MRC-Holland MLPA. Description version 09; 28 April 2016

Transcription

1 SALSA MLPA probemix P244-C1 AIP-MEN1-CDKN1B Lot C1-0815, C Note that the name of the product has been changed to P244 AIP-MEN1-CDKN1B (from lot C onwards). Multiple endocrine neoplasia (MEN) is part of a group of autosomal dominant disorders characterised by the occurrence of tumours in at least two endocrine glands. Two classical syndromes have been both clinically and genetically well characterised: the MEN type 1 (MEN1) and type 2 (MEN2). MEN1 is caused by loss-offunction mutations in the MEN1 gene, and affected patients typically develop multiple parathyroid adenomas, pancreatic islet cell neoplasia, and anterior pituitary adenomas. The MEN1 gene encodes menin, a tumour suppressor gene likely involved in several cellular processes such as DNA repair and apoptosis. MEN2 can be divided in two forms, MEN2A and MEN2B (formerly known as MEN3), and is caused by activating germline mutations in the RET proto-oncogene. The P169 probemix contains probes for each of the RET exons. More recently, mutations in the CDKN1B gene (encoding the cell cycle inhibitor p27) were found in patients with multiple endocrine tumours in the absence of MEN1 and RET mutations, leading to a novel MEN syndrome, named MEN4. Pituitary adenomas are often benign and occur with a frequency of 1:1000. These adenomas are often sporadic but a small part (5%) occurs as part of other familial cancer e.g. MEN1. Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene, which is a tumour suppressor gene, have shown to be a low-penetrant factor for pituitary adenoma predisposition (PAP). The AIP gene comprises 6 exons, spans about 8 kb of genomic DNA and is located at chromosome 11q13.2, about 67 Mb from the p-telomere, the MEN1 gene comprises 10 exons, spans about 7 kb of genomic DNA and is located at chromosome 11q13.1, about 64 Mb from the p-telomere and the CDKN1B gene (3 exons), spans about 5 kb of genomic DNA and is located on chromosome 12p13.1, about 12 Mb from the p- telomere. This P244-C1 AIP-MEN1-CDKN1B probemix contains 11 probes for the 10 exons of the MEN1 gene. In addition, 3 probes for each of the CDKN1B exons are included. Furthermore, this probemix contains probes for all exons of the AIP gene (2 probes for exon 1) and 6 probes for other genes in 11q13 region. In addition, 10 reference probes are included in this probemix, 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 P244 AIP-MEN1-CDKN1B probemix Page 1 of 6

2 Related SALSA probemixes P169 Hirschsprung-1: Contains probes for the RET gene. P017 MEN1: Contains the same MEN1 probes as this P244 probemix, but does not contain probes for the AIP gene, CDKN1B gene and several other genes in the 11q13 region. References Lecoq, A.L. et al., (2016) Very low frequency of germline GPR101 genetic variation and no biallelic defects with AIP in a large cohort of patients with sporadic pituitary adenomas. Eur J Endocrinol. 174(4): Dutta, P. et al., (2015) Clinical profile and outcome of patients with acromegaly according to the 2014 consensus guidelines: Impact of a multi-disciplinary team. Neurol India. 63(3):360-8 Iwata, T. et al., (2014) A novel C-terminal nonsense mutation, Q315X, of the aryl hydrocarbon receptorinteracting protein gene in a Japanese familial isolated pituitary adenoma family. Endocr Pathol. 25(3): Preda, V. et al., (2014) Low rate of germline AIP mutations in patients with apparently sporadic pituitary adenomas before the age of 40: a single-centre adult cohort. Eur J Endocrinol. 171(5): Oriola, J. et al., (2013) Germline mutations of AIP gene in somatotropinomas resistant to somatostatin analogues. Eur J Endocrinol. 168(1):9-13 Data analysis The P244-C1 AIP-MEN1-CDKN1B probemix contains 37 MLPA probes with amplification products between 136 and 445 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 first be normalised intra-sample 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 P244 AIP-MEN1-CDKN1B probemix Page 2 of 6

3 Table 1. SALSA MLPA P244-C1 AIP-MEN1-CDKN1B probemix Length Chromosomal position SALSA MLPA probe (nt) reference AIP MEN1 CDKN1B other 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 136 Reference probe L q CDKN1B probe L23733 Exon ± MEN1 probe L02795 Exon ± BRMS1 probe L q ± MEN1 probe L14680 Exon ± AIP probe L09559 Exon Reference probe L q ± MEN1 probe L01242 Exon ± MEN1 probe L24187 Exon ± MEN1 probe L14681 Exon ± AIP probe L07030 Exon ± MEN1 probe L01243 Exon 2b 229 RELA probe L q ± AIP probe L07028 Exon ± MEN1 probe L00720 Exon Reference probe L q ± AIP probe L09556 Exon Reference probe L q ± MEN1 probe L14816 Exon ± AIP probe L09558 Exon ± MEN1 probe L01245 Exon ± CCND1 probe L q ± AIP probe L09557 Exon SNX15 probe L q Reference probe L q Reference probe L p ± FAM89B probe L q ± AIP probe L09069 Exon Reference probe L q SART1 probe L q CDKN1B probe L23497 Exon1 400 ± MEN1 probe L23498 Upstream 409 Reference probe L p Reference probe L q CDKN1B probe L23500 Exon ± MEN1 probe L23501 Exon Reference probe L q13 ± 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. 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. Notes The MEN1 exon numbering has changed. From description version 09 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for the MEN1 gene (NM_ ). The exon numbering used here may differ from literature! The identity of the genes detected by the reference probes is available on request: info@mlpa.com. SALSA P244 AIP-MEN1-CDKN1B probemix Page 3 of 6

4 Table 2. P244 probes arranged according to chromosomal location Length SALSA Gene / Partial sequence (24 nt Distance to Ligation site (nt) MLPA probe exon adjacent to ligation site) next probe MEN1 NM_ Stop codon (ex 10) 247 ± L00720 Exon 10 (11) CGCCATCAAGCT-GCAACTCACGGC 0.7 kb 154 ± L02795 Exon 9 (10) CGGCATCTGCAA-ATGGGAGGAGGG 0.6 kb 436 ± L23501 Exon 8 (9) CTTTGAAGTAGC-CAATGATGTCAT 0.6 kb 301 ± L01245 Exon 7 (8) ACCCCTACATGT-ACCTGGCTGGCT 0.7 kb 195 ± L24187 Exon 6 (7) CCTCTACCACAA-GGTGGGGGCATC 0.2 kb 202 ± L14681 Exon 5 (6) GCTGCTCTATGA-CCTGGGACATCT 0.4 kb 167 ± L14680 Exon 4 (5) GTGACCGCAAGA-TGGAGGTGGCGT 0.4 kb 283 ± L14816 Exon 3 (4) AGGATCATGCCT-GGGTAGTGTTTG 2.0 kb 220 ± L01243 Exon 2b (3a) CGTGGAGCATTT-TCTGGCTGTCAA 0.7 kb 191 ± L01242 Exon 1 (2b) GAGATCCCAGAA-GCCACAGCGCAG 0.4 kb 400 ± L23498 Upstream (1a) (NM_ ) GCGGAAGTGGGA-AACGAGTGCTGC kb Start codon (ex 2b) L14817 SNX15 gene CGAAGGATGACT-TCCTGCGGCACT kb 355 ± L03512 FAM89B gene ACAAACACCTGT-GCCAAGACCTGA 88.3 kb L00060 RELA gene AAAGGACTGCCG-GGATGGCTTCTA kb L03514 SART1 gene CCGCAAGAAGGA-GAAGGAGGTAGT kb 160 ± L03510 BRMS1 gene CAGAAGAGATGG-AAGCAGAGGGTG kb AIP NM_ Start codon (ex 1) 292 ± L09558 Exon GAGTCCGGAAGT-TGCCGAAAGGGA 0.1 kb 175 ± L09559 Exon AAAACGTGTGAT-ACAGGAAGGCCG 3.9 kb 238 ± L07028 Exon CCATGGAGCTCA-TCATTGGCAAGA 2.3 kb 364 ± L09069 Exon ACAGATGCGTGA-ACACAGCTCCCT 0.7 kb 209 ± L07030 Exon GCAGTGCCACTT-ATCCACCAGGAG 0.3 kb 266 ± L09556 Exon GCTGCTGCTCAA-CTACTGCCAGTG 0.5 kb 319 ± L09557 Exon CAGGCTGACTTT-GCCAAAGTGCTG kb Stop codon (ex 6) L04809 CCND1 gene TCCGCCCTCCAT-GGTGGCAGCGGG Length (nt) SALSA MLPA probe CDKN1B Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe Start codon (ex 1) L23497 Exon GACCCGGGAGAA-AGATGTCAAACG 1.1 kb L23733 Exon ATGCCGGTTCTG-TGGAGCAGACGC 2.3 kb L23500 Exon CCTGTATAAGCA-CTGAAAAACAAC Stop codon (ex 2) ± 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. 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. NM_ , NM_ and NM_ are reference standards in the NCBI RefSeqGene project. Note: The MEN1 exon numbering has changed. From description version 09 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for the MEN1 gene (NM_ ). The exon numbering used here may differ from literature! SALSA P244 AIP-MEN1-CDKN1B probemix Page 4 of 6

5 SALSA MLPA probemix P244-C1 AIP-MEN1-CDKN1B probemix sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P244-C1 AIP-MEN1-CDKN1B (lot C1-0815). SALSA P244 AIP-MEN1-CDKN1B probemix Page 5 of 6

6 Implemented Changes compared to the previous product description version(s). Version April 2016 (55) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Various minor textual changes on page 1. - Exon numbering of the MEN1 gene has been changed in Table 1 and Table 2. Version 08 (53) - Various layout changes. Version 07 (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). - CDKN1B gene information was added on page 1. - New references added on page 1. - Some minor textual changes page 1 and 2. Version 06 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 05 (47) - Two new references added on page 2. - Warnings added to tables about salt sensitivity of probe L01245, L14681, L14680 and L Remark on RefSeqGene standard and transcript variant added below Table 2. - Exon numbering of the MEN1 gene has been changed on page 3 and 4. - Small correction of chromosomal locations in Table 1. - Various minor textual changes on page 2. Version 04 (44) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new picture included). - Warning added to Table 1, salt-sensitive probes. - Some minor textual changes page 1 and 2. SALSA P244 AIP-MEN1-CDKN1B probemix Page 6 of 6