MRC-Holland MLPA. Description version 10; 03 August 2015

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1 SALSA MLPA probemix P278-C1 PCCA Lot C As compared to lot B2-0409, five reference probes have been replaced and two reference probes have been removed. Also, QDX2 control fragments have been added Propionyl-CoA carboxylase (PCC) is comprised of several alpha and beta subunits. The alpha subunits are encoded by the PCCA gene and the beta subunits by the PCCB gene. PCC catalyses the breakdown of certain amino acids as well as lipids and cholesterol. The breakdown product is propionyl-coa, which is further converted into other molecules used for energy. Build-up of propionyl-coa in the body can be toxic and is associated with propionic acidemia. This occurs when there is no or less active PCC available due to mutations in the PCCA or the PCCB gene. Cells from patients with propionic acidemia with mutations in the PCCA gene fall into complementation group pcca. The features of propionic acidemia are episodic vomiting, lethargy and ketosis, neutropenia, periodic thrombocytopenia, hypogammaglobulinemia, developmental retardation, and intolerance to protein. Outstanding chemical features are hyperglycinemia and hyperglycinuria. The PCCA gene (24 exons) spans ~441.4 kb of genomic DNA and is located on chromosome 13q32.3, 99.5 Mb from p-telomere. The P278-C1 probemix contains probes for each of the 24 exons of PCCA (two probes for exons 1, 4, 7). In addition, 10 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 gene in a DNA sample. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak area 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 area, 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 this gene 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 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). References of SALSA probemix P278 PCCA Desviat, L. R. et al., High frequency of large genomic deletions in the PCCA gene causing propionic acidemia. Mol Genet Metab. 96: 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 P278 PCCA Page 1 of 5

2 Data analysis The P278-C1 PCCA probemix contains 37 MLPA probes with amplification products between 142 and 436 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 area of each probe s amplification product by the total area 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 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 made a co-author. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix P278 PCCA Page 2 of 5

3 Table 1. SALSA MLPA P278-C1 PCCA probemix Length (nt) SALSA MLPA probe Chromosomal position reference PCCA 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 142 Reference probe L p PCCA probe L12786 Exon PCCA probe L08698 Exon PCCA probe L08703 Exon PCCA probe L08687 Exon PCCA probe L08710 Exon PCCA probe L08704 Exon Reference probe L q PCCA probe L08702 Exon PCCA probe L08705 Exon Reference probe L p PCCA probe L10283 Exon PCCA probe L09778 Exon PCCA probe L23671 Exon Reference probe L q PCCA probe L08689 Exon PCCA probe L08707 Exon PCCA probe L08712 Exon PCCA probe L08709 Exon PCCA probe L08692 Exon PCCA probe L08700 Exon * Reference probe L q PCCA probe L08688 Exon * Reference probe L q PCCA probe L08706 Exon Reference probe L p PCCA probe L08696 Exon PCCA probe L08693 Exon ± PCCA probe L08697 Exon * Reference probe L q PCCA probe L09777 Exon PCCA probe L08701 Exon * Reference probe L q PCCA probe L08711 Exon PCCA probe L08694 Exon PCCA probe L08695 Exon * Reference probe L p12 More variable. This probe has a high standard deviation. Changed in version C1 (from lot C onwards). Small change in length, no change in sequence detected. * New in version C1 (from lot C onwards). ± SNP (rs ) at -4 nt from ligation site could influence the 364 nt (exon 8) probe signal. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. Note: Exon numbering might be different as compared to literature! Please notify us of any mistakes. The identity of the genes detected by the reference probes is available on request: info@mlpa.com. SALSA probemix P278 PCCA Page 3 of 5

4 Table 2. PCCA probes arranged according to chromosomal location Length (nt) SALSA MLPA probe PCCA exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex1) L08687 Exon GCGGGGACAACA-ATGGCGGGGTTC 0.1 kb L08688 Exon GCGGACCCTGAA-GGTGAGGAGCAA 13.7 kb L08689 Exon AAGACAGTGCTT-AATGGTGTCCCG 9.0 kb L09777 Exon TCTTGTTGCTAA-TAGAGGAGAAAT 0.2 kb L09778 Exon GAAGATGGGCAT-TAAGACAGTTGC 0.1 kb L08692 Exon 4 54 nt after exon 4 GGTCAAGCAGAA-AAAGTGAGACAT 42.9 kb L08693 Exon TCCCACCAGTAA-AAGCTACCTCAA 2.3 kb L08694 Exon TTCAGAAAACAA-AGAATTTGCCAG 52.0 kb L08695 Exon 7 22 nt before exon 7 TTGTTATAAATT-TTGACTTGTTTT 0.1 kb L08696 Exon GGGCGACAAGAT-TGAAAGCAAATT 26.4 kb 364 ± L08697 Exon 8 7 nt before exon 8 CATTTCCTGCTT-TTACAGGATGCA 21.8 kb L08698 Exon AGGCATGCGCAT-TGCTTGGGATGA 5.3 kb L12786 Exon nt after exon 10 GAGTTGTGCCTT-TAGAATCTGTTG 5.8 kb L08700 Exon CTTAATGAAAGA-GAGTGCTCAATT 4.5 kb L08701 Exon AGAACAAGCTGT-AGCTCTTGCCAG 28.2 kb L08702 Exon ACAGAATGCATT-ACTGGCCTGGAC 1.5 kb L08703 Exon CCAAGAACCGTT-ACATCTACCTGG 4.2 kb L08704 Exon GGACAGTGGCAT-CCAACCAGGAAG 2.7 kb L08705 Exon GCACTGAAGAGA-ATGGCAGATGCA 20.8 kb L08706 Exon AAGGAGACATCA-GCACTAAATTTC 9.6 kb L08707 Exon TGTTTGTGGCAT-TCCAGTTAAGAG 28.3 kb L09781 Exon GCTCTCAGTAAA-ATTGCATGATAA 57.1 kb L08709 Exon GGTCGAAACTAA-ATGTGACCAGCA 23.6 kb L08710 Exon AAACATGAGCAT-TCAGTTTCTTGG 66.2 kb L08711 Exon ATTGAACAAATT-TATGCTGGAAAA 12.2 kb L08712 Exon ATTTGTGTGATT-GAAGCCATGAAA 2.4 kb L10283 Exon nt before exon 24 CTGGTTACTAAT-TCTTACTCTCCC stop codon (ex24) ± SNP (rs ) at -4 nt from ligation site could influence the 364 nt (exon 8) 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 and is a reference standard in the NCBI RefSeqGene project. Note: Exon numbering might be different as compared to literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA probemix P278 PCCA Page 4 of 5

5 SALSA MLPA probemix P278-C1 PCCA sample picture Dye Sign al Size (nt) Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P278-C1 PCCA (lot C1-0712) Implemented Changes compared to the previous product description versions Version 10 (49)- 03 August Electropherogram picture(s) using the old MLPA buffer (replaced in December 2012) removed. Version 09 (49) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Various textual changes on page 1. Version 08 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 07 (48) - Remark on RefSeqGene standard and transcript variant added below Table 2. - Various minor textual changes. - Various minor layout changes. Version 06 (46) - A reference deleted on page 1 from disease description because it is not easily accessible. - Warning added in Table 1 and 2, 364 nt probe L Data analysis method has been modified. - Ligation sites of the probes targeting the PCCA gene updated according to new version of the NM_reference sequence. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Partial probe sequences extended to 24 nt. - Various minor textual changes on page 1. - Various minor layout changes. SALSA probemix P278 PCCA Page 5 of 5