MRC-Holland MLPA. Description version 07;

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1 SALSA MLPA probemix P267-A3 Dandy-Walker Malformation Lot A 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 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 : 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

2 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 nt, three DNA denaturation control fragments (Dfragments) 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 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 Many copy number alterations in healthy individuals are described in the database of genomic variants: 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

3 Table 1. SALSA MLPA P267-A3 DWM probemix Length (nt) SALSA MLPA probe Chromosomal position reference ZIC1 ZIC4 VLDLR 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 148 Reference probe L p SMARCA2 probe L p ZIC4 probe L10326 Exon 4 (7) 166 VLDLR probe L08533 Exon VLDLR probe L08524 Exon ZIC4 probe L10327 Exon 5 (8) 184 VLDLR probe L08529 Exon VLDLR probe L08538 Exon Reference probe L p ZIC4 probe L26134 Exon 2 (4) 210 VLDLR probe L08534 Exon ZIC1 probe L08543 Exon SMARCA2 probe L p VLDLR probe L10328 Exon ZIC4 probe L08550 Exon 5 (8) 247 Reference probe L p VLDLR probe L08539 Exon VLDLR probe L10329 Exon ZIC4 probe L08546 Exon VLDLR probe L08535 Exon * Reference probe L p ZIC1 probe L08541 Exon VLDLR probe L08523 Intron VLDLR probe L08532 Exon ZIC1 probe L08544 Exon VLDLR probe L08525 Exon Reference probe L q VLDLR probe L08537 Exon ZIC1 probe L08542 Exon VLDLR probe L08527 Exon VLDLR probe L08540 Exon ZIC1 probe L08545 Exon Reference probe L q VLDLR probe L26044 Exon ZIC4 probe L08548 Exon 3 (5) 427 VLDLR probe L08526 Exon VLDLR probe L08536 Exon * Reference probe L q * Reference probe L q25 * New in version A3 (from lot A onwards). Small change in length in version A3 (from lot A 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

4 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_ stop codon (ex 5) L10327 ZIC4 exon 5 (8) AAGGTTTGGTCT-ACAACACAGTGA 1.4 kb L08550 ZIC4 exon 5 (8) ATTTAATACTGT-CACTGCGCTCTC 3.0 kb L10326 ZIC4 exon 4 (7) reverse GAATGCTTCTTA-CGGTCGCTGCTG 5.0 kb L08548 ZIC4 exon 3 (5) reverse ATGAGCTCCTGT-TTGATGGGCTGG 6.6 kb L26134 ZIC4 exon 2 (4) TGGTGATGAGGA-AACGATTACGGC 3.7 kb L08546 ZIC4 exon ATACATTCCCTA-ACTGCTTCCTGA 3.2 kb start codon (ex 2) ZIC1 NM_ start codon (ex 1) L08541 ZIC1 exon reverse TAGCATAGAGGA-ATGTGAGCGCCA 1.2 kb L08542 ZIC1 exon ATGCACGAGCTA-GTTACGCACGTC 1.7 kb L08543 ZIC1 exon reverse CACATCTTGCAA-AGATAGGGCTTG 0.8 kb L08544 ZIC1 exon reverse AGGAGGGCGATA-AGGAGCTTGTGG 2.2 kb L08545 ZIC1 exon ATGCACTCTATG-TGTTCAGGAAGC stop codon (ex 3) The NM_ sequence represents transcript variant 3 and is a reference standard in the NCBI RefSeqGene project. The NM_ 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

5 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_ start codon (ex 2) L10330 SMARCA2 exon AAGGGCACTGGT- ATGCGACCACCT 71.0 kb L06228 SMARCA2 exon GAGCTGCTTGAT- CGTATTCTGCCA kb stop codon (ex 34) VLDLR NM_ start codon (ex 1) L08523 VLDLR intron kb after exon 1 TGAAGAGGACGA-ATGTGTAGTGAA 11.7 kb L08524 VLDLR exon reverse TACAGCGACCAT-TTGTGCACTGGA 4.4 kb L08525 VLDLR exon CAGCCGATGGAA-GTGTGATGGAGA 1.5 kb L08526 VLDLR exon GAAAATGATTGT-GACAGTGGAGAA 2.0 kb L08527 VLDLR exon reverse CACTGCCATCCT-TGCAGTCAGGGT 0.2 kb L08529 VLDLR exon TGACCAATTTGA-ATGTGAGGATGG 0.2 kb L10328 VLDLR exon AGAATGCATAGA-TATCAGCAAAGT 0.9 kb L10329 VLDLR exon AGCTGGGTTTGA-ACTGATAGATAG 0.8 kb No probe VLDLR exon L26044 VLDLR exon AATCGAAGAGAC-ATCAGGAAGATT 0.8 kb L08532 VLDLR exon TGTACAAGACCA-TCTACTGGACTG 1.2 kb L08533 VLDLR exon nt after exon 12 reverse ACACTATGAAGA-TAGTTGAGTGGG 0.7 kb L08534 VLDLR exon CAGCGTGGACTT-GAATGGCCAAGA 0.6 kb L08535 VLDLR exon nt after exon 14 GCACAGTCCTTA-ATAACTACTTTA 1.5 kb L08536 VLDLR exon reverse CACATCCTCCAT-TCTCCATGTCTT 1.1 kb L08537 VLDLR exon TAGTGGACTAGT-TCCTGGAGGTAT 0.4 kb L08538 VLDLR exon TATCAGAGGTCA-GTGTTCCCCCAA 0.9 kb L08539 VLDLR exon ACATGAAAAGCA-TGAACTTTGACA 1.3 kb L08540 VLDLR exon CTAGAAAGCCAT-ATTCCAGCAGTG stop codon (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_ sequence represents transcript variant 1. The NM_ 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

6 SALSA MLPA probemix P267-A3 DWM sample picture D ye S ign al 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