MRC-Holland MLPA. Description version 14; 28 September 2016

Transcription

1 SALSA MLPA probemix P279-B3 CACNA1A Lot B As compared to version B2 (lot B2-1012), one reference probe has been replaced and the length of several probes has been adjusted. Voltage-dependent calcium channels mediate the entry of calcium ions into excitable cells, and are also involved in a variety of calcium-dependent processes, including muscle contraction, hormone or neurotransmitter release and gene expression. Calcium channels are multi-subunit complexes composed of alpha-1, beta, alpha-2/delta, and gamma subunits. The channel activity is directed by the pore-forming alpha-1 subunit whereas the others act as auxiliary subunits regulating this activity. The distinctive properties of the calcium channel types are related primarily to the expression of a variety of alpha-1 isoforms, alpha-1a, B, C, D, E, and S. The CACNA1A gene encodes the alpha-1a subunit, which is predominantly expressed in neuronal tissue. Mutations in this gene are associated with two neurological disorders, familial hemiplegic migraine and episodic ataxia 2. Episodic ataxia 1 is caused by mutations in the KCNA1 gene. The CACNA1A gene (47 exons) spans ~300 kb of genomic DNA and is located on 19p13.2, 13.3 Mb from the p-telomere. The P279-B3 CACNA1A probemix contains probes for 30 of the 47 exons of the CACNA1A gene (two probes for exon 1). Furthermore one flanking probe located 2 Mb downstream of CACNA1A has been included. KCNA1 (2 exons) spans ~8.4 kb of genomic DNA and is located on 12p13.32, 4.9 Mb from the p- telomere. Probes for both exons are included. In addition, the P279 probemix contains 11 reference probes detecting several different autosomal chromosomal locations. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned gene(s) 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 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 Acid Research 30, e57 (2002). Related SALSA probemixes P348 ATP1A2-CACNA1A: Contains 17 more probes for the CACNA1A gene, as well as probes for most ATP1A2 exons. All CACNA1A probes in P348 are different from the probes included in P279. 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 P279 CACNA1A probemix Page 1 of 6

2 References Vila-Pueyo M. et al., A loss-of-function CACNA1A mutation causing benign paroxysmal torticollis of infancy. Europ J Paediatr Neurol. 18: Carreño O. et al., Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine: clinical, genetic, and functional studies. Mol Genet Genomic Med. 1: Kipfer S. et al., Novel CACNA1A mutation(s) associated with slow saccade velocities. J Neurol. 260: Wan J. et al., Large genomic deletions in CACNA1A cause episodic ataxia type 2. Front Neurol. 2:51. Labrum R.W. et al., Large-scale calcium gene rearrangements in episodic ataxia and hemiplegic migraine: implications for diagnostic testing. J Med Genet. 46: Data analysis The P279-B3 CACNA1A probemix contains 45 MLPA probes with amplification products between 130 and 494 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 normalised intra-sample 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 the intra-normalised probe ratio in a sample by the average intra-normalised probe ratio of all reference samples. Please note that this type of normalisation 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. Vijzelaar 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 included as a co-author. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P279 CACNA1A probemix Page 2 of 6

3 Table 1. SALSA MLPA P279-B3 CACNA1A probemix Length Chromosomal position SALSA MLPA probe (nt) reference KCNA1 CACNA1A 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 Reference probe L q CACNA1A probe L09236 Exon CACNA1A probe L29524 Exon «CACNA1A probe L29525 Exon * Reference probe L q CACNA1A probe L09243 Exon CACNA1A probe L09248 Exon CACNA1A probe L14993 Exon CACNA1A probe L09231 Exon CACNA1A probe L09245 Exon Reference probe L q CACNA1A probe L14994 Exon KCNA1 probe L12371 Exon CACNA1A probe L23736 Exon «CACNA1A probe L09249 Exon CACNA1A probe L09232 Exon CACNA1A probe L23737 Exon «CACNA1A probe L09250 Exon CACNA1A probe L16631 Exon «CACNA1A probe L14995 Exon CACNA1A probe L09244 Exon Reference probe L q CACNA1A probe L09228 Exon CACNA1A probe L29526 Exon CACNA1A probe L09234 Exon Reference probe L q CACNA1A probe L29527 Exon CACNA1A probe L09242 Exon CACNA1A probe L09247 Exon KCNA1 probe L12374 Exon Reference probe L q Reference probe L p CACNA1A probe L09237 Exon CACNA1A probe L09225 Exon LDLR probe L02443 Downstream 409 CACNA1A probe L14997 Exon CACNA1A probe L09239 Exon Reference probe L p CACNA1A probe L09246 Exon «CACNA1A probe L09253 Exon Reference probe L p CACNA1A probe L14998 Exon CACNA1A probe L14999 Exon Reference probe L p Reference probe L p15 * New in version B3 (from lot B onwards). Changed in version B3 (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. 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. SALSA P279 CACNA1A probemix Page 3 of 6

4 Notes The CACNA1A exon numbering has changed. From description version 14 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for the CACNA1A 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 2. The identity of the genes detected by the reference probes is available on request: Table 2. P279 probes arranged according to chromosomal location Table 2a. CACNA1A gene Length (nt) SALSA MLPA probe CACNA1A exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (exon 1) L29526 Exon GATGTACAAGCA-GTCAATGGCGCA 0.1 kb L09225 Exon GTGGTGAGAAAA-TACGCCAAAAAG 50.9 kb L14994 Exon 2 13 nt after exon 2 TGAGTGATGTCT-TTTCTCAGGGTC 2.1 kb L14997 Exon TACTTCATTGGA-ATTTTTTGTTTC 81.2 kb L09228 Exon GGAGTTTGACCT-ACGGACGCTGAG 6.3 kb L14993 Exon GTCCTGAAGTCG-ATCATGAAGGCG 5.8 kb L29527 Exon GTGCTGACTGTT-TTCCAGTGCATA 23.7 kb L09231 Exon ACACCCTACAGA-GCAACGATGCCT 1.5 kb L09232 Exon GCAACAACAGAT-TGAACGTGAGCT 4.1 kb L16631 Exon CACCATAAAGAA-AAGCAAGACAGA 13.1 kb L09234 Exon CTCAGGCCTTCT-ACTGGACTGTAC 8.7 kb L23736 Exon GGGCTGTCATAA-AACCTGGCACAT 4.6 kb L09236 Exon GAACGAGGTCAT-GTACGACGGGAT 3.2 kb L09237 Exon TTGCCCTACAGA-AAGCCAAGGAGG 1.3 kb L29524 Exon GGACCAGTGAGA-TGCGAAAGCAGA 12.4 kb L09239 Exon ACAACATGAAGA-ACAACAAGCTGG 3.5 kb L14999 Exon GCCATTACATCC-TGAACCTGCGCT 7.5 kb L23737 Exon CCTCTGGAATAT-TCTCGACTTCAT 13.1 kb L09242 Exon GAAAAGACATCA-ACACGATTAAAT 3.2 kb L09243 Exon AGAAGTATGAAT-TCCATTACGACA 4.5 kb L09244 Exon CGCCTTTCGAGT-ACACGATCATGG 9.9 kb L09245 Exon TTCTAGAATTAT-TTCCGCGATGCC 9.6 kb L14998 Exon 32 (33) TGTGCAGTCCTT-CAAGGTGAGTCC 0.4 kb L09246 Exon 33 (34) GATCGCCATGCT-CTTCTTCATCTA 3.4 kb L09247 Exon 35 (36) TTGGCACAACAT-CATGCTTTCCTG 7.1 kb L09248 Exon 38 (40) GTCCACTTCAAT-TCCACCCTCATG 10.2 kb 226 «09080-L09249 Exon 39 (41) ATGATGGCGATT-TGGCCCAATCTG 0.3 kb 251 «09081-L09250 Exon 40 (42) CATGATGATCAT-GGAGTACTACCG 1.7 kb 265 «13535-L14995 Exon 42 (44) 25 nt before exon 42 GCGCCCACTGCT-ACCCCGGCCTCT 0.4 kb 148 «09082-L29525 Exon 43 (45) ATGGGCAGAGAT-GGCTACTCCGAC 4.1 kb 445 «09084-L09253 Exon 47 (49b) TGTAGGGCAGTA-GTTCCGTAAGTG kb stop codon (exon 47) L02443 LDLR gene TCAGTGCCAACC-GCCTCACAGGTT «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. 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. The NM_ sequence represents transcript variant 3 and is a reference standard in the NCBI RefSeqGene project. SALSA P279 CACNA1A probemix Page 4 of 6

5 Notes The CACNA1A exon numbering has changed. From description version 14 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for the CACNA1A gene. The exon numbering used in previous versions of this product description can be found between brackets in Table 2. Probes for most of the lacking exons of CACNA1A in the P279 probemix are present in the P348 probemix. Table 2b. KCNA1 gene Length (nt) SALSA MLPA probe KCNA1 exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (exon 2) L12371 Exon GAGAGTGCTGTT-TATCGTCATTTG 4.4 kb L12374 Exon AACTAAACCAAT-TGATTTAATAGT stop codon (exon 2) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Note: Exon numbering used here may differ from literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA MLPA probemix P279-B3 CACNA1A sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P279-B3 CACNA1A (lot B3-0816). SALSA P279 CACNA1A probemix Page 5 of 6

6 Implemented Changes compared to the previous product description versions. Version September 2016 (55) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new picture included). - Various minor textual changes on pages 1 and 2. - New references added. - Exon numbering of the CACNA1A gene has been changed the Table 1 and Table 2a. Version August 2015 (49) - Electropherogram picture(s) using the old MLPA buffer (replaced in December 2012) removed. Version 12 (49) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). Version 11 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 10 (48) - Various minor textual and layout changes. - Remark on RefSeqGene standard and transcript variant added below Table 2. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products Version 09 (46) - Various minor textual and layout changes. Version 08 (46) - Warning added in Table 2, 373 nt probe L Exon numbering of the CACNA1A gene has been changed on page 3 and 4. Version 07 (46) - Added a note about additional probes for CACNA1A gene in the P348 probemix on page 5. - Minor textual changes under Related SALSA MLPA probemixes on page 1. Version 06 (46) - Various minor textual changes on page 1. - Link to the database of genomic variants added to the product description. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. SALSA P279 CACNA1A probemix Page 6 of 6