MRC-Holland MLPA. Description version 30; 06 June 2017

Transcription

1 SALSA MLPA probemix P081-C1/P082-C1 NF1 P081 Lot C1-0517, C As compared to the previous B2 version (lot B2-0813, B2-0912), 11 target probes are replaced or added, and 10 new reference probes are included. P082 Lot C As compared to the previous B2 version (lot B and B2-0912), 13 new target probes are replaced or added, and 8 new reference probes are included. NEUROFIBROMATOSIS is an autosomal dominant disorder characterised particularly by café-au-lait spots and fibromatous tumours of the skin. Neurofibromatosis type I (MIM162200) is caused by defects in the NF1 gene on 17q11.2. Type II is caused by defects in the NF2 gene on chromosome 22, for which the P044 NF2 MLPA probemix can be used. These P081 and P082 MLPA probemixes can be used to identify copy number changes of the NF1 gene. Deletions of part of the NF1 gene as well as deletions and duplications of the complete NF1 gene have been described. Relatively common (5-10% of NF1 cases) is a deletion of a 1.4 Mb chromosomal region that includes the complete NF1 gene. This interstitial 17q11.2 microdeletion arises from unequal crossover between two highly homologous 60 kb duplications. The phenotype of the 17q11.2 microdeletion is usually much more severe than most other NF1 cases and may include severe developmental delay. The P122 NF1 area MLPA probemix can be used to determine the extent of the deletion as it contains many probes for other genes in the frequently deleted 1.4 Mb region. The NF1 gene (58 exons) spans ~300 kb of genomic DNA and is located on 17q11.2, 30 Mb from the p- telomere. The probemixes P081-C1 and P082-C1 together contain one probe for each exon, three probes for exon 1, one probe for intron 1, and two probes for the exons 15, 23, 51 and 58 of the NF1 gene. These probemixes furthermore contain two probes for the OMG gene which is located within intron 36 of the NF1 gene. In addition, 11 reference probes in probemix P081-C1 and 9 reference probes in probemix P082-C1 are included, detecting several different autosomal chromosomal locations. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned gene(s) 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). 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 P081/082 NF1 probemixes Page 1 of 8

2 Related SALSA probemixes P044 NF2: Contains probes for the NF2 gene, involved in Neurofibromatosis type 2. P122 NF1 area: Contains probes for the 17q11.2 region, involved in Neurofibromatosis type 1. P295 SPRED1: Contains probes for the SPRED1 gene at 15q14, involved in Neurofibromatosis type I-like syndrome. References Conforti R et al. (2014). Giant thrombosed intracavernous carotid artery aneurysm presenting as Tolosa Hunt syndrome in a patient harboring a new pathogenic neurofibromatosis type 1 mutation: a case report and review of the literature. Neuropsychiatr Dis Treat. 10: Ben-Salem S et al. (2014). The mutational spectrum of the NF1 gene in neurofibromatosis type I patients from UAE. Childs Nerv Syst. 30: Callum P et al. (2012). Gonosomal mosaicism for an NF1 deletion in a sperm donor: evidence of the need for coordinated, long-term communication of health information among relevant parties. Hum. Reprod. 27: Thomas L et al. (2012). Exploring the somatic NF1 mutational spectrum associated with NF1 cutaneous neurofibromas. Eur J Hum Genet. 20: Valero MC et al. (2011). A highly sensitive genetic protocol to detect NF1 mutations. J Mol Diagn. 13: Havlovicova M et al. (2007). A girl with neurofibromatosis type 1, atypical autism and mosaic ring chromosome 17. Am J Med Genet A. 143: Wimmer K et al. (2006). Spectrum of single- and multi-exon NF1 copy number changes in a cohort of 1,100 unselected NF1 patients. Genes Chromosomes Cancer. 45: Data analysis The P081-C1 NF1 probemix-1 contains 45 MLPA probes with amplification products between 130 nt and 463 nt. The P082-C1 NF1 probemix-2 contains 44 MLPA probes with amplification products between 130 nt and 481 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 intra-sample normalised 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 P081/082 NF1 probemixes Page 2 of 8

3 Table 1a. SALSA MLPA P081-C1 NF1 probemix Length Chromosomal position SALSA MLPA probe (nt) reference NF1 OMG 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 92 Ligation-dependent control fragment at 2q X-fragment: Specific for the X chromosome 105 Y-fragment: Specific for the Y chromosome 130 Reference probe L q * «NF1 probe L23328 Downstream 142 NF1 probe L01922 Exon * NF1 probe L23329 Exon NF1 probe L03309 Exon NF1 probe L01924 Exon NF1 probe L01944 Exon * Reference probe L q NF1 probe L02617 Exon * NF1 probe L23332 Exon * Reference probe L q * NF1 probe L23333 Upstream 202 NF1 probe L03706 Exon * «NF1 probe L26126 Exon NF1 probe L26127 Exon * NF1 probe L22398 Exon * NF1 probe L25737 Exon NF1 probe L26128 Exon NF1 probe L01950 Exon NF1 probe L03300 Exon * Ж NF1 probe SP0601- L22399 Exon * Reference probe L q NF1 probe L22646 Exon * Reference probe L q NF1 probe L01954 Exon NF1 probe L22647 Exon * Reference probe L q NF1 probe L22649 Exon NF1 probe L22650 Exon * Reference probe L p * Ж NF1 probe SP0652- L23404 Exon NF1 probe L01957 Exon NF1 probe L22658 Exon * Reference probe L q NF1 probe L01959 Exon * Ж NF1 probe SP0647- L23336 Exon «NF1 probe L01961 Exon NF1 probe L03709 Exon * Reference probe L q * Ж NF1 probe SP0653- L23405 Exon NF1 probe L26426 Exon NF1 probe L26427 Exon * Reference probe L q OMG probe L03310 Intron 36 of NF1 463 * Reference probe L p13 SALSA P081/082 NF1 probemixes Page 3 of 8

4 Table 1b. SALSA MLPA P082-C1 NF1 probemix Length Chromosomal position SALSA MLPA probe (nt) reference NF1 OMG 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 92 Ligation-dependent control fragment at 2q X-fragment: Specific for the X chromosome 105 Y-fragment: Specific for the Y chromosome 130 Reference probe L q NF1 probe L03316 Exon NF1 probe L01943 Exon NF1 probe L12866 Exon NF1 probe L01925 Exon NF1 probe L01945 Exon * NF1 probe L22738 Exon * Reference probe L q * Reference probe L q NF1 probe L12867 Intron * NF1 probe L26502 Exon NF1 probe L22716 Exon NF1 probe L01949 Exon * Reference probe L q * NF1 probe L26201 Exon NF1 probe L26202 Exon NF1 probe L26200 Exon NF1 probe L26199 Exon NF1 probe L26198 Exon NF1 probe L01953 Exon * Reference probe L q * Ж NF1 probe SP0809- L25738 Exon NF1 probe L26817 Exon NF1 probe L22720 Exon * NF1 probe L22721 Exon * Ж NF1 probe SP0646- L23334 Exon NF1 probe L22725 Exon * NF1 probe L23335 Exon NF1 probe L01960 Exon * Reference probe L p * Ж NF1 probe SP0619- L22739 Exon * Reference probe L q * NF1 probe L22401 Exon * NF1 probe L23330 Exon * Reference probe L q NF1 probe L26175 Exon NF1 probe L23156 Exon NF1 probe L23157 Exon * NF1 probe L22765 Exon OMG probe L03311 Intron 36 of NF1 454 NF1 probe L03307 Exon * Ж NF1 probe SP0602- L22403 Exon * NF1 probe L26174 Exon * Reference probe L q21 * New in version C1 (from lot C onwards). Changed in version C1 (from lot C onwards). Small change in length, no change in sequence detected. SALSA P081/082 NF1 probemixes Page 4 of 8

5 Ж This probe consists of three parts and has two ligation sites. A low signal of this probe can be due to depurination of the sample DNA, e.g. due to insufficient buffering capacity or a prolonged denaturation time. Flanking probe. Included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition tested. Note: We have adopted the NCBI exon numbering that is present in the NM_ and NM_ sequences for this gene. This 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. Table 2. NF1 probes arranged according to chromosomal location Length (nt) P081 P082 SALSA MLPA probe Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (exon 1) 196 * L23333 Upstream 8.0 kb before exon 1 CAAAGCAAGTTC-AGCATCAGAGGA 7.7 kb L01922 Exon nt before exon 1 GCAGAGATCCGC-GCGCTGGGAGAA 0.4 kb 227 * L26201 Exon AAGGATCCCACT-TCCGGTGGGGTG 0.4 kb L03316 Exon reverse TGACCACGGCCT-GGACCCATTCCA 0.6 kb L12867 Intron nt after exon 1 TCGTCTCATCCT-GCCCCGAGAGCT 60.1 kb L01924 Exon GCAGAACACACA-TACCAAAGTCAG 3.0 kb L01925 Exon ATATCTCTCTCA-GTTGATTATATT 4.2 kb L02617 Exon TGCCAGAAATCT-GCCATTTTCTTC 6.7 kb 172 * L22738 Exon AAAATTAAAACG-ACTCCTGAAGGG 11.5 kb L03706 Exon AGCCCTAAAGAA-GGTTGCGCAGTT 0.3 kb 220 * L22398 Exon TAGGCATTTTGG-AACTGGGTAGAA 0.9 kb L22716 Exon AAAGCACCAAAC-GTAAAGCAGCAG 17.9 kb 463 * Ж SP0602 TGACAGAAAGTG-31 nt spanning Exon ; L22403 oligo-aagtacttacat 0.6 kb L26202 Exon GATGTGGATCTA-ATGATTGACTGC 0.7 kb L26128 Exon nt after exon 11 TGAGAAAAATGT-CACTGAAAATAC 4.5 kb L26198 Exon TTGGTGAAACAC-TTCATAAAGCAG 8.2 kb L22647 Exon GACAAGAAGCTA-TAAGTATCTTCT 4.6 kb L26817 Exon CAACTGGTCCCT-CAGTCACACATG 2.5 kb L22649 Exon nt before exon 15 reverse TAACTGGCATGT-ACATATAAAGCT 0.3 kb 281 * Ж SP0809 TCAGTTAGATAG-30 nt spanning Exon ; L25738 oligo-agaaacattttg 1.5 kb 317 * Ж SP nt before exon 16; TTAGGTTATTGA-38 nt spanning Exon 16 -L oligo-acaaatgctttt 1.8 kb L22658 Exon GGATCATGAAGA-ATTACTACGTAC 1.4 kb L26426 Exon CTTGCCCAACTA-TAACACATTCAT 0.7 kb L23157 Exon 19 2 nt after exon 19 AACACTGAGGTA-TGCCCTTAGCAA 0.2 kb 337 * L23335 Exon nt before exon 20 AGCTCTAGACTA-AGTTGCTTTCAA 1.9 kb 382 * Ж SP0647 ACGGACCAATGT-43 nt spanning Exon ; L23336 oligo-atgctatttaac 0.5 kb 436 * L22765 Exon CAATTTGTAGAA-CAAACCATAGCT 0.5 kb 418 * Ж SP0653 AAACTGTGTCAA-34 nt spanning Exon ; L23405 oligo-tctcattttgcc 0.2 kb 337 * Ж SP nt and 213 nt after GCCTGTGACAAT-34 nt spanning Exon 23 -L23404 exon 23 oligo-taagaatttgat 0.2 kb 256 * Ж SP nt before exon 24; GGCTTCAAAAAC-39 nt spanning Exon 24 -L oligo-ataagatggtag 1.4 kb 362 * Ж SP ; TGGAAGCCAAAT-51 nt spanning Exon 25 -L nt after exon 25 oligo-gcaaataaagcc 0.7 kb L03300 Exon TGAGGCACTGTA-CGGTCCTTGCAA 0.3 kb L26175 Exon ATCGGTTTGAGA-GATTGGTGGAAC 2.6 kb 148 * L23329 Exon GCAGACTCCATG-CAGACTCTCTTC 0.3 kb L03709 Exon CATCCTCTGATT-GGCAACATGTTA 13.0 kb SALSA P081/082 NF1 probemixes Page 5 of 8

6 Length (nt) P081 P082 SALSA MLPA probe Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe L01943 Exon TGAGGAAAACCA-GCGGAACCTCCT 3.9 kb 472 * L26174 Exon 31 NM_ : TTCTGTAGGCAA-CTTGCCACTCCC 5.5 kb L01944 Exon CATCGGTGCAGT-AGGAAGTGCCAT 0.7 kb 391 * L23330 Exon TGTGAAAAGCAA-CTTTGATGCAGC 1.3 kb L01945 Exon TCTTTCCTTCAT-AAGTGACGGCAA 1.3 kb 184 * L23332 Exon CTTGCATACCTG-GGTCCTCCAGAG 3.4 kb 197 * L26502 Exon nt before exon 36 ATTACTCTGTTA-TTTTTCTTTTAG 30.3 kb L03311 NM_ : OMG gene; , Exon 2 within NF1 intron 36 GCAGACAGTGGA-CACCATTAACTC 0.6 kb L03310 OMG gene; Exon 2 NM_ : reverse, within NF1 intron 36 CACAGAGACCGA-GGTAAGTGAGCA 30.1 kb L26127 Exon GCCTCAAAGGTA-GCAAAAGGCTTG 1.5 kb L01949 Exon AACCAGTTCACC-TTAACCATTGCA 2.8 kb L01950 Exon CTAGAGACATCA-GGTTTATGTATC 4.5 kb L26200 Exon GACTCCATGGCT-GTCAAATCTAGT 1.5 kb L22646 Exon CAGGTGGCTTGG-GATCAATAAAAG 0.4 kb L26427 Exon GCTGTCCTTCAA-CAATTCCCTTGA 0.7 kb L23156 Exon TCATTACCCAAA-TTTTACTTGCTG 0.4 kb L26199 Exon ATTCCAACGTGC-AAGTGGCTGGAC 0.2 kb L03307 Exon TCTTGTTGTCTT-TGGGTGTATTAG 0.7 kb L01953 Exon TTGCTTAAAAGG-ACCTGACACTTA 1.8 kb L01954 Exon AAGCCCTCTTTT-GGGTAGCTGTGG 2.5 kb L22720 Exon ATCCTCTGGAGT-GGCACTGCAAGC 6.1 kb L22650 Exon TCACCTGCTATT-GTTGCAAGAACA 1.0 kb L01957 Exon AAGAAGTTCGAA-GTCGCTGCAGCC 2.1 kb L22725 Exon GAGACTCAGCCA-TGGTCCTCTCCC 0.1 kb 226 * L25737 Exon CTGTCGGCCAGA-CCAGTCCCCGAG 4.2 kb L01959 Exon AGGCAAGAAATG-GAATCAGGGATC 0.5 kb L12866 Exon TTTACGTAAAGT-TTCAGTGTCTGA 0.3 kb 307 * L22721 Exon AGTTTGATCAAC-GAATTCTTTATG 1.3 kb L01960 Exon GCAGAGTGTGGT-GTACCATGAAGA 0.4 kb 382 * L22401 Exon 56 2 nt before exon 56 TTGATTTGTTGC-AGGTTTTGGTTT 1.6 kb L03309 Exon TGGAATTGATGA-AGAAACCAGTGA 13.5 kb 391 «02530-L01961 Exon GCCACTGTAACA-GTGGACGAACTC 2.0 kb 208 * «19361-L26126 Exon AGTGCCAAGGAT-GCCAAGCTGCCA 6.4 kb 136 * «18363-L23328 Downstream 4.8 kb after exon 58 GGGAAGGAGCTC-AGGCTGTAATGT stop codon (ex 58) * New in version C1 (from lot C onwards). Changed in version C1 (from lot C onwards). Small change in length, no change in sequence detected. «This probe is 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. Ж This probe consists of three parts and has two ligation sites. A low signal of this probe can be due to depurination of the sample DNA, e.g. due to insufficient buffering capacity or a prolonged denaturation time. Flanking probe. Included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition tested. The NM_ represents transcript variant 1 and the NM_ sequence represents transcript variant 2, both sequences are a reference standard in the NCBI RefSeqGene project. Exon 31 is not present in the NM_ transcript variant. Note: We have adopted the NCBI exon numbering that is present in the NM_ and NM_ sequences for this gene. This 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 P081/082 NF1 probemixes Page 6 of 8

7 SALSA MLPA probemix P081-C1/P082-C1 NF1 sample pictures Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P081-C1 NF1 mix-1 (lot C1-0517) D ye S ign al Size (nt) Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P082-C1 NF1 mix-2 (lot C1-0114). SALSA P081/082 NF1 probemixes Page 7 of 8

8 Implemented Changes compared to the previous product description versions. Version June 2017 (55) - Product description adapted to a new product lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Information about NF1 gene and common microdeletion adjusted on page 1. - Minor textual and layout changes. Version July 2015 (54) - Warnings added in Table 1 and Table 2. Low signals for probes that consist of three parts can be due to depurination of the sample DNA. Version 28 (53) - 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). - New references added on page 1. Version 27 (53) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). Version 26 (53) - Warning added to NF1 probe L02619 under table 1 and 2. This probe is more sensitive to experimental variation, due to a less stable binding of the probe to the DNA. Version 25 (48) - Warning added to NF1 probe L01952 under table 1. This probe is more sensitive to experimental variation, due to a less stable binding of the probe to the DNA. Version 24 (48) - Warning added below Table 1b and 2, probe 178 nt L Version 23 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 22 (48) - Product description adapted to a new version (lot number added, new pictures included, small textual changes, more reference articles added). Version 21 (46) - Exon numbering mistakes corrected in Table 1 and 2. Version 20 - Product description adapted to a new lot (lot number added, new picture included). - Ligation sites updated according to new version of the NM_reference sequence. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. Version 19 - Mistake in exon numbering has been corrected as indicated in the tables. - Various minor textual changes on page 1, minor changes in the data analysis section on page 2. - Tables have been numbered. - Sentence when only small numbers of samples are tested, visual comparison of peak profiles should be sufficient removed from data analysis section. SALSA P081/082 NF1 probemixes Page 8 of 8