MRC-Holland MLPA. Description version 12; 27 November 2015

Transcription

1 SALSA MLPA probemix P079-A3 OTC Lot A As compared to previous version (lot A2-0211), 5 reference probes have been replaced. Also, 8 reference probes and the TSPAN7 flanking probe have been removed. Ornithine transcarbamylase deficiency (OTCD) is characterized by a frequent inborn error of the urea cycle, a partially dominant X-linked trait, causing hyperammonemia and and orotic aciduria. Missense, nonsense, and frameshift mutations in the gene ornithine cabamoyltransferase (OTC) lead to OTCD. The protein encoded by this gene is a nuclear-encoded mitochondrial matrix enzyme that catalyses the second step of the urea cycle in mammals. Severe deficiency or total absence of OTC results in the accumulation of ammonia and other precursor metabolites during the first few days of life. The OTC gene (10 exons) spans ~69 kb of genomic DNA and is located on Xp11.4, ~38 Mb from the p- telomere. The P079-A3 probemix contains one probe for each exon of the gene. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned gene(s) in a DNA sample. Deletions of a probe s recognition sequence on the X-chromosome will lead to a complete absence of the corresponding probe amplification product in males, whereas female heterozygotes are recognisable by a 35-50% reduction in relative peak height. 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 References for SALSA probemix P079 OTC Brassier et al., Long-term outcomes in Ornithine Transcarbamylase deficiency: a series of 90 patients. Orphanet J Rare Dis. 10:58. Choi et al., Clinical outcomes and the mutation spectrum of the OTC gene in patients with ornithine transcarbamylase deficiency. J Hum Genet. 60: Lee et al., OTC Gene in Ornithine Transcarbamylase Deficiency: Clinical Course and Mutational Spectrum in Seven Korean Patients. Pediatr Neurol. 51: Storkanova et al., Ornithine carbamoyltransferase deficiency: molecular characterization of 29 families. Clin Genet. 84: SALSA P079 OTC probemix Page 1 of 5

2 Data analysis The P079-A3 OTC probemix contains 20 MLPA probes with amplification products between 147 nt and 382 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 be normalised intra-sample by dividing the peak height of each amplification product by the total peak height of only the reference probes in the 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 that no changes occurred in the genomic regions recognised by the reference probes. It is recommended to use reference and patient samples of the same sex to minimize variation, but this is not strictly necessary. Sex determination can also be done by visual examination of the electropherogram. Data normalization 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 at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P079 OTC probemix Page 2 of 5

3 Table 1. SALSA MLPA P079-A3 OTC probemix Length (nt) SALSA MLPA probe Chromosomal position Reference OTC 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 147 * Reference probe L07374 Xp OTC probe L02099 Exon * Reference probe L06570 Xp OTC probe L02101 Exon OTC probe L02103 Exon Reference probe L01009 Xp OTC probe L02105 Exon * Reference probe L09375 Xp Reference probe L00833 Xq OTC probe L02107 Exon OTC probe L02100 Exon * Reference probe L07367 Xp OTC probe L02102 Exon OTC probe L02104 Exon * Reference probe L13868 Xq OTC probe L02106 Exon Reference probe L00338 Xq OTC probe L02108 Exon Reference probe L00965 Xq Reference probe L01456 Xq28 * New from version A3 (from lot A3-1015) onwards. Note: Exon numbering used here may differ from literature! Please notify us of any mistakes. The identity of the genes detected by the reference probes is available on request: info@mlpa.com. Table 2. OTC probes arranged according to chromosomal location Length SALSA Ligation site Partial sequence (24 nt Distance to OTC (nt) MLPA probe NM_ adjacent to ligation site) next probe start codon (ex 1) L02099 Exon TGAACACATTTC-TTAGTTTTTAGG 14.7 kb L02100 Exon reverse TTTAGAGTGAGA-AGGTCACGGCCC 2.5 kb L02101 Exon AGAAAAGAAGTA-CTCGAACAAGAT 11.6 kb L02102 Exon TGTGAATGAAAG-TCTCACGGACAC 20.0 kb L02103 Exon CTGTCAGATTTG-TACCATCCTATC 2.3 kb L02104 Exon reverse ATTCCGAATTTC-GCTGCGCTCATC 5.1 kb L02105 Exon AGTGTAACCAAG-TTGGCAGAGCAG 0.1 kb L02106 Exon GCTGACAAATGA-TCCATTGGAAGC 3.0 kb L02107 Exon TGCCTCTGACTG-GACATTTTTACA 9.2 kb L02108 Exon reverse GCTTCTGGAGCT-GAGGTGAGTAAT stop codon (ex 10) 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 P079 OTC probemix Page 3 of 5

4 SALSA MLPA probemix P079-A3 OTC sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA probemix P079-A3 OTC (lot A3-1015). Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng human female control DNA analysed with SALSA probemix P079-A3 OTC (lot A3-1015). SALSA P079 OTC probemix Page 4 of 5

5 Implemented Changes compared to the previous product description versions. Version November 2015 (55) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - References updated. - Manufacturer s address adjusted. Version August 2015 (54) - Various minor textual changes. - Figure(s) based on the use of old MLPA buffer (replaced in December 2012) removed. - Peak area replaced with peak height. Version 10 (48) - Warning added in Table 1, 222 nt probe L Version 09 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 08 (48) - Remark added below Table 1 for TSPAN7 probe. - Remark on RefSeqGene standard added below Table 2. - Various minor textual changes. - Various minor layout changes. Version 07 (46) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Various minor textual changes throughout the document. - Various minor layout changes. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Warning added in Table 1, 174 nt probe L00075 and 293 nt probe L Female sample picture added. Version 06 (43) - Minor changes in the product description on page 1. - Minor changes in the data analysis section on page 2. - Minor changes on table titles. SALSA P079 OTC probemix Page 5 of 5