SALSA MLPA probemix P466-A1 CDC73 Lot A1-0415. Germ-line inactivating point mutations and deletions of CDC73 gene (also known as HRTP2) underlie hyperparathyroidism-jaw tumour syndrome (HPT-JT) (OMIM: 145001) and CDC73 aberrations are also detected in familial primary hyperparathyroidism (OMIM: 145000). HPT-TJ is an autosomal dominant syndrome, characterized by the predisposition to develop parathyroid adenoma or carcinoma, and by the prevalence to develop malignant neoplastic lesions also in jaw bones, kidneys or uterus. Defects in the tumour suppressor gene CDC73 on chromosome 1q, are identified in approximately 60% of HPT-JT cases (Carpten JD et al. 2002, Nat Genet. 32:676-80) and both intragenic and gross germline deletions of CDC73 are also detected (Cascon A et al. 2011, Genes Chromosomes Cancer. 50:922-9; Bricaire L et al. 2013, J Clin Endocrinol Metab. 98:E403-8; Korpi-Hyövälti E et al. 2014, J Clin Endocrinol Metab. 99:3044-8). Moreover, bi-allelic inactivation or mutations of CDC73 leading to dysfunction of the parafibromin protein has been reported in the majority of sporadic parathyroid carcinomas and also in subsets of parathyroid adenomas (Shattuck TM et al. 2003, N Engl J Med. 349:1722-9; Howell VM et al. 2003, J Med Genet. 40:657-63). The CDC73 gene has altogether 17 exons, encodes for the protein parafibromin, spans ~133 kb of genomic DNA and is located on 1q31.2, 193 Mb from the p-telomere. The P466-A1 probemix contains probes for each exon of the CDC73 gene (two probes for exon 17), one probe for the B3GALT2 gene that is located in reverse orientation in intron 10 of the CDC73 gene, and five probes for flanking regions of the CDC73 gene to aid in the determination of the extent of the possible copy number aberration. In addition, 14 reference probes have been included in this probemix, detecting autosomal chromosomal locations which are reported to be relatively silent in tumours of parathyroid gland. However, it should be noticed that tumour genomes can harbour multiple numerical and structural aberrations, which can complicate interpretation of these reference probes. This SALSA probemix is designed to detect copy number changes of one or more sequences in the above mentioned CDC73 gene and its flanking chromosomal region 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 this 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 probemix P466 CDC73 Page 1 of 5
Data analysis The P466-A1 CDC73 probemix contains 38 MLPA probes with amplification products between 124 and 409 nt. In addition, it contains nine 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 should be normalised with a more robust method, as the target sites of the reference probes maybe gained or lost. (1) Intra-sample normalisation should be performed by dividing the signal of each target-specific probe by the signal of every single reference probe in that sample, thus creating as many ratios per target-specific probe as there are reference probes. Subsequently, the median of all these produced ratios per probe should be taken; this is the probe s Normalisation Constant. (2) Secondly, inter-sample comparison should be performed by dividing the Normalisation Constant of each probe in a given sample by the average Normalisation Constant of that probe in all the reference samples. Data normalisation should be performed within one experiment. Always use sample and reference DNA extracted with the same method and derived from the same source of tissue. Confirmation of deletions, duplications 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. Warning: If this assay is used on tumour samples, MLPA analysis on tumour samples provides information on the average situation in the cells from which the DNA sample was purified. Gains or losses of genomic regions or genes may not be detected if the percentage of tumour cells is low. Furthermore, although reference probes are located in silent regions that are not frequently altered in copy number in tumours of parathyroid gland, there is always a possibility that one or more reference probes do show a copy number alteration in a sample. Normal copy number variation in healthy individuals is described in the database of genomic variants: http://dgv.tcag.ca/dgv/app/home. When in doubt, users should always verify the latest updates of the database and scientific literature when interpreting their findings. This probemix was developed by I. Zondervan and S. Savola at. In case the results obtained with this probemix lead to a scientific publication, it would be very much appreciated if the probemix designers could be included as co-authors. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix P466 CDC73 Page 2 of 5
Table 1. SALSA MLPA P466-A1 CDC73 probemix Length (nt) SALSA MLPA probe Chromosomal position Reference CDC73 Other targets 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 124 Reference probe S0863-L28580 10q22.2 130 Reference probe 19551-L26105 2p13.2 136 GLRX2 probe 20515-L28105 1q31.2 142 CDC73 probe 20516-L28106 Exon 13 148 CDC73 probe 20517-L28107 Exon 5 154 CDC73 probe 20518-L28108 Exon 10 162 Reference probe 10694-L26548 6p12.3 167 CDC73 probe 20519-L28375 Exon 12 172 CDC73 probe 20520-L28110 Exon 8 178 Reference probe 07032-L28099 14q11.2 185 CDC73 probe 20521-L28111 Exon 14 190 Reference probe 12780-L28100 2q12.3 196 CDC73 probe 20522-L28461 Exon 1 205 LINC01031 probe 20523-L28113 1q31.2 214 CDC73 probe 20524-L28114 Exon 9 220 Reference probe 17444-L21200 16p13.2 229 CDC73 probe 20525-L28115 Exon 3 236 Reference probe 19652-L26684 4p13 244 CDC73 probe 20526-L28116 Exon 17 252 KCNT2 probe 20527-L28117 1q31.3 258 Reference probe 16472-L26940 17q23.2 267 CDC73 probe 20528-L28118 Exon 15 274 ABCA4 probe 17140-L20332 1p22.1 283 Reference probe 13796-L26436 3q25.31 292 CDC73 probe 20529-L28119 Exon 2 301 CDC73 probe 20530-L28120 Exon 16 310 Reference probe 08739-L28101 9q21.13 319 Ø B3GALT2 probe 20531-L28121 1q31.2 (Exon 2) 328 Reference probe 18779-L28102 3p22.2 337 CDC73 probe 20533-L28123 Exon 4 346 CDC73 probe 20534-L28124 Exon 7 352 Reference probe 06708-L24718 10p11.1 363 CDC73 probe 20535-L28125 Exon 11 372 TROVE2 probe 20536-L28126 1q31.2 382 CDC73 probe 20537-L28127 Exon 6 391 Reference probe 05914-L28104 18p11.21 400 CDC73 probe 20538-L28128 Exon 17 409 Reference probe 10638-L28103 8q12.2 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. Ø Intron probe. This probe detects the B3GALT2 gene that is located in reverse orientation in intron 10 of the CDC73 gene. This probe is only included to facilitate the determination of the extent of a deletion / duplication of CDC73 gene. Copy number alterations of a single intron probe are unlikely to be related to the condition tested. Note: Exon numbering used here may differ from literature! Please notify us of any mistakes: info@mlpa.com. The identity of the genes detected by the reference probes is available in table 2. SALSA probemix P466 CDC73 Page 3 of 5
Table 2. P466 probes arranged according to chromosomal location Length (nt) SALSA MLPA probe Gene / Exon Location / Ligation site Partial sequence (24 nt adjacent to ligation site) Distance to next probe MV location (HG18) Flanking probes upstream of CDC73. 274 17140-L20332 ABCA4 1p22.1 ACAGCGACCCAT-TCATCCTCTTCC 97 Mb 01-094.294892 372 20536-L28126 TROVE2 1q31.2 CCTTTGCTGGAG-GTGTCCATCCTG 15.0 kb 01-191.318383 136 20515-L28105 GLRX2 1q31.2 CTTGAATATGGA-AACCAGTTCCAA 24.7 kb 01-191.333338 CDC73, at 1q31.2. Indicated ligation sites are in NM_024529.4 (NG_012691.1), unless otherwise specified. start codon 244-246 (ex 1) 196 20522-L28461 Exon 1 322-323 ACGAAGTGATCT-TCGGGGAGTTCT 2.9 kb 01-191.358004 292 20529-L28119 Exon 2 444-443 reverse TGAGAAAGGTGC-ACGTTATTAAGT 5.0 kb 01-191.360887 229 20525-L28115 Exon 3 508-509 TGGTTAGAAGAC-CTGATCGAAAAG 5.2 kb 01-191.365919 337 20533-L28123 Exon 4 585-586 AGCGCTCCCTTA-GAAATAGGTCTT 0.1 kb 01-191.371147 148 20517-L28107 Exon 5 643-644 AAGTTTTAGCAG-AAGCAAAGAAAC 2.6 kb 01-191.371287 382 20537-L28127 Exon 6 718-719 CCCGTTTGGAGG-GTCACAAAGAAG 3.8 kb 01-191.373864 346 20534-L28124 Exon 7 863-864 TGACATAACTGC-CCTTAAACAGAG 6.0 kb 01-191.377671 172 20520-L28110 Exon 8 1064-1063 reverse TTACCACAGGTG-CTGCATTTGGGG 2.4 kb 01-191.383676 214 20524-L28114 Exon 9 1137-1136 reverse TCTTTTCCTTTG-AATCTTTCCTGA 2.1 kb 01-191.386073 154 20518-L28108 Exon 10 1202-1201 reverse TTACAGATTTCA-GTGTCATACCAT 28.2 kb 01-191.388146 319 Ø 20531-L28121 B3GALT2; NM_003783.3; Exon 2 1615-1614 reverse GGGTAGAGGTCT-GGTGGCATGTAC 23.2 kb 01-191.416382 363 20535-L28125 Exon 11 1245-1244 reverse TGGGCTGCAGGA-GTCTGAGTCTTC 8.3 kb 01-191.439545 167 20519-L28375 Exon 12 1296-1297 AGACCTCCCCCA-AATCAGAAGAAA 0.3 kb 01-191.447811 142 20516-L28106 Exon 13 1334-1335 CATTATCATAAT-TCCTGCAGCTAC 20.7 kb 01-191.448130 185 20521-L28111 Exon 14 1506-1505 reverse TAAGGTACTGTA-ACACTAATTGCA 3.2 kb 01-191.468818 267 20528-L28118 Exon 15 1614-1615 TTCAAAGGTTGG-CCATGGCTTTTG 13.5 kb 01-191.472034 301 20530-L28120 Exon 16 1724-1725 GAAATGGGATGT-AACAGTATTAGA 0.9 kb 01-191.485515 400 20538-L28128 Exon 17 1835-1836 GCACTTGAGATT-CTGAATTATTTG 0.8 kb 01-191.486423 244 20526-L28116 Exon 17 2623-2622 reverse GTTATACTGCAA-TAGGGCTGATAC 114.3 kb 01-191.487211 stop codon 1837-1839 (ex 17) Flanking probes downstream of CDC73. 205 20523-L28113 LINC01031 1q31.2 GGACATGAATTC-AGTAAGCTCCTT 3.1 Mb 01-191.601528 252 20527-L28117 KCNT2 1q31.3 TTGGCAAGCCAA-CAGTTCAGAAAG 01-194.703459 Reference probes. 130 19551-L26105 DYSF 2p13.2 CCATTGCCAAGA-AGGTCAGTGTCC 37 Mb 02-071.750350 190 12780-L28100 EDAR 2q12.3 TGGACATTGCAA-TGATCAGGGCAG 02-108.893894 328 18779-L28102 ACVR2B 3p22.2 TTGCCCACAGGT-ACCTGGGTCAGC 119 Mb 03-038.496297 283 13796-L26436 KCNAB1 3q25.31 CTTTTCCAGAGA-GAGAAAGTGGAG 03-157.715622 236 19652-L26684 ATP8A1 4p13 CAGATTCTTCTT-CGAGGAGCTCAG 04-042.278439 162 10694-L26548 PKHD1 6p12.3 CAGTTTCTGTAT-TTCCTAAAACAG 06-051.717147 409 10638-L28103 CHD7 8q12.2 GACTCAATGGTA-ATGGTGTGGGTT 08-061.913286 310 08739-L28101 PCSK5 9q21.13 TCTCACAGGTCT-GTGTGCTTTGCT 09-078.143100 352 06708-L24718 ZNF25 10p11.1 CAGGTGATTCCT-GGGGCTGCCAGC 38 Mb 10-038.300693 124 S0863-L28580 KAT6B 10q22.2 GATCCCATTCCA-ATATGTAGCTTC 10-076.389726 178 07032-L28099 RPGRIP1 14q11.2 GCTGAATGGACA-AGATCCTGATCA 14-020.883152 220 17444-L21200 GRIN2A 16p13.2 CGTGGATTCCAT-ACGCCAGGATTC 16-009.765794 258 16472-L26940 MED13 17q23.2 TTCTGGTCAGCA-TGTCCTCAAGCA 17-057.382966 391 05914-L28104 RNMT 18p11.21 TACAATGAACTT-CAGGAAGTTGGT 18-013.724482 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. Ø Intron probe. This probe detects the B3GALT2 gene that is located in reverse orientation in intron 10 of the CDC73 gene. This probe is only included to facilitate the determination of the extent of a deletion / duplication of CDC73 gene. Copy number alterations of a single intron probe are unlikely to be related to the condition tested. The NM_024529.4 (NG_012691.1) sequence is a reference standard in the NCBI RefSeqGene project. Note: Exon numbering may differ from literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA probemix P466 CDC73 Page 4 of 5
SALSA MLPA probemix P466-A1 CDC73 sample picture Figure. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P466-A1 CDC73 (lot A1-0415). Implemented Changes compared to the previous product description version(s). Version 01 28 April 2015 (T07) - Not applicable, new document. SALSA probemix P466 CDC73 Page 5 of 5