SALSA MLPA probemix P371-A1 Microdeletion Syndromes 5 Lot A1-0509

Transcription

1 mix P371-A1 Microdeletion Syndromes 5 Lot A The purpose of the P371 mix is to further investigate results found with the P245 Microdeletion mix. The P245 mix provides a possibility to screen samples for 21 different microdeletion syndromes in a single reaction. For confirmation of results obtained with this P245 mix, four different mixes are now available with additional s in these 21 regions: P371, P372, P373 and P374 Microdeletion Syndromes. The P371-A1 mix contains s for the 2p16 microdeletion syndrome region (8 s), the Langer- Giedion region on 8q24 (10 s), the 9q22 microdeletion syndrome region (5 s), the WAGR syndrome region on 11p13 (7 s), the 15q24 microdeletion syndrome region (9 s) and the 17q21 microdeletion syndrome region (8 s). This SALSA mix is designed to detect deletions/duplications of one or more sequences in the aforementioned regions in a DNA sample. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak area 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 area, 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 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 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 P245 Microdeletion syndromes 1: s for 21 different microdeletion syndromes. P297 Microdeletion syndromes 2: s for several recently described microdeletion syndromes (1q21.1- TAR; 2p16.1; 15q13; 16p11; 17q12). P372 Microdeletion syndromes 6 (confirmation P245): DiGeorge 22q11, DiGeorge-2 10p15, Rubinstein- Taybi (CREBBP), NF1 region, Sotos. P373 Microdeletion syndromes 7 (confirmation P245): 1p36, 3q29, Cri-du-chat, Wolf-Hirschhorn region, 22q13 Phelan-McDermid. P374 Microdeletion syndromes 8 (confirmation P245): Williams, Prader-Willi/Angelman, Miller-Dieker, Smith-Magenis, Xq28-MECP2-RETT syndrome. P064 MR-1: 1p36, Sotos, Williams, Prader-Willi/Angelman, Miller-Dieker, Smith-Magenis, Alagille, DiGeorge. P096 MR-2: Cri-du-chat, Wolf-Hirschhorn, Langer-Giedion, WAGR, Rubinstein-Taybi. More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands SALSA mix P371 Microdeletion Syndromes 5 Page 1 of 8

2 Data analysis The P371-A1 Microdeletion-5 mix contains 47 MLPA s with amplification products between 130 and 481 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 be normalized intra-sample by dividing the peak area of each amplification product by the combined peak area of all peaks in that sample (global normalization). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalized ratio in a sample by the average intra-normalized ratio of all reference samples. 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 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 mix P371 Microdeletion Syndromes 5 Page 2 of 8

3 Table 1. P371-A1 Microdeletion-5 mix Chromosomal position 2p16 8q24 9q22 11p13 15q24 17q 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 EIF3H L q PAX L p DCDC L p TRPS L q CSK L q CRHR L q MAPT L q BCL11A L p REL L p SEMA7A L q EIF3H L q TRPS L q XPO L p MAPT L q TGFBR L q TRPS L q CRHR L q CLK L q PAX L p TGFBR L q PEX L p CYP1A L q TGFBR L q EXT L q PAX L p EXT L q TRPS L q TGFBR L q MAPT L q PAX L p FANCL L p EXT L q TGFBR L q MAPT L q TRPS L q PAX L p PAX L p MAPT L q CYP1A L q REL L p CYP1A L q SEMA7A L q CCT L p MAPT L q PML L q PEX L p PML L q24.1 SALSA mix P371 Microdeletion Syndromes 5 Page 3 of 8

4 Table 2. P371 s arranged according to chromosomal location Table 2a. 2p16 region Gene L08247 FANCL ATTGTAGGCCAA-AGTCCAGTTGAA kb P L11411 FANCL kb from L L14411 BCL11A GCCACCTCTCCA-TGGGATTCATAT kb L14412 REL CAGGGGTTGGGA-AGGTGTGAGCCG 36.8 kb L09009 REL AACGATTGGGAA-GCAAAAGGCATC kb P L10628 REL kb from L L08260 PEX TTTCTTCTGGAT-ATGGTGCCTATG 14.2 kb L14419 PEX TGAGGATGACCA-TGTAGTTGCCAG kb L14556 XPO CATCCCATTGTA-AAGCGACTTCAA kb L09022 CCT AGTTGTATTGAA-ACAATTTAATGA The interstitial 2p16.1 microdeletion syndrome has been described by Rajcan-Separovic, E. et al. (2007, J Med Genet. 44: ). Phenotype is characterised by moderate to severe mental retardation. Table 2b. Langer-Giedion region L07025 TRPS1 exon GCGATCAAGCAT-TCCAGACCTGGG 4.0 kb L07023 TRPS1 exon TCTGGCGAAAGA-ATGCAAATGGCG kb L14413 TRPS1 exon CAAGGAAAGCAA-AGAACACTCATG 15.3 kb L07405 TRPS1 exon CCGTTCTGTGTT-TTCTGGTGTGCT 4.5 kb L14423 TRPS1 exon TAGCCTCCTGCT-AGTTGACTAATA kb P L07411 TRPS1 exon kb from L14423 P L00679 EIF3S3 gene kb from L L14786 EIF3H exon GCAATTCATGTT-TATCTGCAACAG 99.8 kb L14781 EIF3H exon 2 (1) TAGATGGCCTTG-TGAGTGCTGTTC kb L02480 EXT1 exon TCCCAGTGCAGA-TCCACTCATCAG 37.5 kb L06418 EXT1 exon ACAATGCCACTT-TCTGTCTGGTTC kb L06416 EXT1 exon CAAAAAACGCTA-TTTCATCCTGCT Most LGS patients have a microdeletion that includes the TRPS1 and EXT1 genes. More s for the Langer-Giedion region are present in the P215 EXT1 mix and the P228 LGS mix. More info in OMIM Table 2c. 9q22 region L14416 TGFBR1, exon TCTGCCACCTCT-GTACAAAAGACA 3.8 kb L14418 TGFBR1, exon GAGGGTACTACG-TTGAAAGACTTA 9.9 kb L04034 TGFBR1, exon TTGGTGTCAGAT-TATCATGAGCAT 2.3 kb L04035 TGFBR1, exon GATTCAGCCACA-GATACCATTGAT 4.4 kb P L04036 TGFBR1, exon kb from L04035 P L10006 TGFBR1, exon kb from L L04038 TGFBR1, exon GCCAATGGAGCA-GCTAGGCTTACA An interstitial 9q22.3 microdeletion syndrome that includes the TGFBR1 gene has been described by Redon, R. et al. (2006, Eur J Hum Genet. 14:759-67). Clinical phenotype is characterised by mental retardation, macrocephaly, overgrowth and trigonocephaly. More s for the TGFBR1 gene are present in the P148 TGFBR mix. SALSA mix P371 Microdeletion Syndromes 5 Page 4 of 8

5 Table 2d. 11p13 WAGR region L07345 DCDC1, exon TGGAGTGGCGTT-TACTACCAGTAA kb L05486 PAX6, exon 14 (13) TGAGCCACTGCT-TTCTGCAGGCTG 4.0 kb L05484 PAX6, exon 10 (9) AGTTCCAACGGA-GAAGATTCAGAT 6.1 kb L07346 PAX6, exon 9 (8) TCTTCGCAACCT-GGCTAGCGAAAA 0.8 kb L05483 PAX6, exon 8 (7) TGGGAAATCCGA-GACAGATTACTG 4.8 kb P L02690 PAX6, exon 6 (5) kb from L L07190 PAX6, exon 5 (4) GCCTCTGGTCTT-TCTGGGACTTCG 0.5 kb L07348 PAX6, exon 4 (3) GTCCACTCTCAC-AATAAAAGGCCT Most patients with WAGR syndrome have a chromosomal deletion that includes the PAX6 and WT1 genes. More s for the PAX6 gene / WAGR region are present in the P219 PAX6 mix and the P118 WT1 mix. More info in OMIM Aniridia is an easy to detect feature of the WAGR syndrome phenotype. Table 2e. 15q24 region L05759 PML CGGTGCGTGAGT-TCCTGGACGGCA 47.9 kb L09341 PML GTCAACAGCAGA-ACAGAGGCAGGT kb L14793 SEMA7A CTGCATCCTGTT-CATCGAGAACCT 8.2 kb P L10003 SEMA7A kb from L L15053 SEMA7A GGGACCTGGCTT-CAATGTTTCTAC kb L14417 CLK ATACCGGTGTGA-AGAGCGGAGCCC kb L14420 CYP1A GAGAACGCCAAT-GTCCAGCTGTCA 3.9 kb P L06406 CYP1A1, exon kb from L L07007 CYP1A TCCTTGGAACCT-TCCCTGATCCTT 26.3 kb L07638 CYP1A CTCATGTACCTT-GTGACCAAGCCT 50.2 kb L08229 CSK TTTCGGAATCCT-TCTCTGGGAAAT The 15q24 microdeletion syndrome has been described by Sharp, A. J. et al. (2007, Hum Mol Genet. 16: ). Phenotype is characterised by mental retardation and growth retardation. The PML gene is outside the commonly deleted area. Deletion of PML has been described in J Med Genet. 43: (2006). Table 2f. 17q21 region L08221 CRHR1, exon 4 (1) CGAGACGGAGCT-GCGGGTGCCCTC 49.0 kb P L07620 CRHR1, exon 8 (7) kb from L L14410 CRHR1, exon 14 (11) CTCAGACGTGGT-GGATGCCCGGAG kb L08205 MAPT, exon GGGGCTACACCA-TGCACCAAGACC 9.4 kb L14414 MAPT, exon ATGTGTTCCAGA-ATCTCCCCTGCA 11.8 kb L14424 MAPT, exon TTCCTCTCCAAA-GTTTCCACAGAG 3.4 kb L08210 MAPT, exon CAGTAAAAGCAA-AGACGGGACTGG 9.3 kb L14422 MAPT, exon 11 (10) CCTTCCTTCCCA-GGTGAACCTCCA 27.7 kb P L08385 MAPT, exon kb from L14422 P L08501 MAPT, exon kb from L L08219 MAPT, exon 15 (14) GTCGCCAGTGGT-GTCTGGGGACAC More s for the MAPT gene and other genes in this 17q21.31 microdeletion area are present in the P275 MAPT-17q21 mix. More info on this microdeletion syndrome in OMIM The cause of the phenotype is suspected to be a deletion of the MAPT and CRHR1 genes. A duplication of the same region has recently been described in a girl with severe psychomotor developmental delay, facial dysmorphism and microcephaly (Kirchhoff, M. et al., 2007, Eur J Med Genet. 50: ). SALSA mix P371 Microdeletion Syndromes 5 Page 5 of 8

6 Note: The exon numbering of the PAX6 and CRHR1 genes has changed. From description version 02 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for these genes. This exon numbering used here may differ from literature! The exon numbering used in previous versions of this product description can be found between brackets in Table 2. Exon numbering might be different as compared to literature! Complete sequences are available on request: Please notify us of any mistakes: SALSA mix P371 Microdeletion Syndromes 5 Page 6 of 8

7 mix P371-A1 Microdeletion-5 sample picture D ye Sign al Size Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA mix P371-A1 Microdeletion-5 (lot A1-0509). The old MLPA buffer (replaced in December 2012) was used. Vials with the old MLPA buffer have a white label Dye Signal Size Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA mix P371-A1 Microdeletion-5 (lot A1-0509). The new MLPA buffer (introduced in December 2012) was used. Vials with the new MLPA buffer have a yellow label. SALSA mix P371 Microdeletion Syndromes 5 Page 7 of 8

8 Implemented Changes compared to the previous product description version. Version 05 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 04 (48) - Various minor textual and layout changes. - Exon numbering of the CRHR1 and MAPT genes has been changed on page 3 and 4. Version 03 (46) - Unclear remark 301 nt removed. Version 02 (46) - Warning added in Table 1, 301 nt L Exon numbering of the PAX6 and CRHR1 genes has been changed on page 3 and 4. - Data analysis method has been modified. - Small changes of lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Various minor textual changes. - Various minor layout changes. SALSA mix P371 Microdeletion Syndromes 5 Page 8 of 8