SALSA MLPA probemix P267-A3 Dandy-Walker Malformation Lot A3-0813. As compared to the previous lot A2-0209, two reference probes have been replaced and one added. Also, the control fragments have been adjusted (QDX2). Dandy-Walker malformation (DWM) is a form of cerebellar hypoplasia. It was first described by Dandy and Blackfan in 1914. In 1942, Taggart and Walker defined the main clinical and pathological features after which Benda designated this disorder as Dandy-Walker syndrome. DWM is characterized by hypoplasia and upward rotation of the cerebellar vermis, cystic dilation of the fourth ventricle and hydrocephalus. Patients often suffer from delayed motor development, ataxia and hypotonia. Approximately half of them show mental retardation. In addition, other anomalies have been described including agenesis of the corpus callosum, visual deficits and epilepsy. In some individuals diagnosed with DWM deletions of 3q2 were found, encompassing the ZIC1 and ZIC4 genes. Grinberg et al. (2004, Nat Genet) investigated the contributions of these genes in a mouse model that showed heterozygous loss of ZIC1 and ZIC4 which resulted in a DWM-like phenotype. A deletion of the VLDLR gene results in a different form of cerebellar hypoplasia that is also characterized by ataxia, disturbed equilibrium and mental retardation. The ZIC1 gene (3 exons) spans ~7.3 kb of genomic DNA and is located on chromosome 3q24, ~147 Mb from the p-telomere. The ZIC4 gene (8 exons) spans ~21 kb of genomic DNA and is located on chromosome 3q24, ~147 Mb from the p-telomere. The VLDLR gene (19 exons) spans ~33 kb of genomic DNA and is located on chromosome 9p24.2, ~2.6 Mb from the p-telomere. The P267-A3 probemix contains five probes for the ZIC1 gene, six probes for the ZIC4 gene, and 18 probes for the VLDLR gene. This probemix furthermore contains two probes for the SMARCA2 gene, which is also located in the 9p24 region, downstream of VLDLR. In addition, eight 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 genes 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 these genes are expected to be small (point) mutations, most of 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 6, 1057 DN Amsterdam, the Netherlands SALSA probemix P267 DWM Page 1 of 6
Data analysis The P267-A3 DWM probemix contains 39 MLPA probes with amplification products between 148 and 454 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 (Dfragments) 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 intra-sample normalised 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 this intra-normalised probe ratio in a sample by the average intra-normalised probe ratio of all reference samples. Please note that this type of normalization 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 by R.J.L. Schuit at. In case the results obtained with this probemix lead to a scientific publication, it would be very much appreciated if the probemix designer could be made a co-author. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix P267 DWM Page 2 of 6
Table 1. SALSA MLPA P267-A3 DWM probemix Length (nt) SALSA MLPA probe Chromosomal position reference ZIC1 ZIC4 VLDLR 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 148 Reference probe 03564-L02930 3p22 154 SMARCA2 probe 06655-L06228 9p24 160 ZIC4 probe 08548-L10326 Exon 4 (7) 166 VLDLR probe 08532-L08533 Exon 12 172 VLDLR probe 08523-L08524 Exon 2 178 ZIC4 probe 08550-L10327 Exon 5 (8) 184 VLDLR probe 08528-L08529 Exon 7 190 VLDLR probe 08537-L08538 Exon 17 197 Reference probe 03547-L26133 11p15 204 ZIC4 probe 08546-L26134 Exon 2 (4) 210 VLDLR probe 08533-L08534 Exon 13 220 ZIC1 probe 08542-L08543 Exon 2 227 SMARCA2 probe 06654-L10330 9p24 232 VLDLR probe 08527-L10328 Exon 6 238 ZIC4 probe 08549-L08550 Exon 5 (8) 247 Reference probe 07037-L06648 17p13 256 VLDLR probe 08538-L08539 Exon 18 265 VLDLR probe 08529-L10329 Exon 8 274 ZIC4 probe 08545-L08546 Exon 1 283 VLDLR probe 08534-L08535 Exon 14 294 * Reference probe 11900-L23408 6p12 301 ZIC1 probe 08540-L08541 Exon 1 310 VLDLR probe 08522-L08523 Intron 1 319 VLDLR probe 08531-L08532 Exon 11 328 ZIC1 probe 08543-L08544 Exon 3 337 VLDLR probe 08524-L08525 Exon 3 346 Reference probe 08684-L08696 13q32 355 VLDLR probe 08536-L08537 Exon 16 364 ZIC1 probe 08541-L08542 Exon 1 373 VLDLR probe 08526-L08527 Exon 5 382 VLDLR probe 08539-L08540 Exon 19 391 ZIC1 probe 08544-L08545 Exon 3 400 Reference probe 04737-L04154 7q21 409 VLDLR probe 08530-L26044 Exon 10 418 ZIC4 probe 08547-L08548 Exon 3 (5) 427 VLDLR probe 08525-L08526 Exon 4 436 VLDLR probe 08535-L08536 Exon 15 445 * Reference probe 15086-L16849 12q14 454 * Reference probe 12526-L23849 4q25 * New in version A3 (from lot A3-0813 onwards). Small change in length in version A3 (from lot A3-0813 onwards). No change in sequence detected. SNP at -2 nt from ligation site could influence the probe signal. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. Note: The exon numbering of the ZIC4 gene has changed. From description version 07 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for this gene. This exon numbering used here may differ from literature! The exon numbering used in previous versions of this product description can be found between brackets in Table 1 and 2. The identity of the genes detected by the reference probes is available on request: info@mlpa.com. SALSA probemix P267 DWM Page 3 of 6
Table 2. P267 probes arranged according to chromosomal location Table 2a. 3q24 Length (nt) SALSA MLPA probe Gene / exon Ligation site Partial sequence (24 nt adjacent to ligation site) Distance to next probe ZIC4 NM_032153.5 stop codon 1465-1467 (ex 5) 178 08550-L10327 ZIC4 exon 5 (8) 3572-3573 AAGGTTTGGTCT-ACAACACAGTGA 1.4 kb 238 08549-L08550 ZIC4 exon 5 (8) 2175-2176 ATTTAATACTGT-CACTGCGCTCTC 3.0 kb 160 08548-L10326 ZIC4 exon 4 (7) 1215-1214 reverse GAATGCTTCTTA-CGGTCGCTGCTG 5.0 kb 418 08547-L08548 ZIC4 exon 3 (5) 837-836 reverse ATGAGCTCCTGT-TTGATGGGCTGG 6.6 kb 204 08546-L26134 ZIC4 exon 2 (4) 493-494 TGGTGATGAGGA-AACGATTACGGC 3.7 kb 274 08545-L08546 ZIC4 exon 1 297-298 ATACATTCCCTA-ACTGCTTCCTGA 3.2 kb start codon 463-465 (ex 2) ZIC1 NM_003412.3 start codon 720-722 (ex 1) 301 08540-L08541 ZIC1 exon 1 320-319 reverse TAGCATAGAGGA-ATGTGAGCGCCA 1.2 kb 364 08541-L08542 ZIC1 exon 1 1475-1476 ATGCACGAGCTA-GTTACGCACGTC 1.7 kb 220 08542-L08543 ZIC1 exon 2 1808-1807 reverse CACATCTTGCAA-AGATAGGGCTTG 0.8 kb 328 08543-L08544 ZIC1 exon 3 1984-1983 reverse AGGAGGGCGATA-AGGAGCTTGTGG 2.2 kb 391 08544-L08545 ZIC1 exon 3 4175-4176 ATGCACTCTATG-TGTTCAGGAAGC stop codon 2061-2063 (ex 3) The NM_032153.5 sequence represents transcript variant 3 and is a reference standard in the NCBI RefSeqGene project. The NM_003412.3 sequence is a reference standard in the NCBI RefSeqGene project. Note The exon numbering of the ZIC4 gene has changed. From description version 07 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for this gene. This exon numbering used here may differ from literature! The exon numbering used in previous versions of this product description can be found between brackets in Table 1 and 2. SALSA probemix P267 DWM Page 4 of 6
Table 2b. 9p24 Length (nt) SALSA MLPA probe Gene / exon Ligation site Partial sequence (24 nt adjacent to ligation site) Distance to next probe SMARCA2 NM_003070.3 start codon 100-102 (ex 2) 227 06654-L10330 SMARCA2 exon 3 393-394 AAGGGCACTGGT- ATGCGACCACCT 71.0 kb 154 06655-L06228 SMARCA2 exon 23 3267-3268 GAGCTGCTTGAT- CGTATTCTGCCA 519.8 kb stop codon 4870-4872 (ex 34) VLDLR NM_003383.3 start codon 398-400 (ex 1) 310 08522-L08523 VLDLR intron 1 1.5 kb after exon 1 TGAAGAGGACGA-ATGTGTAGTGAA 11.7 kb 172 08523-L08524 VLDLR exon 2 522-521 reverse TACAGCGACCAT-TTGTGCACTGGA 4.4 kb 337 08524-L08525 VLDLR exon 3 666-667 CAGCCGATGGAA-GTGTGATGGAGA 1.5 kb 427 08525-L08526 VLDLR exon 4 817-818 GAAAATGATTGT-GACAGTGGAGAA 2.0 kb 373 08526-L08527 VLDLR exon 5 1191-1190 reverse CACTGCCATCCT-TGCAGTCAGGGT 0.2 kb 184 08528-L08529 VLDLR exon 6 1248-1249 TGACCAATTTGA-ATGTGAGGATGG 0.2 kb 232 08527-L10328 VLDLR exon 7 1395-1396 AGAATGCATAGA-TATCAGCAAAGT 0.9 kb 265 08529-L10329 VLDLR exon 8 1557-1558 AGCTGGGTTTGA-ACTGATAGATAG 0.8 kb No probe VLDLR exon 9 409 08530-L26044 VLDLR exon 10 1747-1748 AATCGAAGAGAC-ATCAGGAAGATT 0.8 kb 319 08531-L08532 VLDLR exon 11 1976-1977 TGTACAAGACCA-TCTACTGGACTG 1.2 kb 166 08532-L08533 VLDLR exon 12 39 nt after exon 12 reverse ACACTATGAAGA-TAGTTGAGTGGG 0.7 kb 210 08533-L08534 VLDLR exon 13 2283-2284 CAGCGTGGACTT-GAATGGCCAAGA 0.6 kb 283 08534-L08535 VLDLR exon 14 25 nt after exon 14 GCACAGTCCTTA-ATAACTACTTTA 1.5 kb 436 08535-L08536 VLDLR exon 15 2532-2531 reverse CACATCCTCCAT-TCTCCATGTCTT 1.1 kb 355 08536-L08537 VLDLR exon 16 2724-2725 TAGTGGACTAGT-TCCTGGAGGTAT 0.4 kb 190 08537-L08538 VLDLR exon 17 2765-2766 TATCAGAGGTCA-GTGTTCCCCCAA 0.9 kb 256 08538-L08539 VLDLR exon 18 2885-2886 ACATGAAAAGCA-TGAACTTTGACA 1.3 kb 382 08539-L08540 VLDLR exon 19 3308-3309 CTAGAAAGCCAT-ATTCCAGCAGTG stop codon 3017-3019 (ex 19) SNP at -2 nt from ligation site could influence the probe signal. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. The NM_003070.3 sequence represents transcript variant 1. The NM_003383.3 sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com SALSA probemix P267 DWM Page 5 of 6
SALSA MLPA probemix P267-A3 DWM sample picture 70000 60000 50000 40000 96.46 86.18 91.18 105.58 146.74 152.50 159.05 171.63 165.15 182.43 177.32 188.28 227.00 232.40 220.50 203.91 238.09 209.67 247.35 255.78 264.38 274.10 282.34 293.90 300.85 310.06 317.44 327.79 335.40 344.84 354.39 371.95 382.88 400.80 390.60 408.63 417.76 426.95 453.97 445.48 435.91 30000 196.94 364.43 100.72 20000 D ye S ign al 10000 0 50 100 150 200 250 300 350 400 450 500 Size (nt) Figure. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P267-A3 DWM (lot A3-0813). Implemented Changes compared to the previous product description versions Version 07 (53) - Description adjusted to new version of the probemix. - ZIC4 exon numbering changed. Version 06 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 05 (47) - Remark on RefSeqGene standard and transcript variant added below Table 2. - Various minor lay-out changes. Version 04 (46) - Exon numbering of the ZIC4 gene has been changed on page 3 and 4. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Data analysis method has been modified. - Various minor textual changes on page 1. - Various minor layout changes. SALSA probemix P267 DWM Page 6 of 6