SALSA MLPA probemix P244-C1 AIP-MEN1-CDKN1B Lot C1-0815, C1-1112. Note that the name of the product has been changed to P244 AIP-MEN1-CDKN1B (from lot C1-1112 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 : 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 P244 AIP-MEN1-CDKN1B probemix Page 1 of 6
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):523-30 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):273-81 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):659-66 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 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 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 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 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
Table 1. SALSA MLPA P244-C1 AIP-MEN1-CDKN1B probemix Length Chromosomal position SALSA MLPA probe (nt) reference AIP MEN1 CDKN1B other 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 136 Reference probe 16416-L18869 18q21 148 CDKN1B probe 18425-L23733 Exon 2 154 ± MEN1 probe 03404-L02795 Exon 9 160 ± BRMS1 probe 04155-L03510 11q13 167 ± MEN1 probe 13158-L14680 Exon 4 175 ± AIP probe 07380-L09559 Exon 1 184 Reference probe 09724-L10074 12q24 191 ± MEN1 probe 01663-L01242 Exon 1 195 ± MEN1 probe 18765-L24187 Exon 6 202 ± MEN1 probe 13159-L14681 Exon 5 209 ± AIP probe 07383-L07030 Exon 4 220 ± MEN1 probe 01664-L01243 Exon 2b 229 RELA probe 01120-L00060 11q13 238 ± AIP probe 07381-L07028 Exon 2 247 ± MEN1 probe 01164-L00720 Exon 10 257 Reference probe 01055-L00628 17q21 266 ± AIP probe 07384-L09556 Exon 5 274 Reference probe 02766-L02195 9q22 283 ± MEN1 probe 01665-L14816 Exon 3 292 ± AIP probe 07379-L09558 Exon 1 301 ± MEN1 probe 01666-L01245 Exon 7 310 ± CCND1 probe 05403-L04809 11q13 319 ± AIP probe 07385-L09557 Exon 6 328 SNX15 probe 01667-L14817 11q13 337 Reference probe 12276-L13219 22q13 346 Reference probe 04943-L04329 1p22 355 ± FAM89B probe 04157-L03512 11q13 364 ± AIP probe 07382-L09069 Exon 3 373 Reference probe 03919-L03374 15q21 382 SART1 probe 04159-L03514 11q13 391 CDKN1B probe 18426-L23497 Exon1 400 ± MEN1 probe 18427-L23498 Upstream 409 Reference probe 10681-L11263 6p12 417 Reference probe 12559-L21017 3q13 427 CDKN1B probe 18429-L23500 Exon 3 436 ± MEN1 probe 18430-L23501 Exon 8 445 Reference probe 12002-L23932 8q13 ± 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_000244.3). 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
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_000244.3 Stop codon 1956-1958 (ex 10) 247 ± 01164-L00720 Exon 10 (11) 1855-1856 CGCCATCAAGCT-GCAACTCACGGC 0.7 kb 154 ± 03404-L02795 Exon 9 (10) 1390-1391 CGGCATCTGCAA-ATGGGAGGAGGG 0.6 kb 436 ± 18430-L23501 Exon 8 (9) 1228-1229 CTTTGAAGTAGC-CAATGATGTCAT 0.6 kb 301 ± 01666-L01245 Exon 7 (8) 1092-1093 ACCCCTACATGT-ACCTGGCTGGCT 0.7 kb 195 ± 18765-L24187 Exon 6 (7) 1036-1037 CCTCTACCACAA-GGTGGGGGCATC 0.2 kb 202 ± 13159-L14681 Exon 5 (6) 931-932 GCTGCTCTATGA-CCTGGGACATCT 0.4 kb 167 ± 13158-L14680 Exon 4 (5) 825-826 GTGACCGCAAGA-TGGAGGTGGCGT 0.4 kb 283 ± 01665-L14816 Exon 3 (4) 672-673 AGGATCATGCCT-GGGTAGTGTTTG 2.0 kb 220 ± 01664-L01243 Exon 2b (3a) 250-251 CGTGGAGCATTT-TCTGGCTGTCAA 0.7 kb 191 ± 01663-L01242 Exon 1 (2b) 34-35 GAGATCCCAGAA-GCCACAGCGCAG 0.4 kb 400 ± 18427-L23498 Upstream (1a) (NM_130804.2) 242-243 GCGGAAGTGGGA-AACGAGTGCTGC 216.0 kb Start codon 111-113 (ex 2b) 328 01667-L14817 SNX15 gene CGAAGGATGACT-TCCTGCGGCACT 546.0 kb 355 ± 04157-L03512 FAM89B gene ACAAACACCTGT-GCCAAGACCTGA 88.3 kb 229 01120-L00060 RELA gene AAAGGACTGCCG-GGATGGCTTCTA 306.0 kb 382 04159-L03514 SART1 gene CCGCAAGAAGGA-GAAGGAGGTAGT 375.0 kb 160 ± 04155-L03510 BRMS1 gene CAGAAGAGATGG-AAGCAGAGGGTG 1141.0 kb AIP NM_003977.2 Start codon 126-128 (ex 1) 292 ± 07379-L09558 Exon 1 97-98 GAGTCCGGAAGT-TGCCGAAAGGGA 0.1 kb 175 ± 07380-L09559 Exon 1 178-179 AAAACGTGTGAT-ACAGGAAGGCCG 3.9 kb 238 ± 07381-L07028 Exon 2 312-313 CCATGGAGCTCA-TCATTGGCAAGA 2.3 kb 364 ± 07382-L09069 Exon 3 511-512 ACAGATGCGTGA-ACACAGCTCCCT 0.7 kb 209 ± 07383-L07030 Exon 4 668-669 GCAGTGCCACTT-ATCCACCAGGAG 0.3 kb 266 ± 07384-L09556 Exon 5 832-833 GCTGCTGCTCAA-CTACTGCCAGTG 0.5 kb 319 ± 07385-L09557 Exon 6 989-990 CAGGCTGACTTT-GCCAAAGTGCTG 2157.0 kb Stop codon 1116-1118 (ex 6) 310 05403-L04809 CCND1 gene TCCGCCCTCCAT-GGTGGCAGCGGG Length (nt) SALSA MLPA probe CDKN1B Exon Ligation site NM_004064.4 Partial sequence (24 nt adjacent to ligation site) Distance to next probe Start codon 571-573 (ex 1) 391 18426-L23497 Exon 1 568-569 GACCCGGGAGAA-AGATGTCAAACG 1.1 kb 148 18425-L23733 Exon 2 1120-1121 ATGCCGGTTCTG-TGGAGCAGACGC 2.3 kb 427 18429-L23500 Exon 3 1297-1298 CCTGTATAAGCA-CTGAAAAACAAC Stop codon 1165-1167 (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_000244.3, NM_003977.2 and NM_004064.4 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_000244.3). The exon numbering used here may differ from literature! SALSA P244 AIP-MEN1-CDKN1B probemix Page 4 of 6
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
Implemented Changes compared to the previous product description version(s). Version 09 28 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 01666-L01245, 13159-L14681, 13158- L14680 and 01665-L14816. - 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