SALSA MLPA probemix P372-B1 Microdeletion Syndromes 6 Lot B1-1016, B

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1 SALSA MLPA probemix P372-B1 Microdeletion Syndromes 6 Lot B1-1016, B The purpose of the P372 probemix is to further investigate results found with the P245 Microdeletion Syndromes-1A probemix. The P245 probemix provides a possibility to screen samples for various different microdeletion syndromes in a single reaction. For confirmation of results obtained with this P245 probemix, four different probemixes are available with additional probes in these regions: P371, P372, P373 and P374 Microdeletion Syndromes. The P372-B1 probemix contains probes for the Sotos syndrome 1 region on 5q35 (7 probes), the DiGeorge region on 22q11 (18 probes), the Rubinstein-Taybi CREBBP gene on 16p13 (7 probes), the DiGeorge 2 region on 10p14 (6 probes) and the NF1 microdeletion region on 17q (10 probes). This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the above mentioned regions 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. 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 Acids 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 Related SALSA probemixes P245 Microdeletion Syndromes-1A: Contains probes for several microdeletion/microduplication syndromes and can be used for primary screening of microdeletion/microduplication syndromes. P064 Microdeletion Syndromes-1B: Contains probes for 1p36 deletion, Wolf-Hirschhorn, Cri-du-Chat, Sotos, Saethre-Chotzen, Williams-Beuren, Langer-Giedion, WAGR, Prader-Willi/Angelman, Rubinstein- Taybi, Miller-Dieker, Smith-Magenis, Alagille, DiGeorge, and Phelan-McDermid syndrome. P297 Microdeletion Syndromes-2: Contains probes for microdeletion syndromes on 1q21.1, 3q29, 7q36.1, 12p11.23, 15q13, 15q24.1, 16p11, 17q12, 18q21.2, and 20p12.2. P371 Microdeletion Syndromes 5: One of four different P245 follow-up probemixes. This probemix contains more probes for a subset of the microdeletion/microduplication syndromes covered by P245. P373 Microdeletion Syndromes 7: One of four different P245 follow-up probemixes. This probemix contains more probes for a subset of the microdeletion/microduplication syndromes covered by P245. P374 Microdeletion Syndromes 8: One of four different P245 follow-up probemixes. This probemix contains more probes for a subset of the microdeletion/microduplication syndromes covered by P245. Several MLPA probemixes for specific syndromes are available, such as P026 Sotos, P250 DiGeorge, P313 CREBBP, P081 & P082 NF1, P122 NF1-area. Please see Table 2 or our website. SALSA P372 Microdeletion Syndromes 6 probemix Page 1 of 7

2 Data analysis The P372-B1 Microdeletion Syndromes 6 probemix contains 48 MLPA probes with amplification products between 128 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 probemix can be normalised intra-sample by dividing the peak height of each amplification product by the combined peak height of all peaks in that sample (global 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. 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 P372 Microdeletion Syndromes 6 probemix Page 2 of 7

3 Table 1. SALSA MLPA P372-B1 Microdeletion 6 probemix Length Chromosomal position (hg18) SALSA MLPA probe (nt) 5q35 10p14 16p13 17q11 22q 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 128 TCEB1P3 probe L p VPREB1 probe L q NSD1 probe L q «DGCR8 probe L q ATAD5 probe L q NSD1 probe L q GATA3 probe L p PROP1 probe L q TCEB1P3 probe L p SUZ12 probe L q CLTCL1 probe L q NF1 probe L q «ZNF74 probe L q NSD1 probe L q GATA3 probe L p «RNF135 probe L q NSD1 probe L q RAB36 probe L q TXNRD2 probe L q BCR probe L q GNAZ probe L q UTP6 probe L q SMARCB1 probe L q CREBBP probe L p «KLHL22 probe L q ADAP2 probe L q RIMBP3C probe L q HIC2 probe L q CREBBP probe L p GNB1L probe L q NF1 probe L q CREBBP probe L p FGFR4 probe L q CREBBP probe L p COMT probe L q DGCR8 probe L q «NF1 probe L q NSD1 probe L q CREBBP probe L p LZTR1 probe L q «CREBBP probe L p GATA3 probe L p NF1 probe L q TCEB1P3 probe L p CDC45 probe L q NF1 probe L q CREBBP probe L p MAPK1 probe L q11.21 SALSA P372 Microdeletion Syndromes 6 probemix Page 3 of 7

4 Table 2. P372 probes arranged according to chromosomal location Table 2a. Sotos syndrome, 5q L00859 FGFR GCGATTCTGTCT-TCAGCCACGACC kb L11228 NSD1 exon AGATCCTTCTGA-GAGAGCCTGGGT 42.5 kb L14390 NSD1 exon GCCAAGGAAGCG-AAAACGACAGAG 13.3 kb L20962 NSD1 exon TACCACGCCAAT-GACTTTTGCCTG 9.7 kb L02068 NSD1 exon GAGCAGCAAGGA-TAAGATGGGCAA 11.0 kb L02069 NSD1 exon GTGCTTTTCCAA-GCGCCAATATCC kb L06894 PROP TGAGGTCAAACA-AGTACCACCAAG More probes for the NSD1 gene (Sotos syndrome 1) and NFIX gene (Sotos syndrome 2) are present in the P026 Sotos probemix. The frequency of complete gene deletions has been reported as 10% (United Kingdom) to 45% (Japan) of all NSD1 mutations detected. More information on Sotos syndrome can be found in OMIM # The distance from the NSD1 gene to the 5q telomeric probes in P036 and P is approximately 3950 kb. The most common causes of Sotos syndrome are point mutations in the NSD1 gene that will not be detected by these MLPA probes. Table 2b. DiGeorge syndrome-2, 10p13-p L00776 GATA3 exon GAGTGCCTCAAG-TACCAGGTGCCC 5.4 kb L14385 GATA3 exon GGGGCAACCTCG-ACCCCACTGTGG 5.6 kb L14382 GATA3 exon TACTACAAGCTT-CACAATGTAAGT 1.9 Mb L14783 TCEB1P3 area GTCTTGATTCCA-TTCTGACACTGC kb L14794 TCEB1P3 area TGCCAGTTCAGA-CCAGTATTGACA 53.5 kb L14785 TCEB1P3 area TCATCCAGAAGA-GTCCATCAACTG More probes for the 10p14 DiGeorge region 2 are present in the P250 DiGeorge probemix. More information on DiGeorge region 2 can be found in OMIM # Besides this DGS2 region, deletion of the 17p terminal region can also cause a DiGeorge-like phenotype. These 17p deletions should be detected by the P036/P069/P070/P249 Human Telomere probemixes. The great majority of DiGeorge syndrome patients have a 22q11 deletion. SALSA P372 Microdeletion Syndromes 6 probemix Page 4 of 7

5 Table 2c. Rubinstein-Taybi syndrome, 16p L10319 CREBBP exon AAAAAGATGCTG-GACAAGGCGTTT 21.2 kb L14386 CREBBP exon CTGGCTCATGTT-CAACAATGCCTG 1.0 kb L14388 CREBBP exon CCCAGAGTCATT-ACCTTTCCGGCA 11.9 kb L10306 CREBBP exon ACTCAGCCATCA-ACTCCTGTGTCG 8.0 kb L10301 CREBBP exon AGCCTATGCTAA-GAAAGTGGAAGG 13.3 kb L10297 CREBBP exon CAAATCATCTCT-CATTGGAAGAAC 87.8 kb 427 «09880-L10293 CREBBP exon GCTCGCCCGGTT-TCTCGGCGAATG More probes for the CREBBP gene are present in the P313 CREBBP probemix. Only a minority of Rubinstein-Taybi patients can be detected with the use of these probes; most patients have point mutations in CREBBP or EP300. More information on the Rubinstein-Taybi syndrome can be found in OMIM # The 16p13.3 deletion syndrome (OMIM #610543) is caused by larger deletions that include the CREBBP gene. Table 2d. NF1 microdeletion syndrome region, 17q L25048 ATAD GCAGGTACGCTT-TAAGACAGTTAC 91.8 kb L03291 ADAP TGAAGGCCAAGT-TCGAAGCCAGAG 57.8 kb 220 «03783-L03292 RNF GGAACATCTTGT-AGACATTGTCAG kb L14389 NF1 intron TCGTCTCATCCT-GCCCCGAGAGCT kb L14387 NF1 exon TAACTGGCATGT-ACATATAAAGCT 5.0 kb L22703 NF1 exon CTTGCCCAACTA-TAACACATTCAT 0.8 kb L23335 NF1 exon AGCTCTAGACTA-AGTTGCTTTCAA kb 391 «02530-L01961 NF1 exon GCCACTGTAACA-GTGGACGAACTC kb L14394 UTP TCCCAGAGTCTC-TAAACAATTCAG kb L14384 SUZ CAATGATAAATC-TACGGCTCCTAT More probes for the NF1 gene are present in the P081 & P082 NF1 probemixes. More probes for other genes in this area are present in the P122 NF1 area probemix. Approximately 5-10% of all NF1 patients carry a heterozygous deletion of approximately 1.5 Mb that includes the NF1 gene. This NF1 microdeletion results in a more severe phenotype that often includes mental retardation, facial dysmorphism and developmental delay. More information on the NF1 region can be found in OMIM # SALSA P372 Microdeletion Syndromes 6 probemix Page 5 of 7

6 Table 2e. DiGeorge / 22q11.2 duplication / Distal 22q11.2 deletion syndrome Lengt h (nt) SALSA MLPA probe Gene Partial sequence (24 nt adjacent to ligation site) Distance to 22p telomere in kb (hg18) Distance to next probe Start DiGeorge region; LCR22-A L05809 CLTCL1 TGTTGCCTTGGT-GACCGAGACCGC kb kb L05808 CDC45 ATGTTCGTGTCC-GATTTCCGCAAA kb kb L14393 GNB1L CGGGATCGCCGA-GGTCACGATCCG kb kb L05814 TXNRD2 GGAGGGTCAGGA-GAGGAGCTGCAG kb 64.0 kb L10761 COMT TTGACACCTACT-GCGAGCAGAAGG kb kb 148 «08475-L08486 DGCR8 GGTAATGGACGT-TGGCTCTGGTGG kb 24.2 kb L10765 DGCR8 GACTCAGCGACT-GCACCAGTGGCA kb kb LCR22-B 202 «05927-L07395 ZNF74 CAGGCAGATTAT-TCCTCGATGCTG kb 93.9 kb 283 «01227-L05815 KLHL22 TCTTCGATGTTG-TGCTGGTGGTGG kb kb LCR22-C L25375 LZTR1 GACTTCCAGACC-TGGGAGGTCGTC kb kb LCR22-D; End of the commonly deleted DiGeorge region L15009 HIC2 GTTCCAGCAGAT-CTTGGACTTCAT kb kb L05796 RIMBP3C CTGGGCCCAAGG-CCTAATAGGTGA kb kb L17403 MAPK1 CCAGAGAGAGAT-TCTTCCCCAATT kb kb L09689 VPREB1 CTGAGCCACTCA-GCATCTCCTGGT kb kb LCR22-E L08489 GNAZ TCACCATCTGCT-TTCCCGAGTACA kb 29.8 kb L08496 RAB36 AAGGCCACCATT-GGGGTGGACTTT kb kb L09686 BCR GGAGATGAGAAA-ATGGGTCCTGTC kb kb LCR22-F L08100 SMARCB1 GCTGGAGAACTA-AGGCGGAATCAG kb LCR22-G More probes for the 22q11 DiGeorge region are present in the P250 DiGeorge probemix. Deletions in 22q11 are the most frequent cause of DiGeorge syndrome. The extent of the 22q11 deletion is variable, although the majority extend from the first (LRC22-A) until the fourth (LRC22-D) repeat. More information on the DiGeorge region can be found in OMIM # The chromosome 22q11.2 distal deletion syndrome (OMIM #611867) has been described by e.g. Ben- Shachar S et al. (2008) Am J Hum Genet. 82: This syndrome frequently results in developmental delay, growth delay and mild skeletal abnormalities. Note: Exon numbering might be different as compared to literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA P372 Microdeletion Syndromes 6 probemix Page 6 of 7

7 SALSA MLPA probemix P372-B1 Microdeletion-6 sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P372-B1 Microdeletion-6 (lot B1-1016). Implemented Changes compared to the previous product description versions. Version November 2017 (55) - Information added on the 22q11.2 distal deletion syndrome under Table 2e. - Titles changed in Table 2. - Several textual and layout changes throughout the document. Version June 2017 (55) - Adjusted chromosomal band for RAB36. Version May 2017 (55) - Product description adapted to a new lot (lot number added, new picture included). - Various minor textual changes. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Warnings removed in Table 1 and Table 2: 142 nt probe L11228 and 292 nt probe L Warning added in Table 1 and Table 2: 202 nt probe L Removed P245 probes from Table 2. Version January 2017 (55) - Warning added in Table 1 and Table 2, 283 nt probe L05815 and 391 nt probe L Version 07 (52) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new picture included). Version 06 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 05 (48) - Warning about salt sensitivity of 427 nt probe L10293 added to tables. SALSA P372 Microdeletion Syndromes 6 probemix Page 7 of 7