MRC-Holland MLPA. Description version 10; 8 March 2018

Transcription

1 mix P264-C1 Human Telomere-9 Lot C As compared to version B1, one in the 3q region and two s in the 4q region have been replaced. Furthermore, 32 s have been changed in length, but not in sequence This P264-C1 mix can be used to confirm and further characterise abnormalities detected by SALSA mix P036 and/or SALSA mix P070 used for broad human sub screening. This SALSA P264-C1 Human Telomere-9 mix contains: 15 s in the terminal 9.1 Mb of chromosome arm 1q 13 s in the terminal 5.0 Mb of chromosome arm 2q 11 s in the terminal 3.9 Mb of chromosome arm 3q 10 s in the terminal 5.1 Mb of chromosome arm 4q SALSA mixes P036 and P070 each contain one MLPA for each subtelomeric region and are frequently used for initial screening for subtelomeric deletions and duplications. Most of the s in these mixes detect a well-characterised gene close to the. The s in P036 and P070 are different, allowing results obtained with one mix to be confirmed by the other. For the p-s of the acrocentric chromosomes 13, 14, 15, 21 and 22, it is not possible to design reliable s. The P036 and P070 mixes contain a second for these q arms, which target a DNA sequence close to the. We recommend using SALSA mix P036 Subs mix 1 and SALSA mix P070 Subs mix 2B for primary screening of the subs. Results obtained with these mixes can be confirmed and further characterized with one of the specific follow-up mixes for analysis, such as this P264-C1 Human Telomere-9 mix. This SALSA mix 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. Note that a mutation or polymorphism in the sequence detected by a can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some 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 mixes 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 mixes and reagents includes a limited license to use these products for research purposes. The use of a SALSA mix 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 mix P264 Human Telomere-9 Page 1 of 7

2 Related SALSA mixes for specific subtelomeric analysis 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 P277 Human Telomere-10: 5q, 6q, 7q, 8q 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 Data analysis 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 The P264-C1 Human Telomere-9 mix contains 49 MLPA s with amplification products between 126 and 483 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 mix should be normalised with a robust method. (1) Intra-sample normalisation should be performed by dividing the signal of each by the signal of every other in that sample, thus creating as many ratios per as there are other s. Subsequently, the median of all these produced ratios per should be taken; this is the s Normalisation Constant. (2) Secondly, intersample comparison should be performed by dividing the Normalisation Constant of each in a given sample by the average Normalisation Constant of that 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, long range PCR, qpcr or FISH. 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 mix 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 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 mix P245 Microdeletion Syndromes-1A but many rare interstitial deletions will not be More information on copy number changes in the human genome can be found in the Database of Genomic Variants ( This mix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA mix P264 Human Telomere-9 Page 2 of 7

3 Table 1. P264-C1 Human Telomere-9 mix Chromosomal position 1q 2q 3q 4q Chr + distance to p- 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 126 ATG4B L q , SH3BP5L L q , DLG L q , EFCAB L q , LSG L q , FRG L q , ZNF L q , «TNK L q , «ZBTB18 (ZNF238) L q , UBXN L q , DLG L q , ZFP L q , CEP L q , KIF26B L q , NEU L q , PDLIM L q , BDH L q , FAT L q , «PDCD L q , CHRM L q , TRAF3IP L q , * OPA L q , CYP4V L q , ACAP L q , D2HGDH L q , HNRNPU L q , HELT L q , PASK L q , TRIM L q , SH3BP5L L q , * LINC L q , CAPN L q , NDUFA L q , LRRFIP L q , LMLN L q , * F L q , GP L q , AKT L q , HDAC L q , NLRP L q , STK L q , TFB2M L q , AGXT L q , «SLC25A L q , TRIML L q , SMYD L q , COL6A L q , MELTF (MFI2) L q , ADSS L q ,654 SALSA mix P264 Human Telomere-9 Page 3 of 7

4 * New in version C1 (from lot C onwards). Notes: names of MFI2 and ZNF238 have changed from product description version 09 onwards. The gene names used in previous versions can be found between brackets in Table 1 and Table 2. Many s in this mix are sensitive to salt, the peak height of the denaturation control fragments (88 nt and 96 nt) should be checked carefully for lower signals. Please notify us of any mistakes: info@mlpa.com. Table 2. P264 s arranged according to chromosomal location Table 2a. 1q to 1q L30077 CHRM3 ATTGGCTCCGAG-ACGAGAGCCATC 9111 kb kb L30046 CEP170 AAGCAACACGCT-GTCATCAACTAT 5798 kb kb L30115 AKT3 TTGCCTCTGCAG-TCTGTCTGCTAC 5374 kb kb 178 «21284-L18054 ZBTB18 (ZNF238) GAGTCTCTCCGA-TGGCAGCAGCCA 4966 kb kb L11452 ADSS TTCTGACTTTGA-TGGCTTCTCTGA 4596 kb kb L18057 HNRNPU AAGCGACGCCTT-TCTGACAAGGGT 4156 kb kb L29664 EFCAB2 GTGTGCTCACCA-TCCAGAGTCTCT 3938 kb kb L29669 KIF26B GCCGTGTCAACT-TCTGCAAGGCCC 3317 kb kb L11451 SMYD3 AAGCTGATGCGA-TGCTCTCAGTGC 2665 kb kb L29688 TFB2M GCTGGAGTGCAA-TCCAGGTGAGTC 2454 kb kb L29666 ZNF669 TTAATTACAGAA-GTCTTTCCCTAT 1918 kb kb L29686 NLRP3 GACCTCCAGCCA-GAGCCTGCGAAA 1575 kb kb L11151 TRIM58 GCAGCGCTTCAG-ATTGGAGTTTGA 1155 kb kb L22368 SH3BP5L, exon 7 CAAGGTGACAGA-ACTGGAGCAGCA 177 kb 12.7 kb L17627 SH3BP5L, exon 2 AACCTAGTGTGA-GGTTCTTCCTGT 164 kb Deletions of the 1q have been reported in several studies screening for subtelomeric rearrangements (de Vries et al., 2003, J Med t. 40:385-98; Flint and Knight, 2003, Curr Opin t Dev. 13:310-6). Subtelomeric deletion of the 1q region causes a recognizable phenotype, including intellectual disability, microcephaly, growth retardation and corpus callosum abnormalities. Patients with microscopic deletion have the same, albeit more severe, phenotype as patients with submicroscopic deletions in this region. This implies that presentation of the 1q deletion syndrome is mainly caused by haploidy of genes in the subtelomeric region (van Bon et al., 2008, J Med t. 45:346-54; de Vries et al., 2001, J Med t. 38:175-8). Van Bever et al. suggest that genes in the distal 6 Mb to the are associated with intellectual disability (van Bever et al., 2005, Am J Med t. 135:91-5). More information on the 1q43-q44 deletion syndrome can be found in OMIM # Please note that a subtelomeric 1q duplication of 2.9 Mb has been observed in healthy individuals (Rooms et al., 2006, Clin t. 69:58-64; Balikova et al., 2007, Hum Mutat. 28:958-67). SALSA mix P264 Human Telomere-9 Page 4 of 7

5 Table 2b. 2q to 2q L09292 COL6A3 AGAAACTCCCAA-TTGGAACTCAGC 4990 kb kb L30080 LRRFIP1 CTGCAGAACCAA-AGAGCGAAGACG 4614 kb kb L30048 TRAF3IP1 AGGAGATAGAGA-AGCTCCGCACGT 3980 kb kb L30116 HDAC4 GAGTTTGGAGCT-CGTTGGAGCTAT 3012 kb kb L09288 NDUFA10 CCTTGGAGCACT-TGCTGACCACAG 2342 kb kb L17633 CAPN10 ATGTGTTCTGGC-TCCCCTTACTGG 1772 kb kb L29689 AGXT CTGGACATCAAG-TGGCTGGCCAAC 1488 kb kb L30079 PASK TCCTATTGTCTA-TCATCACTGGCT 1222 kb kb L30114 STK25 GAGCAGGGTGAC-GTGAAGCTGGCG 864 kb kb L25601 ATG4B AGCGTTCCCTGT-GCAGGCGCCACT 696 kb 78.2 kb L17632 D2HGDH TCACCACGGTGT-CCATCTTGTGTC 618 kb 74.0 kb L29675 NEU4 TCCTTTTGTACA-TTCTCCCTGCGT 544 kb 34.9 kb 238 «15664-L29677 PDCD1 GTCTGCAGAACA-CTGGTGGCCAAG 509 kb Monosomy 2q37 patients show significant variability in clinical presentation which is mainly dependent on the size of the deletion. Clinical features include intellectual disability, short stature and facial dysmorphism. In approximately 50% of cases, brachymetaphalangism has been reported (Aldred et al., 2004, J Med t. 41:433-9). More information about this condition can be found in OMIM # Please note that a subtelomeric 2q deletion of 1.6 Mb has been observed in a healthy individual (Balikova et al., 2007, Hum Mutat. 28:958-67). Table 2c. 3q to 3q 257 * L30078 OPA1 AAGAGGTAGGCA-TCATTTATCACC 4621 kb 718,8 kb L09301 GP5 GGAGCTCGCTCT-GAACCAGAATCA 3902 kb kb L29665 LSG1 CAATGATGGCTA-TGATTGGGGTCG 3630 kb kb L30049 ACAP2 GACCTCAGGCTT-TGCACAGTGAAA 2993 kb kb 171 «21286-L29670 TNK2 AGGAACCGGACA-AGCTGCACATCC 2412 kb kb L29667 UBXN7 AGGAATCGCTGA-AGAGCCCAGTAC 1883 kb kb L24857 MELTF (MFI2) TTGACACCCTGA-AAGGCGTGAAGT 1266 kb kb L09294 DLG1, exon 2 GTTTATTCCGTA-ACTCTGTTGTCG 993 kb 1.4 kb L30076 DLG1, exon 1 TGGATCTGGTGT-AGGCGAGGTCAC 992 kb kb L18055 BDH1 CTAAACAGTGAC-CGATTGAGAACC 768 kb kb L10543 LMLN ATGAATGGCTGG-ATTCACGATGGA 255 kb * New in version C1 (from lot C onwards). SALSA mix P264 Human Telomere-9 Page 5 of 7

6 Several patients with 3q29 microdeletion syndrome have been reported. The clinical phenotype was variable despite an almost identical deletion size and may include mild-to-moderate intellectual disability, delayed psychomotor development, and slightly dysmorphic facial features (Willatt et al., 2005, Am J Hum t. 77:154-60; Digilio et al., 2009, Am. J. Med. t. 149A: ; Li et al., 2009, Europ. J. Med. t. 52: ; Quintero-Rivera et al., 2010, Am. J. Med. t. 152A: ). Lisi et al. reported a family in which 5 members had a 3q29 microduplication. They presented with mild-tomoderate intellectual disability, dysmorphic features, and other variable clinical features (Lisi et al., 2008, Am. J. Med. t. 146A: ). More information about these syndromes can also be found in OMIM # (3q29 microdeletion) and # (3q29 microduplication). Table 2d. 4q to 4q L29684 HELT CAAGGAACGCAA-AGTGAGTCGGCT 5096 kb kb 436 «09053-L30081 SLC25A4 GGAAGATTGCAA-AAGACGAAGGAG 4968 kb kb L30047 PDLIM3 GTCATCCTGGCT-ATTGACGGCTTT 4590 kb kb L09305 CYP4V2 GGATAGAATCCA-ACCAGAAAAGAG 3904 kb 55.7 kb 364 * L30050 F11 GTAAGCCAACAA-GGTCTTTTCAGG 3848 kb kb L18056 FAT1 CTGCAGTTTACA-CACCTCGAGTAC 3405 kb kb L11447 ZFP42 TTCAAGGCTGCA-ACAGGAGGTTTA 2111 kb 97.5 kb L17626 TRIML2 TTGAACCTGAGA-GAAACCCTTCTG 2014 kb kb 328 * L30113 LINC01596 GGGAAGTGGTAA-CCATGGAGGCCA 233 kb 59.3 kb L30033 FRG1 GTGCTGCCCCGA-CTCACATACTCG 174 kb * New in version C1 (from lot C onwards). Terminal deletion of the long arm of chromosome 4 is a rare genetic event associated with a distinctive phenotype dependent on the deletion size. Deletions spanning the 4q32 band include intellectual disability and mild craniofacial anomalies (Sills et al., 2007, Orphanet J Rare Dis. 2:9). All patients with a confirmed diagnosis of facioscapulohumeral muscular dystrophy (FSHD) and for whom detailed molecular studies have been performed, carry a chromosomal rearrangement within the subtelomeric region of 4q (4q35). The abnormalities found in FSHD patients are located in a complex chromosomal area telomeric of the FRG1 gene. These cannot be detected using MLPA. More information about this condition can be found in OMIM # Please note that benign subtelomeric duplications as well as deletions of 4q of more than 1 Mb have previously been reported (Ravnan et al., 2005, J Med t. 43:478-89; Balikova et al., 2007, Hum Mutat. 28:958-67). Notes: More information on copy number changes in the human genome can be found in the Database of Genomic Variants ( Complete sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA mix P264 Human Telomere-9 Page 6 of 7

7 mix P264-C1 Human Telomere-9 sample picture Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA mix P264-C1 Human Telomere-9 (lot C1-0517). Implemented Changes compared to the previous product description versions. Version 10 8 Match 2018 (10) - Information about the Telomere Follow-up Set has been removed on page 1. - References to P069 have been removed. Version July 2017 (10) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new picture included). - Updated gene name MFI2 to MELTF and ZNF238 to ZBTB18 in Table 1 and 2. - Various textual changes. - Information added on 3q29 duplication syndrome on page 6. - Warnings added about s sensitive to salt in Table 1 and Table 2, and on page 4. Version December 2015 (10) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Link to Database of Genomic Variants updated. - Various minor textual changes. - Peak area replaced with peak height. - Small changes of lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. Version 07 (02) - Electropherogram picture of old buffer (introduced Dec. 2012) removed. Version 06 (02) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 05 (02) - Various minor layout changes. SALSA mix P264 Human Telomere-9 Page 7 of 7