SALSA MLPA probemix P277-B2 Human Telomere-10 Lot B As compared to version B1-1210, the control fragments have been replaced (QDX2).

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1 SALSA MLPA probemix P277-B2 Human Telomere-10 Lot B As compared to version B1-1210, the control fragments have been replaced (QDX2). This P277-B2 probemix can be used to confirm and further characterise abnormalities detected by SALSA probemix P036 and/or SALSA probemix P070 used for broad human subtelomere screening. This SALSA P277-B2 Human Telomere-10 probemix contains: 14 probes in the terminal 6.8 Mb of chromosome arm 5q 11 probes in the terminal 4.5 Mb of chromosome arm 6q 11 probes in the terminal 6.8 Mb of chromosome arm 7q 11 probes in the terminal 4.6 Mb of chromosome arm 8q SALSA probemixes P036 and P070 each contain one MLPA probe for each subtelomeric region and are frequently used for initial screening for subtelomeric deletions and duplications. Most of the probes in these probemixes detect a well-characterised gene close to the telomere. The probes in P036 and P070 are different, allowing results obtained with one probemix to be confirmed by the other. For the p-telomeres of the acrocentric chromosomes 13, 14, 15, 21 and 22, it is not possible to design reliable probes. The P036 and P070 probemixes contain a second probe for these q arms, which target a DNA sequence close to the centromere. We recommend using SALSA probemix P036 Subtelomeres mix 1 and SALSA probemix P070 Subtelomeres mix 2B for primary screening of the subtelomeres. Results obtained with these probemixes can be confirmed and further characterized with one of the specific follow-up probemixes for telomere analysis, such as this P277-B2 Human Telomere-10 probemix. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the above mentioned chromosomal 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 SALSA P277 Human Telomere-10 probemix Page 1 of 6

2 Related SALSA probemixes P147 1p36 deletions: 1p P208 Human Telomere-6: 2p, 3p, 6p, 8p P230 Human Telomere-7: 9p, 10p, 11p, 12p P249 Human Telomere-8: 17p, 18p, 19p, 20p P264 Human Telomere-9: 1q, 2q, 3q, 4q P286 Human Telomere-11: 9q, 10q, 11q, 12q P291 Human Telomere-12: 13q, 14q, 15q, 16q P320 Human Telomere-13: 17q, 18q, 19q, 20q P365 Human Telomere-14: 7p,15q-cen, 16p, 21q-cen, 21q P096 MR-2: 4p, 5p P140 HBA: 16p P163 GJB-WSF1: 13q-cen P188 22q13: 22q P250 DiGeorge: 22q P340 EHMT1: 9q P356 Chromosome 22q: 22q-cen, 22q ME028 PWS/AS: 15q Data analysis The P277-B2 Human Telomere-10 probemix contains 47 MLPA probes with amplification products between 130 and 492 nt. In addition, it contains nine 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 should be normalised with a robust method. (1) Intra-sample normalisation should be performed by dividing the signal of each probe by the signal of every other probe in that sample, thus creating as many ratios per probe as there are other probes. Subsequently, the median of all these produced ratios per probe should be taken; this is the probe s Normalisation Constant. (2) Secondly, intersample 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. Only samples purified by the same method should be compared. Confirmation of most deletions and amplifications can be done by e.g. Southern blotting or long range PCR. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website Subtelomeric abnormalities The detection of abnormal copy numbers in subtelomeric regions is very complicated. Compared to other regions of the genome, these regions are rich in sequences with variable copy numbers. Copy number changes of sequences within these regions can also occur in unaffected individuals and the effect of a deletion or duplication will depend on the genes present in the affected region. The choice of genes targeted by this probemix has in part been guided by their chromosomal location. Not all copy number changes identified will result in phenotypic abnormalities! For all abnormalities detected, we strongly recommend testing the patient s parents to determine whether a copy number aberration found in the patient is truly de novo. Please be aware that a considerable number of abnormalities detected by a single probe may not have a phenotypic effect, but can be due to a rare polymorphism or a copy number change which is also present in one of the parents. For some chromosome arms, even a large subtelomeric deletion of more than 1 Mb can be inherited without a phenotypic effect. No method will be capable of detecting all chromosomal aberrations. MLPA will not detect inversions or balanced translocations. Frequent microdeletion syndromes can be detected with SALSA MLPA probemix P245 Microdeletion Syndromes but many rare interstitial deletions will not be detected. More information on copy number changes in the human genome can be found in the Database of Genomic Variants ( This probemix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P277 Human Telomere-10 probemix Page 2 of 6

3 Table 1. SALSA MLPA P277-B2 Human Telomere-10 probemix Length (nt) SALSA MLPA probe Chromosomal position 5q 6q 7q 8q Chr + distance to p-telomere in kb (hg18) 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 130 MAPK9 probe L q ± GRINA probe L q ± MNX1 probe L q ± GLI4 probe L q CNOT6 probe L q DPP6 probe L q RNF32 probe L q T probe L q ± SHH probe L q ± MGAT1 probe L q LYNX1 probe L q PDE10A probe L q TBP probe L q ± RECQL4 probe L q ADAMTS2 probe L q DLL1 probe L q NCAPG2 probe L q ± DBN1 probe L q WDR60 probe L q SMOC2 probe L q ± GPT probe L q HTR5A probe L q MAML1 probe L q ± SCRT1 probe L q BTNL3 probe L q PDCD2 probe L q COL23A1 probe L q PTK2 probe L q TCTE3 probe L q VIPR2 probe L q PTPRN2 probe L q FGFR4 probe L q ZC3H3 probe L q MLLT4 probe L q TRIM41 probe L q PROP1 probe L q ± FLT4 probe L q SCRIB probe L q UBE3C probe L q PTPRN2 probe L q PTP4A3 probe L q THBS2 probe L q NSD1 probe L q TSNARE1 probe L q RPS6KA2 probe L q ZNF354A probe L q FAM120B probe L q Changed in version B2 (from lot B 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. SALSA P277 Human Telomere-10 probemix Page 3 of 6

4 Table 2. P277 probes arranged according to chromosomal location Table 2a. 5q L00859 FGFR4 GCGATTCTGTCT-TCAGCCACGACC 4401 kb 191 kb L02071 NSD1 GGAAAGACTGTT-TGCAAATGTGGA 4209 kb 179 kb L09633 DBN1 GACACTTTGAGA-ACCAGAAGGTGA 4030 kb 526 kb L18982 PROP1 AGCACTCGAGTC-TGAGAACGGAGA 3504 kb 261 kb L10914 COL23A1 GGTGATCGACTA-TGATGGCAGGAT 3243 kb 458 kb L15356 ZNF354A CAGAATTCACAA-TTACTTAATCAA 2785 kb 468 kb L10909 ADAMTS2 TGGGTGCCCACA-TCAACGTGGTCC 2317 kb 594 kb L09636 MAML1 ACACCGATCTGA-TCGACTCCCTGC 1723 kb 462 kb L15347 MAPK9 TAGAAATAGCAA-ACCTGTCAGCAT 1262 kb 338 kb L28684 CNOT6 TCTTACTGCCTT-TCCTGCCCCAAG 924 kb 58 kb L00695 FLT4 AGGAGTCACACG-TCATCGACACCG 867 kb 160 kb L09638 MGAT1 CTGGACCTGTCT-TACCTGCAGCGG 706 kb 214 kb L17825 BTNL3 CACTCCCACATA-CCACCCTACATT 493 kb 229 kb L18981 TRIM41 TACAACGCAGAG-ACTCTAGCCCAC 264 kb 191 kb 5q telomere Reported cases of distal monosomy of the long arm of chromosome 5 show developmental delay, defects of cardiac septation, malformations of the CNS and facial dysmorphisms (Schafer et al. (2001) Am J Med t. 103:63-68). Partial duplication of the distal long arm of chromosome 5 is associated with a specific phenotype that includes growth and mental retardation, microcephaly, strabismus, a prominent widened nasal bridge and a long philtrum (Abuelo et al. (2000) Am J Med t. 94: ; Chen et al. (2006) Am J Med t. 140: ). The phenotype of microcephaly and short stature, observed in subtelomeric trisomy 5q patients is thought to be caused by overdose of the NSD1 gene. This syndrome has a reverse phenotype, compared with the 5q deletion syndrome, including cerebral gigantism, macrocephaly, advanced bone age and overgrowth (Chen et al. (2006) Am J Med t. 140: ). Table 2b. 6q L09651 PDE10A AATCCCAACATT-TAACAGGTGAGT 4990 kb 590 kb L11456 T TGCTGAAGGTGA-ACGTGTCTGGCC 4400 kb 695 kb L17027 RPS6KA2 TTGCTGGAACTA-TGGAAAAAGATC 3704 kb 900 kb L18980 MLLT4 TCCAGGAGTCGA-AGCAGAAGGACG 2804 kb 714 kb L11459 SMOC2 GTGCTTACAGCA-TTGTGGAATCAT 2091 kb 554 kb L17069 THBS2 ACCCCAGGAACA-TTGGCTGGAAGG 1537 kb 530 kb L17821 TCTE3 CCTAGGAAAGAA-AGGAGGCCTAGC 1007 kb 543 kb L15348 DLL1 ATCCGCTATCCA-GGCTGTCTCCAT 459 kb 119 kb L15357 FAM120B AACTTGGTTTCA-AAACACTCTTGC 344 kb 162 kb L09641 TBP ATCATGAGGATA-AGAGAGCCACGA 182 kb 17 kb L11460 PDCD2 TGCCACAAAGCA-TATTACTGCAGC 165 kb 6q telomere Frequently observed symptoms in subtelomeric 6q deletion include developmental delay, seizures, hypoplasia of the corpus callosum, dimpling on elbows and knees and specific dysmorphic features (Stevenson et al. (2004) Clin Dysmorphol. 13: ). Rooms et al. have demonstrated that TBP is a likely candidate gene for mental retardation in the 6q subtelomeric deletion phenotype but a possible contribution to the phenotype of other genes in the deleted region cannot be ruled out, notably PSMB1 and PDCD2 (Rooms et al. (2006) Eur J Hum t. 14: ). Pure partial duplications of the long arm of chromosome 6 are rare. Despite the variable sizes of the involved regions, a recognizable 6q duplication phenotype can be delineated, which is characterized by distinct facial dysmorphism with hypertelorism, downslanting palpebral fissures, low nuchal hairline, a carp shaped mouth and a broad, short neck. Further symptoms are profound mental retardation, joint contractures, pre- and/or postnatal growth retardation, microcephaly and a broad spectrum of malformations such as cerebral or heart defects or anomalies of the renal and urogenital systems, and early lethality in cases with larger deletions (Zweier et al. (2008) Eur J Med t. 51: ). Please note that a terminal deletion of 6q of approximately 0.7 Mb in size has been observed in a normal individual (Balikova et al. (2007) Hum Mutat. 28: ). SALSA P277 Human Telomere-10 probemix Page 4 of 6

5 Table 2c. 7q L28683 DPP6 GAAGACATGATG-GCATGAAATGTG 5507 kb 1180 kb L09652 HTR5A TCGTGTACTGGA-AGATCTACAAGG 4327 kb 803 kb L06396 SHH CTCGGAAGTCAT-CAGTTCCATGGG 3523 kb 843 kb L28734 RNF32 CTCGAATTCTTC-TTTACAGATGGG 2681 kb 355 kb L17819 MNX1 TGATCCTGCCTA-AGATGCCCGACT 2326 kb 239 kb L00697 UBE3C CAGTGCCGACGT-GGACATTCACCA 2087 kb 373 kb L17823 PTPRN2 ACCACCTGAAGA-ACAAGAACCGGC 1714 kb 513 kb L15352 PTPRN2 ACATGGAGAGGA-AGAAGTCCGAGC 1202 kb 568 kb L10911 NCAPG2 CAAGCCGTGTCT-AAGGAGCTGGTT 634 kb 189 kb L16362 WDR60 TCTCCATTAGCA-TGGCCACGAGGA 445 kb 212 kb L17822 VIPR2 AAATAATCCTCT-GAGTACCAATGC 232 kb 7q telomere Information about clinical manifestations of subtelomeric 7q deletion is scarce. Horn et al. investigated four patients with 7q deletion. They found that in all four patients, the Sonic Hedgehog gene (SHH) and the homeobox gene MNX1, among others, are included in the deletions (Horn et al. (2004) Am J Med t. A 128A:85-92). Mutations and deletions affecting the SHH gene have been identified earlier as one of the causes of holoprosencephaly (Belloni et al. (1996) Nat t. 14: ; Roessler et al. (1996) Nat. t. 14: ). The homeobox gene MNX1 is located 1.2 Mb telomeric to SHH and has been shown to be involved in Currarino syndrome (Lynch et al. (1995) Nat t. 11:93 95). In the group studied by Horn et al., all patients showed besides mental retardation and short stature- only minimal manifestations of the holoprosencephaly (HPE) spectrum and only one displayed symptoms of the Currarino syndrome (Horn et al. (2004) Am J Med t. 128A:85-92). Table 2d. 8q L10915 PTK2 CTGGTTCACATG-GAATCACAGCCA 4493 kb 728 kb L15334 PTP4A3 CACACACGCACA-AGACCCGGTGCT 3765 kb 781 kb L15355 TSNARE1 GCACCCGAATAA-ACCGCTGTGGCT 2983 kb 563 kb L09657 LYNX1 TGCTCACCCTGA-TCCTGGTGGTCC 2421 kb 576 kb L17818 GLI4 GCGGCAAGAGTT-TCAAGTATAACT 1845 kb 263 kb L01302 ZC3H3 GCATGTCCTTGA-GAGACAGGTCCA 1583 kb 253 kb L15354 SCRIB AAAGTCCGGTGA-CTGTGATGGGGA 1329 kb 193 kb L28596 GRINA TTCTCTGCATCT-TCATCCGGAACC 1136 kb 392 kb L10913 SCRT1 CAAGAAGGTCAA-ACTTGACGCGTT 744 kb 173 kb L28605 GPT AGCTGAGCAGGT-TCCATGCCAAGT 572 kb 7 kb L11457 RECQL4 AGGCAAACGTTT-TCAAAACCTCGA 565 kb 728 kb 8q telomere Knowledge on chromosomal aberrations involving the subtelomere of 8q is limited. In 2005, Bonaglia et al. detected a 2.3 Mb duplication of chromosome 8q24.3 in a patient with severe mental retardation and epilepsy. They hypothesized that the GRINA gene, a glutamate binding subunit of NMDA receptor ion channel lying within the duplicated segment, may be responsible for the epilepsy (Bonaglia et al. (2005) Eur J Hum t. 13: ). Note: Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA P277 Human Telomere-10 probemix Page 5 of 6

6 SALSA MLPA probemix P277-B2 Human Telomere-10 sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P277 (lot B2-1115). Implemented Changes compared to the previous product description versions. Version 10 8 March 2018 (10) - Information about the Telomere Follow-up Set has been removed on page 1. - References to P069 have been removed. - Probes present in P036 or P070 removed from Table 2. Version January 2015 (10) - 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. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. Version 08 (06) - Various minor textual and layout changes. Version 07 (02) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 06 (02) - Warning about salt sensitivity of 184 nt probe L06396 added to Table 1. - Various minor textual changes. - Various minor layout changes. Version 05 (01) - Information about the Telomere Follow-up Set has been added on page 1. SALSA P277 Human Telomere-10 probemix Page 6 of 6