mix P169-C2 HIRSCHSPRUNG-1 Lot C2-0915. 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 : www.mlpa.com E-mail : 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
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 64-70-76-82 nt, three DNA Denaturation control fragments (D-fragments) at 88-92-96 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 www.mlpa.com. Many copy number alterations in healthy individuals are described in the database of genomic variants: http://dgv.tcag.ca/dgv/app/home. 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
Table 1. P169-C2 Hirschsprung-1 mix Chromosomal position reference ZEB2 GDNF RET EDN3 64-70-76-82 Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA 88-92-96 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 16316-L18705 3q21 136 ± ZEB2 05478-L04901 Exon 1 141 RET 18025-L06390 Exon 4 148 Reference 16544-L19035 11q13 154 ± RET 05505-L05592 Exon 11 160 ZEB2 05485-L04908 Exon 6 166 ± GDNF 05515-L04938 Exon 1 172 ± ZEB2 18078-L23060 Exon 2 178 ± RET 05506-L04929 Exon 12 183 ± RET 18079-L23061 Exon 19a 190 ZEB2 18080-L22486 Exon 3 196 RET 05497-L04920 Exon 3 202 RET 18026-L22380 Exon 6 208 Reference 10730-L11312 6p12 214 ZEB2 05486-L22381 Exon 7 220 ± RET 05507-L22379 Exon 13 226 ± GDNF 05516-L22382 Exon 2 232 ± RET 05508-L20172 Exon 14 238 ± RET 18027-L23566 Exon 1 244 ZEB2 05481-L22731 Exon 3 250 ZEB2 05487-L22732 Exon 8 256 RET 05499-L23570 Exon 5 262 Reference 17579-L23911 7q11 268 ± RET 05509-L22733 Exon 15 275 ZEB2 05483-L23064 Exon 4 281 EDN3 18028-L23147 Exon 1 286 EDN3 05492-L22734 Exon 3 292 RET 18081-L23071 Exon 2 300 Reference 16027-L18204 12p13 307 Ж EDN3 18082-SP0606-L22488 Exon 2 317 ZEB2 05488-L22735 Exon 9 324 GDNF 12957-L22736 Exon 2 331 Ж RET 18331-SP0609-L23070 Exon 7 338 ± RET 07288-L22737 Exon 17 347 ZEB2 05484-L22740 Exon 5 355 EDN3 05493-L23065 Exon 4 364 Ж RET 18084-SP0607-L22490 Exon 9 373 RET 18546-L28617 Exon 8 381 Reference 12940-L23154 13q14 389 RET 18029-L04927 Exon 10 397 ZEB2 05489-L23067 Exon 10 406 GDNF 05518-L23068 Exon 3 416 ± ZEB2 06123-L22745 Exon 1 422 Reference 10394-L22748 9q34 433 ± RET 18030-L23567 Exon 18 444 EDN3 05494-L23569 Exon 5 452 ± RET 05514-L23069 Exon 20 460 ± Ж GDNF 18085-SP0608-L22491 Exon 2 469 ± RET 18330-L23098 Exon 16 479 Reference 13539-L23528 19p13 490 Reference 12461-L21828 22q12 Changed in version C2 (from lot C2-0915 onwards). Small change in length, no change in sequence detected. SALSA P169 Hirschsprung-1 mix Page 3 of 6
SNP rs200289472 could influence the 183 nt signal (18079-L23061). SNP rs199572076 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_014795.3 next start codon 523-525 (ex 2) 136 ± 05478-L04901 Exon 1 77-78 CAGAGAGAAACT-TGGCGATCACGT 0.3 kb 416 ± 06123-L22745 Exon 1 388-389 CTCCCCCACACT-TCGCGGCTTCTT 2.6 kb 172 ± 18078-L23060 Exon 2 474-475 GCGTGCTGCCGA-AGCAGGGCGCCG 87.4 kb 244 05481-L22731 Exon 3 (4) 627-628 GTGGACACAGGT-TCTGAAACAGAT 0.1 kb 190 18080-L22486 Exon 3 (4) 722-723 TAGTGTGCCCAA-CCATGAGTCCTC 5.1 kb 275 05483-L23064 Exon 4 (5) 891-892 ATGGGGCCAGAA-GCCACGATCCAG 19.9 kb 347 05484-L22740 Exon 5 (6) 1054-1055 TTATTTACCCAG-AAGCCCCTGAGG 0.9 kb 160 05485-L04908 Exon 6 (7) 1291-1292 GCACCCAGCTCG-AGCGGCATATGG 2.7 kb 214 05486-L22381 Exon 7 (8) 1356-1357 CAAGGAGCAGGT-AATCGCAAGTTC 2.5 kb 250 05487-L22732 Exon 8 (9) 2935-2936 ACACTCCAAACA-GCTTCTCTTCTG 2.3 kb 317 05488-L22735 Exon 9 (10) 3542-3543 ATGTGACAAGAC-ATTCCAGAAAAG 7.8 kb 397 05489-L23067 Exon 10 (11) 4915-4916 TGTTAAGAGGGT-AACATGGGTTAC stop codon 4165-4167 (ex 10) The NM_014795.3 sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2b. GDNF GDNF NM_000514.3 next start codon 201-203 (ex 2) 166 ± 05515-L04938 Exon 1 102-103 ATCAGCCCGGAT-GGGTCTCCTGGC 3.8 kb 460 ± Ж 18085-SP0608-49-50 and 79-80 GACGGGGGCGCG-30 nt spanning Exon 2 L22491 (NM_001190468.1) oligo-ggattagggcca 0.3 kb 226 ± 05516-L22382 Exon 2 (3) 28-29 (NM_199231.2) CTCCAAGTCCCT-GCTAACTTCTTG 0.7 kb 324 12957-L22736 Exon 2 (4b) 210-209 reverse CACGACATCCCA-TAACTTCATCTT 19.0 kb 406 05518-L23068 Exon 3 (5) 721-722 GAGTGACAAAGT-AGGGCAGGCATG stop codon 834-836 (ex 3) The NM_000514.3 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_001190468.1 transcript variant 3, and in the NM_199231.2 transcript variant 2 (and NM_001190469.1 transcript variant 4). SALSA P169 Hirschsprung-1 mix Page 4 of 6
Table 2c. RET RET NM_020975.4 next start codon 191-193 (ex 1) 238 ± 18027-L23566 Exon 1 166-167 CAGTCCCTCCAG-CCGTGGCCCCAG 23.4 kb 292 18081-L23071 Exon 2 482-483 TCCTCTACCTTA-ACCGGAGCCTGG 1.9 kb 196 05497-L04920 Exon 3 773-774 TGCCTGTGCAGT-TCTTGTGCCCCA 2.4 kb 141 18025-L06390 Exon 4 836-837 TGCCCTTCCGCT-GCGCCCCGGACA 1.5 kb 256 05499-L23570 Exon 5 1201-1202 TGGCCCAACGAG-ACCTCGGTCCAG 2.6 kb 202 18026-L22380 Exon 6 1299-1300 GAACCGCACCAT-GCAGCTGGCGGT 2.4 kb 331 Ж 18331-SP0609-1689-1690 and 8 nt CCAGGCCCAGCT-32 nt spanning Exon 7 L23070 after 7 oligo-tgctccagggag 0.7 kb 373 18546-L28617 Exon 8 1758-1759 TGCAGTCAGCAA-GAGACGGCTGGA 0.7 kb 364 Ж 18084-SP0607-50 nt and 20 nt GGTGGTGGGGGC-30 nt spanning Exon 9 L22490 before 9 oligo-ctgctgtgtgtc 0.8 kb 389 18029-L04927 Exon 10 2024-2025 CCTGCAACTGCT-TCCCTGAGGAGG 0.9 kb 154 ± 05505-L05592 Exon 11 2153-2154 TGCTGTCTGCCT-TCTGCATCCACT 2.0 kb 178 ± 05506-L04929 Exon 12 2351-2352 GGGAATTCCCTC-GGAAGAACTTGG 1.8 kb 220 ± 05507-L22379 Exon 13 2514-2515 CCTGCTGTCAGA-GTTCAACGTCCT 1.3 kb 232 ± 05508-L20172 Exon 14 2752-2753 CTCATCTCATTT-GCCTGGCAGATC 0.4 kb 268 ± 05509-L22733 Exon 15 2823-2824 CTTGGCAGCCAG-AAACATCCTGGT 1.9 kb 469 ± 18330-L23098 Exon 16 2983-2984 TACACCACGCAA-AGTGATGTGTAA 1.7 kb 338 ± 07288-L22737 Exon 17 2999-3000 GCAGATGGTCTT-TTGGTGTCCTGC 1.2 kb 433 ± 18030-L23567 Exon 18 3155-3156 TGCAATGCTGGA-AGCAGGAGCCGG 1.8 kb 183 ± 18079-L23061 Exon 19a 3354-3355 CCTCCCTTCCAC-ATGGATTGAAAA 2.4 kb 452 ± 05514-L23069 Exon 20 4383-4384 CCCAGAATTGCT-GACAGCAGAGGC stop codon 3533-3535 (ex 20) The NM_020975.4 sequence represents transcript variant 2 and is a reference standard in the NCBI RefSeqGene project. Table 2d. EDN3 EDN3 NM_207034.2 next start codon 387-389 (ex 1) 281 18028-L23147 Exon 1 208-209 GCAGCGCGCTCT-GAAAGTTTATGA 0.8 kb 307 Ж 18082-SP0606-494-493 and 470-469 GCTGCAGTGGGG-24 nt spanning Exon 2 L22488 reverse oligo-gcatccccagac 19.7 kb 286 05492-L22734 Exon 3 820-821 TGCGGGGCCACT-TCCAGGGAATCT 1.3 kb 355 05493-L23065 Exon 4 (4a) 962-963 ACAGACAAAGAA-GAGGAAGGGAAG 3.5 kb 444 05494-L23569 Exon 5 (5d) 2544-2545 GTTTGGCACCGT-GGCAAGATGGTA stop codon 1101-1103 (ex 5) The NM_207034.2 sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. SNP rs200289472 could influence the 183 nt signal (18079-L23061). SNP rs199572076 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
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 12 12 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 11 01 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