPA probemix P169-C2 HIRSCHSPRUNG-1 Lot C As compared to version C1 (lot C1-0612), the length of one probe has been adjusted.

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1 mix P169-C2 HIRSCHSPRUNG-1 Lot C As compared to version C1 (lot C1-0612), the length of one has been adjusted. Hirschsprung disease (HSCR), or aganglionic megacolon, is a congenital disorder characterised by the absence of enteric ganglia along a variable length of the intestine. The disease can be classified according to the length of the aganglionosis into short segment (accounts for 80% of the HSCR cases), long segment and total colonic aganglionosis forms. Dominant mutations in the RET gene, as well as recessive mutations in several other genes including the EDN3, GDNF and ZEB2 genes, are associated with HSCR. The RET gene encodes for one of the cell-surface receptor tyrosine kinases, which plays a role in cell growth and differentiation. The RET gene (20 s) spans ~53 kb of genomic DNA and is located on 10q11.21, ~43 Mb from the p-telomere. The P169-C2 mix contains one for each of the gene. The ZEB2 gene encodes for a member of the Zfh1 family and functions as a DNA-binding transcriptional repressor that interacts with activated SMADs. The ZEB2 gene (10 s) spans ~136 kb of genomic DNA and is located on 2q22.3, ~144 Mb from the p-telomere. This mix contains one for each of the gene and two s for 1 and 4. The EDN3 gene encodes for a member of the endothelin family. The EDN3 gene (5 s) spans ~26 kb of genomic DNA and is located on 20q13.32, ~59 Mb from the p-telomere. This mix contains one for each of the gene. The GDNF gene encodes for a neurotrophic factor. The GDNF gene (3 s) spans ~27 kb of genomic DNA and is located on 5p13.2, ~38 Mb from the p-telomere. This mix contains one for each of the gene and three s for 2. In addition, nine reference s are included in this mix, detecting several different autosomal chromosomal locations. This SALSA mix 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. Note that a mutation or polymorphism in the sequence detected by a can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some 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 s. 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 mixes 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 mixes and reagents includes a limited license to use these products for research purposes. The use of a SALSA mix 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 mixes P318 Hirschsprung-2: Contains s for the PHOX2B, GFRA3, GFRA2, GFRA1, EDNRB, NRTN, PSPN and SOX10 genes. 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 P169 Hirschsprung-1 mix Page 1 of 6

2 References Sánchez-Mejías, A. et al. (2010) Novel MLPA procedure using self-designed s allows comprehensive analysis for CNVs of the genes involved in Hirschsprung disease. BMC Med Genet. 11: 71. Núñez-Torres, R. et al. (2009) A novel study of Copy Number Variations in Hirschsprung disease using the Multiple Ligation-dependent Probe Amplification (MLPA) technique. BMC Med Genet. 10: 119. Data analysis The P169-C2 Hirschsprung-1 mix contains 51 MLPA s with amplification products between 130 and 490 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. Data generated by this mix can first be normalised intra-sample by dividing the peak height of each s amplification product by the total peak height of only the reference s in this mix (block normalisation). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalised ratio in a sample by the average intra-normalised ratio of all reference samples. Please note that this type of normalisation assumes no changes occurred in the genomic regions recognised by the reference s. Data normalisation should be performed within one experiment. Only samples purified by the same method should be compared. Confirmation of most s 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 s are unlikely to be the cause of the condition tested for. Users should always verify the latest scientific literature when interpreting their findings. This mix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P169 Hirschsprung-1 mix Page 2 of 6

3 Table 1. P169-C2 Hirschsprung-1 mix Chromosomal position reference ZEB2 GDNF RET EDN 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 ± ZEB L04901 Exon RET L06390 Exon Reference L q ± RET L05592 Exon ZEB L04908 Exon ± GDNF L04938 Exon ± ZEB L23060 Exon ± RET L04929 Exon ± RET L23061 Exon 19a 190 ZEB L22486 Exon RET L04920 Exon RET L22380 Exon Reference L p ZEB L22381 Exon ± RET L22379 Exon ± GDNF L22382 Exon ± RET L20172 Exon ± RET L23566 Exon ZEB L22731 Exon ZEB L22732 Exon RET L23570 Exon Reference L q ± RET L22733 Exon ZEB L23064 Exon EDN L23147 Exon EDN L22734 Exon RET L23071 Exon Reference L p Ж EDN SP0606-L22488 Exon ZEB L22735 Exon GDNF L22736 Exon Ж RET SP0609-L23070 Exon ± RET L22737 Exon ZEB L22740 Exon EDN L23065 Exon Ж RET SP0607-L22490 Exon RET L28617 Exon Reference L q RET L04927 Exon ZEB L23067 Exon GDNF L23068 Exon ± ZEB L22745 Exon Reference L q ± RET L23567 Exon EDN L23569 Exon ± RET L23069 Exon ± Ж GDNF SP0608-L22491 Exon ± RET L23098 Exon Reference L p Reference L q12 Changed in version C2 (from lot C onwards). Small change in length, no change in sequence detected. SALSA P169 Hirschsprung-1 mix Page 3 of 6

4 SNP rs could influence the 183 nt signal (18079-L23061). SNP rs could influence the 331 nt signal (18331-SP0609-L23070). In case of apparent deletions, it is recommended to sequence the region targeted by this. ± This is located within, or close to, a very strong CpG island. A low signal of this can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. Ж This 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 s is available on request: info@mlpa.com. Table 2. P169 s arranged according to chromosomal location Table 2a. ZEB2 ZEB2 NM_ next start codon (ex 2) 136 ± L04901 Exon CAGAGAGAAACT-TGGCGATCACGT 0.3 kb 416 ± L22745 Exon CTCCCCCACACT-TCGCGGCTTCTT 2.6 kb 172 ± L23060 Exon GCGTGCTGCCGA-AGCAGGGCGCCG 87.4 kb L22731 Exon 3 (4) GTGGACACAGGT-TCTGAAACAGAT 0.1 kb L22486 Exon 3 (4) TAGTGTGCCCAA-CCATGAGTCCTC 5.1 kb L23064 Exon 4 (5) ATGGGGCCAGAA-GCCACGATCCAG 19.9 kb L22740 Exon 5 (6) TTATTTACCCAG-AAGCCCCTGAGG 0.9 kb L04908 Exon 6 (7) GCACCCAGCTCG-AGCGGCATATGG 2.7 kb L22381 Exon 7 (8) CAAGGAGCAGGT-AATCGCAAGTTC 2.5 kb L22732 Exon 8 (9) ACACTCCAAACA-GCTTCTCTTCTG 2.3 kb L22735 Exon 9 (10) ATGTGACAAGAC-ATTCCAGAAAAG 7.8 kb L23067 Exon 10 (11) TGTTAAGAGGGT-AACATGGGTTAC stop codon (ex 10) The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2b. GDNF GDNF NM_ next start codon (ex 2) 166 ± L04938 Exon ATCAGCCCGGAT-GGGTCTCCTGGC 3.8 kb 460 ± Ж SP and GACGGGGGCGCG-30 nt spanning Exon 2 L22491 (NM_ ) oligo-ggattagggcca 0.3 kb 226 ± L22382 Exon 2 (3) (NM_ ) CTCCAAGTCCCT-GCTAACTTCTTG 0.7 kb L22736 Exon 2 (4b) reverse CACGACATCCCA-TAACTTCATCTT 19.0 kb L23068 Exon 3 (5) GAGTGACAAAGT-AGGGCAGGCATG stop codon (ex 3) The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Probes for alternative s 2 are present in the NM_ transcript variant 3, and in the NM_ transcript variant 2 (and NM_ transcript variant 4). SALSA P169 Hirschsprung-1 mix Page 4 of 6

5 Table 2c. RET RET NM_ next start codon (ex 1) 238 ± L23566 Exon CAGTCCCTCCAG-CCGTGGCCCCAG 23.4 kb L23071 Exon TCCTCTACCTTA-ACCGGAGCCTGG 1.9 kb L04920 Exon TGCCTGTGCAGT-TCTTGTGCCCCA 2.4 kb L06390 Exon TGCCCTTCCGCT-GCGCCCCGGACA 1.5 kb L23570 Exon TGGCCCAACGAG-ACCTCGGTCCAG 2.6 kb L22380 Exon GAACCGCACCAT-GCAGCTGGCGGT 2.4 kb 331 Ж SP and 8 nt CCAGGCCCAGCT-32 nt spanning Exon 7 L23070 after 7 oligo-tgctccagggag 0.7 kb L28617 Exon TGCAGTCAGCAA-GAGACGGCTGGA 0.7 kb 364 Ж SP nt and 20 nt GGTGGTGGGGGC-30 nt spanning Exon 9 L22490 before 9 oligo-ctgctgtgtgtc 0.8 kb L04927 Exon CCTGCAACTGCT-TCCCTGAGGAGG 0.9 kb 154 ± L05592 Exon TGCTGTCTGCCT-TCTGCATCCACT 2.0 kb 178 ± L04929 Exon GGGAATTCCCTC-GGAAGAACTTGG 1.8 kb 220 ± L22379 Exon CCTGCTGTCAGA-GTTCAACGTCCT 1.3 kb 232 ± L20172 Exon CTCATCTCATTT-GCCTGGCAGATC 0.4 kb 268 ± L22733 Exon CTTGGCAGCCAG-AAACATCCTGGT 1.9 kb 469 ± L23098 Exon TACACCACGCAA-AGTGATGTGTAA 1.7 kb 338 ± L22737 Exon GCAGATGGTCTT-TTGGTGTCCTGC 1.2 kb 433 ± L23567 Exon TGCAATGCTGGA-AGCAGGAGCCGG 1.8 kb 183 ± L23061 Exon 19a CCTCCCTTCCAC-ATGGATTGAAAA 2.4 kb 452 ± L23069 Exon CCCAGAATTGCT-GACAGCAGAGGC stop codon (ex 20) The NM_ sequence represents transcript variant 2 and is a reference standard in the NCBI RefSeqGene project. Table 2d. EDN3 EDN3 NM_ next start codon (ex 1) L23147 Exon GCAGCGCGCTCT-GAAAGTTTATGA 0.8 kb 307 Ж SP and GCTGCAGTGGGG-24 nt spanning Exon 2 L22488 reverse oligo-gcatccccagac 19.7 kb L22734 Exon TGCGGGGCCACT-TCCAGGGAATCT 1.3 kb L23065 Exon 4 (4a) ACAGACAAAGAA-GAGGAAGGGAAG 3.5 kb L23569 Exon 5 (5d) GTTTGGCACCGT-GGCAAGATGGTA stop codon (ex 5) The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. SNP rs could influence the 183 nt signal (18079-L23061). SNP rs could influence the 331 nt signal (18331-SP0609-L23070). In case of apparent deletions, it is recommended to sequence the region targeted by this. ± This is located within, or close to, a very strong CpG island. A low signal of this can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. Ж This consists of three parts and has two ligation sites. Notes: The NM_sequence (ZEB2) and numbering (ZEB2, GDNF and EDN3) has changed! From description version 12 onwards, we have adopted the NCBI numbering that is present in the NM_ sequences for these genes. The numbering used here may differ from literature! The numbering used in previous versions of this product description can be found between brackets in Table 2. Complete sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA P169 Hirschsprung-1 mix Page 5 of 6

6 mix P169-C2 Hirschsprung sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with mix P169-C2 Hirschsprung-1 (lot C2-0915). Implemented Changes compared to the previous product description versions. Version November 2015 (55) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new picture included). - Various numberings changed. - Refseq sequence and ligation sites EDN3 adjusted. Version July 2015 (54) - Figure based on the use of old MLPA buffer (replaced in December 2012) removed. Version 10 (49) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new picture included). Version 09 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 08 (47) - Various minor textual and layout changes on page 1 and 2. - Small changes of lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Remark on RefSeqGene standard and transcript variant added below Table 2. - Warning added below Table 2 that the numbering used is different from the NCBI numbering in the NM_ reference sequence. - s of the s targeting the GDNF and ZEB2 genes updated according to new version of the NM_reference sequence. - Data analysis method has been modified. Version 07 (45) - Various minor textual changes on page 1 and 2. - Name changed to Hirschsprung-1. - Sentence when only small numbers of samples are tested, visual comparison of peak profiles should be sufficient removed from data analysis section - Tables have been numbered. - Data analysis method has been modified. - Various minor layout changes. SALSA P169 Hirschsprung-1 mix Page 6 of 6