MRC-Holland MLPA. Description version 12; 16 March 2015

Transcription

1 P128-C1 Cytochrome P450 mix Lot C As compared to version B2 (lot B2-0811), nine reference s have been replaced and one CYP2D6 has been removed. This SALSA mix is for basic research and intended for experienced MLPA users only! This mix enables you to quantify genes or chromosomal regions in which the occurrence of copy number changes is not yet well-established and the relationship between genotype and phenotype is not yet clear. 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. Cytochrome P450 enzymes are important in the biosynthesis and degradation of endogenous compounds such as steroids, lipids and vitamins. These enzymes reduce or alter the pharmacologic activity of many drugs and facilitate their elimination. Differences in drug metabolism in the intestine or liver between patients are major contributors to drug response, including adverse effects. Glutathione transferases are a family of proteins that play an important role in detoxification by catalyzing the conjugation of many compounds with reduced glutathione. This P128-C1 Cytochrome P450 mix is intended for the detection of copy number changes of several Cytochrome P450 and Glutathione S-transferase genes. It contains s for the CYP2D6, CYP2C9, CYP2C19, CYP1B1, CYP3A4, CYP3A5, CYP2E1, CYP1A1, CYP1A2, CYP2A6, CYP2B6, GSTP1, GSTT1 and GSTM1 genes. In the normal population several of these genes differ in copy number. For each gene at least two s are present. In addition, 12 reference s are included in this P128-C1 mix, detecting several different autosomal chromosomal locations. An outline of the literature on these genes can be found in the OMIM database: This P128-C1 mix does not contain s for point mutations in CYP2D6 or the other genes! It will therefore not be possible to detect all poor metabolizers with this product. SALSA mixes are 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. Note that a mutation or polymorphism (e.g. SNP) in the sequence detected by a can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some 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 mixes 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 mixes and reagents includes a limited license to use these products for research purposes. The use of an SALSA mix 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 P128 Cytochrome P450 mix Page 1 of 9

2 References for SALSA mix P128 - Souzeau E et al (2015). CYP1B1 copy number variation is not a major contributor to primary congenital glaucoma. Mol Vis Feb 11;21: Martis S et al (2013). Multi-ethnic Cytochrome-P450 Copy Number Profiling: Novel Pharmacogenetic Alleles and Mechanism of Copy Number Variation Formation. Pharmacogenomics J Dec; 13(6). - Engman M et al (2013). GSTM1 Gene Expression Correlates to Leiomyoma Volume Regression in Response to Mifepristone Treatment. PLoS One Dec 4;8(12). - Kim J et al (2012). Copy number variation and gene rearrangements in CYP2D6 genotyping using multiplex ligation-dependent amplification in Koreans. Pharmacogenomics Jul;13(10): Data analysis The P128-C1 Cytochrome P450 mix contains 54 MLPA s with amplification products between 128 and 504 nt. In addition, it contains nine 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 mix can first be normalised intra-sample by dividing the peak height of each s amplification product by the total peak height of only the reference s in this mix (block normalisation). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalised ratio in a sample by the average intra-normalised ratio of all reference samples. Please note that this type of normalisation assumes no changes occurred in the genomic regions recognised by the reference s. 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 s are unlikely to be the cause of the condition tested for. Users should always verify the latest scientific literature when interpreting their findings. This mix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P128 Cytochrome P450 mix Page 2 of 9

3 Table 1. P128-C1 Cytochrome P450 mix Chromosomal position 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 128 Reference L q GSTT L q11 / CYP1B L p22 / CYP3A L q22 / CYP2E L q26 / CYP3A L q22 / * Reference L p Reference L q GSTP L q13 / CYP1B L p22 / CYP2A L q13 / CYP2A L q13 / CYP2D L q13 / CYP2B L q13 / CYP1A L q24 / * Reference L q CYP2C L q23 / CYP2C L q23 / CYP2D L q13 / CYP2E L q26 / CYP2A L q13 / CYP3A L q22 / CYP1A L q24 / CYP3A L q22 / * Reference L q CYP1A L q24 / * Reference L p * Reference L q CYP2C L q23 / * Reference L q CYP2C L q23 / CYP2B L q13 / GSTM L p13 / CYP1A L q24 / CYP3A L q22 / CYP2A L q13 / CYP2C L q23 / GSTM L p13 / CYP2B L q13 / CYP2E L q26 / GSTT L q11 / CYP2D L q13 / * Reference L q CYP1A L q24 / CYP2C L q23 / CYP1A L q24 / CYP2D L q13 / downstream 445 CYP3A L q22 / * Reference L q CYP2C L q23 / GSTP L q13 / Reference L p CYP2C L q23/ * Reference L q11 * New in version C1 (from lot C onwards). SALSA P128 Cytochrome P450 mix Page 3 of 9

4 Table 2. P128 s arranged according to chromosomal location Table 2a. GSTM1 gene GSTM1 NM_ start codon (ex 1) L16401 exon 3 39 nt after exon 3 GGGGAAAGTGCA-ACGTGTCTCTGA 0.9 kb L10846 exon nt after exon 5 TCTGTCAGCCAG-TTCACATCACCT stop codon (ex 8) The GSTM1 gene (8 exons), spans ~6 kb of genomic DNA and is located on 1p13.3, 110 Mb from the p-telomere. The GSTM1 gene is extremely variable in copy number (Moyer AM et al, 2007, Clin Cancer Res Dec 1;13(23): ). Analysis is complicated further by the presence of many SNPs. In Caucasians, no GSTM1 enzyme activity is present in 50% of individuals, primarily due to gene deletions (Buchard, A. et al, 2007, J Mol Diagn Nov; 9(5): ). The NCBI NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2b. CYP1B1 gene CYP1B1 NM_ start codon (ex 2) L08029 exon CAAGAGACTCGA-GTGGGAGTTAAA 4.7 kb L16393 exon reverse CAAAGCTGGAGA-AGCGCATGGCTT stop codon (ex 3) The CYP1B1 gene (3 exons), spans ~8.6 kb of genomic DNA and is located on 2p22.2, 38 Mb from p-telomere. The CYP1B1 gene is not located in a genomic region known for having frequent germline copy number variants. Germline mutations in CYP1B1 are involved in primary congenital glaucoma. The NCBI NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2c. CYP3A4 / CYP3A5 genes Gene / / Genbank CYP3A4 NM_ start codon (ex 1) L09939 exon AGTAGTGATGGC-TCTCATCCCAGA 14.2 kb L09940 exon 6 45 nt before exon 6 CTTCTGGGACTA-GAGTCTGCACAT 11.8 kb L16403 exon TCTTCAAGAAAT-CTGTGCCTGAGA 82.0 kb stop codon (ex 13) CYP3A5 NM_ start codon (ex 1) L09943 exon CCTCTGCCTTTG-TTGGGAAATGTT 3.5 kb L09944 exon TGAAGGTCAACT-CCCTGTGCTGGC 12.0 kb L16389 exon GCCCAGTCAATA- ATCTTCATTTTT stop codon (ex 13) The CYP3A4 gene (13 exon), spans ~27 kb of genomic DNA and is located on 7q22.1, 99 Mb from the p-telomere. The CYP3A5 gene (13 exons), spans ~32 kb of genomic DNA and is located on 7q22.1, 99 Mb from the p-telomere. The distance between the two genes is ~77 kb. The CYP3A4 and CYP3A5 genes are not located in a genomic region known for having frequent germline copy number variants (Lamba JK et al, 2006, Pharmacogenet Genomics Jun;16(6):415-27). The NCBI NM_ and NM_ sequences are the reference standards in the NCBI RefSeqGene project. SALSA P128 Cytochrome P450 mix Page 4 of 9

5 Table 2d. CYP2C19 / CYP2C9 genes Gene / / Genbank CYP2C19 NM_ start codon (ex 1) L27965 exon AGCTCTCAAAAA-TCTATGGCCCTG 45.5 kb L05221 exon AAACTTGGTAAT-CACTGCAGCTGA 32.3 kb L16394 exon GACACAACTCCT-GTTGTCAATGGA 85.8 kb stop codon (ex 9) CYP2C9 NM_ start codon (ex 1) L07012 exon CAATGGATTCTC-TTGTGGTCCTTG 42.5 kb L05352 exon reverse TTTCTGCCAATC-ACACGTTCAATC 4.8 kb L16349 exon reverse AAGTCAGGGAAA-TTAATATGGTTG 0.1 kb L16350 exon GTTTGACCCTCA-TCACTTTCTGGA 2.8 kb L09934 exon TTGACCCAAAGA- ACCTTGACACCA stop codon (ex 9) The CYP2C19 gene (9 exons), spans ~91 kb of genomic DNA and is located on 10q23.33, 97 Mb from the p-telomere. The CYP2C9 gene (9 exons), spans ~51 kb of genomic DNA and is located on 10q23.33, 97 Mb from the p-telomere. The distance between the genes are ~86 kb. The CYP2C19 and CYP2C9 genes are not located in a genomic region known for having frequent germline copy number variants. Defects in the CYP2C9 gene have effects on the metabolism of the widely used Warfarin drug. The NCBI NM_ and the NM_ sequences are the reference standards in the NCBI RefSeqGene project. Table 2e. CYP2E1 gene CYP2E1 NM_ start codon (ex 1) L01842 exon GGAGCACCATCA-ATCTCTGGACCC 1.0 kb L07633 exon CGCTTGTACACA-ATGGACGGTATC 4.1 kb L07634 exon CCATTTTCCACA-GGTGAGAAAGAT stop codon (ex 9) The CYP2E1 gene (9 exons), spans ~12 kb of genomic DNA and is located on 10q26.3, 135 Mb from the p-telomere. The CYP2E1 gene shows frequent germline copy number changes. Variation 2896 and 8670 of the database of genomic variants indicate a gain in CYP2E1 copy number in 7.4% respectively 5% of the individuals. The NCBI NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2f. GSTP1 gene GSTP1 NM_ start codon (ex 1) L28022 exon CCAGAGCTGGAA-GGAGGAGGTGGT 0.2 kb L07011 exon ACCAGTCCAATA-CCATCCTGCGTC stop codon (ex 7) The GSTP1 gene (7 exons), spans ~3 kb of genomic DNA and is located on 11q13.2, 67 Mb from the p-telomere. Germline copy number changes (gains) of the GSTP1 gene have been described, but are rare. The NCBI NM_ sequence is a reference standard in the NCBI RefSeqGene project. SALSA P128 Cytochrome P450 mix Page 5 of 9

6 Table 2g. CYP1A1 / CYP1A2 genes Gene/ exon / Genbank CYP1A2 NM_ stop codon (ex 7) L07639 exon CCAAGTGGGAGA-TCTTCCTCTTCC 3.1 kb L07638 exon CTCATGTACCTT-GTGACCAAGCCT 1.4 kb L07637 exon CCTCGTGAAGAA-CACTCATGAGTT 24.9 kb start codon (ex 2) CYP1A1 NM_ start codon (ex 2) L26332 exon TCCTTGGAACCT-TCCCTGATCCTT 3.1 kb L16402 exon GTCAACCTGAAT-AATAATTTCGGG 0.8 kb L06407 exon GAGAACGCCAAT-GTCCAGCTGTCA stop codon (ex 7) The CYP1A1 gene (7 exons), spans ~6 kb of genomic DNA and is located on 15q24.1, 75 Mb from the p-telomere. The CYP1A2 gene (spans 7 exons), spans ~5 kb of genomic DNA and is located on 15q24.1, 75 Mb from the p-telomere. The distance between the genes are ~23 kb. Germline copy number changes of CYP1A1 and 1A2 are probably quite rare. Entry 9749 of the database of genomic variants mentions one gain of CYP1A1 in 112 samples and no CNVs that include CYP1A2. The NCBI NM_ and the NM_ sequences are the reference standards in the NCBI RefSeqGene project. Table 2h. CYP2A6 / CYP2B6 genes Gene/ exon / Genbank CYP2A6 NM_ stop codon (ex 9) L07812 exon nt before exon 5 AGCCTCGTTTAA-ATACCTGAAACC 1.6 kb L16404 exon nt before exon 3 TTTGCAGCTCTG-CTGGGCAATGGC 0.8 kb L09928 exon 2 85 nt after exon 2 ACTCTCCTGCCA-ACTGGAGGCTAA 0.7 kb L09927 exon TGGCCTCAGGGA-TGCTTCTGGTGG kb start codon (ex 1) CYP2B6 NM_ start codon 8-10 (ex 1) L16750 exon CGACCCATTCTT-CCGGGGATATGG 0.3 kb L20817 exon CGGATTCAGGAG-GAGGCTCAGTGT 2.6 kb L09932 exon TTTGGAAAACGA-TTCCACTACCAA stop codon (ex 9) The CYP2A6 gene (9 exons), spans ~7 kb of genomic DNA and is located on 19q13.2, 41 Mb from the p-telomere. The CYP2B6 gene (9 exons), spans ~27 kb of genomic DNA and is located on on 19q13.2, 42 Mb from the p-telomere. The distance between the genes are ~140.9 kb. Germ line copy number changes of CYP2A6 are relatively frequent. Entry of the database of genomic variants mentions 2 losses and 2 gains in 112 samples for CYP2A6. Entry 3191 mentions 1 gain and 2 losses in the 270 HapMap samples. The NCBI NM_ and the NM_ sequences are the reference standards in the NCBI RefSeqGene project. SALSA P128 Cytochrome P450 mix Page 6 of 9

7 Table 2i. GSTT1 gene GSTT1 exon NM_ start codon (ex 1) L27966 exon CGTGGATCTGAT-TAAAGGTAGGTC 7.6 kb L16405 exon GGAGGACCTCTT-CCAGGAGGCCCA stop codon (ex 5) The GSTT1 gene (5 exon), spans ~8 kb of genomic DNA and is located on 22q11.23, 24 Mb from the p-telomere. The GSTT1 gene is extremely variable in copy number (Moyer AM et al, 2007, Clin Cancer Res Dec 1;13(23): ). Analysis is complicated by the presence of many SNPs. In Caucasians, no GSTT1 enzyme activity is present in 15% of individuals, primarily due to gene deletions (Buchard, A. et al, 2007, J Mol Diagn Nov; 9(5): ). The NCBI NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2j. CYP2D6 gene SALSA MLPA CYP2D6 exon NM_ start codon (ex 1) L07013 exon AGTGAGGCAGGT-ATGGGGCTAGAA 2.2 kb L16406 exon nt before exon 5 CTGTACCTCCTA-TCCACGTCAGAG 0.3 kb L09938 exon 6 68 nt after exon 6 CCCATGAACTTT-GCTGGGACACCC 1.8 kb L03680 downstream 492 nt after exon 9 reverse CCTGGGCTTCCA-TGGGGCCTTCCC stop codon (ex 9) The CYP2D6 gene (9 exons), spans ~4.4 kb of genomic DNA and is located on 22q13.2, 43 Mb from the p-telomere. Germline copy number changes of CYP2D6 are common due to the presence of a repeated sequence located just before and after the gene. CYP2D6 gene deletions resulting in a poor metabolizer phenotype are known as CYP2D6*5 or CYP2D6(D). CYP2D6 gene duplications resulting in ultrarapid metabolism of certain drugs are also common. Entry 7350 of the database of genomic variants mentions 3 gains and 6 losses in 50 samples. The frequency of the CYP2D6*5 allele has been reported by others to be 0.04 in Caucasians. CYP2D6 is known to metabolize more than 65 commonly used drugs. Patients are usually divided into four different subpopulations (poor, intermediate, extensive and ultra metabolizers) that define the rate of drug metabolism by CYP2D6. 5 to 10 percent of Caucasians have poor ability to metabolize, as do 1 to 2 percent of Southeast Asians while up to 30 percent of people with a North Eastern African background are ultra metabolizers who can carry gene duplications ranging from 3-13 copies of CYP2D6. The NCBI NM_ sequence is a reference standard in the NCBI RefSeqGene project. Note: numbering might be different as compared to literature! Complete sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA P128 Cytochrome P450 mix Page 7 of 9

8 mix P128-C1 Cytochrome P450 sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male DNA analysed with mix P128-C1 Cytochrome P450 (lot C1-1014). Implemented Changes compared to the previous product description version(s). Version March 2015 (54) - For all genes in this P128-C1 mix; gene exon numbers and ligation sites in table 1 and 2 have been adjusted according to NCBI Map Viewer. - Product description adapted to a new product version (version number changed, lot number added, new picture included). - Various textual changes on page 1 and 2. - Changes of lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. Version (53) - of 394 nt (09527-L16406) updated in Table 1 and 2j. Version 10 (48) - Warning added in Table 1, 401 nt L Version 09 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 08 (48) - Information on the specificity of the 299 nt CYP2D6 added below tables 1 and 2j. Version 07 (48) - 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 page 1 and 2. - Various minor layout changes. - Small changes of lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. Version 06 - Sequence of CYP2C19 exon 6 has been corrected SALSA P128 Cytochrome P450 mix Page 8 of 9

9 Description version 12; 16 March 2015 Version 05 - 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). Version 04 - Warning text basic research added on page 1. - Various minor textual changes on page 1. - Various minor layout changes. SALSA P128 Cytochrome P450 mix Page 9 of 9