SALSA MLPA probemix P338-B1 GBA Lot B

Transcription

1 SALSA MLPA probemix P338-B1 GBA Lot B This SALSA probemix is for basic research and intended for experienced MLPA users only! This probemix enables you to quantify genes or chromosomal regions in which the detection of copy number changes is complex. Interpretation of results can be complicated. cannot provide assistance with interpretation of results obtained with this product, and recommends thoroughly screening any available literature. Suggestions from specialists for improvement of this product or product description are highly appreciated. Gaucher disease is an autosomal recessive lysosomal storage disorder characterised by a deficient activity/accumulation of beta-glucocerebrosidase. As a result of this deficiency, there is intracellular accumulation of glucosylceramide (GlcCer, glucosylcerebroside) primarily within cells of mononuclear phagocyte origin, which are the characteristic 'Gaucher cells' identified in most tissues. Defects in the GBA gene on chromosome 1q22 is the main cause of Gaucher Disease. The GBA gene encodes a lysosomal membrane protein that cleaves the beta-glucosidic linkage of glucosylceramide, an intermediate in glycolipid metabolism. The GBA gene (11 exons) spans ~10 kb of genomic DNA and is located on 1q22, ~155 Mb from the p- telomere. The P338-B1 probemix contains one probe for each exon of the gene with the exception of exons 2, 5 and 11. The exon 9 probe will generate a normal signal at the wild type of the 55-bp deletion; the signal will be reduced when the deletion is present. The exon 10 probe will generate a normal signal at the wild type of the L444P mutation (also known as L483P), the signal will be reduced when the mutation is present. In addition, 12 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 GBA gene 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). 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 P338 GBA probemix Page 1 of 5

2 References Amico, G et al. (2016). MLPA-based approach for initial and simultaneous detection of GBA deletions and recombinant alleles in patients affected by Gaucher Disease. Mol Genet Metab, Basgalupp, S et al. (2016). Use of a multiplex ligation-dependent probe amplification method for the detection of deletions/duplications in the GBA1 gene in Gaucher disease patients. Blood Cells Mol Dis, Ortiz-Cabrera, N et al (2016). Nine-year experience in Gaucher disease diagnosis at the Spanish reference center Fundación Jiménez Díaz. Mol Genet Metab Rep 9:79 85 Data analysis The P338-B1 GBA probemix contains 24 MLPA probes with amplification products between 155 nt and 382 nt. This includes the 166 nt probe for exon 9 that will generate a lower signal when the 55-bp deletion (rs ) is present. The 355 nt probe for exon 10 will generate a lower signal when the L444P mutation is present. 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 (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 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 co-author. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P338 GBA probemix Page 2 of 5

3 Table 1. SALSA MLPA P338-B1 GBA probemix Length Chromosomal position SALSA MLPA probe (nt) reference other GBA 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 155 Reference probe L p GBA probe L13669 Exon Reference probe L q Reference probe L q GBA probe L13673 Exon Reference probe L q Reference probe L q GBA probe L23141 Exon GBA probe L13665 Upstream 230 CKS1B probe L q Reference probe L p GBA probe L13663 Exon Reference probe L q GBA probe L13670 Exon GBA probe L13672 Exon PKLR probe L q Reference probe L q Reference probe L p GBA probe L13668 Exon CLK2 probe L q Reference probe L p GBA probe L13666 Exon Reference probe L p Reference probe L q14 This probe detects the wild type sequence. The presence of the 55-bp deletion (rs ) will result in a decreased signal of the 166 nt probe. The presence of the L444P (also known as L483P) mutation will result in a decreased probe signal of the 355 nt probe. Flanking probe. Included to facilitate determination of the extend of a deletion / duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition being tested. 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 P338 GBA probemix Page 3 of 5

4 Table 2. GBA probes arranged according to chromosomal location Length (nt) SALSA MLPA probe GBA exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe L06074 PKLR ACAGTCTCCCAT-TCTCATATGTAG 16.1 kb L14640 CLK2 ATGTGTGAGAAA-TACAAGTTTACT 29.5 kb start codon (exon 1) L13665 upstream 3.3 kb before exon 1 ATCCTGCCTTCA-GAGTCTTACTGC 3.3 kb 283 # L13670 exon 1 9 nt before exon 1 TGTCATGTGACG-CTCCTAGTCATC 1.3 kb No probe exon L13673 exon AGCTCGGTGGTG-TGTGTCTGCAAT 0.3 kb 213 # L23141 exon TTGGAGGGGCCA-TGACAGATGCTG 1.6 kb No probe exon # L13668 exon GGGCCAGATACT-TTGTGAAGTAAG 0.5 kb L13663 exon 7 42 nt before exon 7 reverse CAAGAAAGTGGA-CCAGACCAGCTG 1.2 kb 292 # L13672 exon GAAGCAGCTAAA-TATGTTCATGGC 0.7 kb L13669 exon GGAGGACCCAAT-TGGGTGCGTAAC 0.5 kb 355 # L13666 exon reverse CCACTGCGTCCA-GGTCGTTCTTCT kb No probe exon 11 stop codon (exon 11) L22931 CKS1B TTCTGTTACAGA-CATGTCATGCTG This probe detects the wild type sequence. The presence of the 55-bp deletion (rs ) will result in a decreased signal of the 166 nt probe. The presence of the L444P (also known as L483P) mutation will result in a decreased probe signal of the 355 nt probe. Flanking probe. Included to facilitate determination of the extend of a deletion / duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition being tested. # This probe s specificity relies on a single nucleotide difference compared to a related gene or pseudogene. As a result, an apparent duplication of only this probe can be the result of a non-significant single nucleotide sequence change in the related gene or pseudogene. 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 P338 GBA probemix Page 4 of 5

5 SALSA MLPA probemix P338-B1 GBA sample picture Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P338-B1 GBA (lot B1-0716). Implemented Changes compared to the previous product description versions. Version 08 (55) 26 April Warning added to Table 2 for probe specificity relying on a single nucleotide difference between target gene and related gene or pseudogene. - A reference was added. Version 07 (55) 14 November References added. Version 06 (55) - 18 August Not applicable, new document. SALSA P338 GBA probemix Page 5 of 5