MRC-Holland MLPA. Description version 05;

Transcription

1 MLPA Probemix P235-B2 Retinitis Pigmentosa Lot B2-1013: As compared to the previous version B1 (lot B1-0808), two reference probes have been replaced and one added; in addition, the control fragments have been adjusted (QDX2). Retinitis pigmentosa (RP) is a hereditary degenerative disease of the photoreceptor neurons of the retina. RP is characterized by progressive degeneration of the peripheral retina (leading to night blindness), loss of the peripheral visual field and an abnormal electroretinogram. The genes most frequently involved in RP are RHO, IMPDH1, RP1 and PRPF31. Rhodopsin (RHO) is a highlyspecialized G protein-coupled receptor that detects photons in the rod photoreceptors of vertebrates. The RHO gene (5 exons) spans ~7 kb of genomic DNA and is located on 3q22.1, 129 Mb from the p-telomere. The P235-B2 probemix contains one probe for each exon. Mutations in IMPDH1, a widely expressed rate-limiting enzyme of the de novo pathway of guanine nucleotide biosynthesis, have been shown to cause autosomal dominant RP. The IMPDH1 gene (17 exons) spans ~18 kb of genomic DNA and is located on chromosome 7q32.1, 128 Mb from the p-telomere. The P235-B2 probemix contains 9 probes for IMPDH1. The gene for human oxygen regulated photoreceptor protein (RP1) encodes a protein that is localized in the connecting cilia of both rod and cone receptors. The RP1 protein is required for the morphogenesis of the outer segments of photoreceptor cells. The RP1 gene (4 exons) spans ~15 kb of genomic DNA and is located on chromosome 8q12.1, 56 Mb from the p-telomere. Mutations in RP1 cause at least 7% of autosomal dominant RP. The P235-B2 probemix contains two probes for exon 1 and one probe each for the other exons. In addition, PRPF31 encodes a 61 kda protein which is essential for splicing in all cell types. The pathologic effect of mutations in this gene can be seen in rod photoreceptor. The PRPF31 gene (14 exons) spans ~16 kb of genomic DNA and is located on chromosome 19q13.42, 55 Mb from the p-telomere. The P235-B2 probemix contains one probe for each exon (two probes for exon 1). Furthermore, this probemix contains one probe for RPE65, located on 1p31.2 and can also be linked Retinitis Pigmentosa. Finally, this probemix contains eight reference probes detecting seven different autosomal chromosomal locations. This probemix is 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 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 these genes are expected to be small (point) mutations, most of which will not be detected by this test. probemix are sold by for research purposes and to demonstrate the possibilities of the MLPA technique. This probemix is not CE/FDA certified for use in diagnostic procedures. mix are supplied with all necessary buffers and enzymes. Purchase of the MLPA test probemix includes a limited license to use these products for research purposes. The use of this probemix 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 6, 1057 DN Amsterdam, the Netherlands probemix P235 Retinitis Pigmentosa Page 1 of 6

2 Related probemixes P221 LCA-1: Leber congenital amaurosis, genes included: RPE65, AIPL1, CRB1, CRX. P222 LCA-2: Leber congenital amaurosis, genes included: CEP290, GUCY2D, RDH, RPGRIP1. Data analysis The P235-B2 Retinitis pigmentosa probemix contains 43 s with amplification products between 130 and 454 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 (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 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 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 N. Laddach & J.P. Schouten at. In case the results obtained with this probemix lead to a scientific publication, it would be very much appreciated if the first probemix designer could be made a co-author. Info/remarks/suggestions for improvement: info@mlpa.com. probemix P235 Retinitis Pigmentosa Page 2 of 6

3 Table 1. MLPA P235-B2 Retinitis Pigmentosa probemix Chromosomal position reference PRPF31 RHO RP1 IMPDH 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 PRPF31 probe L PRPF31 probe L Reference probe L q PRPF31 probe L RP1 probe L ± IMPDH1 probe L ± IMPDH1 probe L RHO probe L RP1 probe L ± IMPDH1 probe L PRPF31 probe L Reference probe L p PRPF31 probe L RHO probe L RP1 probe L ± IMPDH1 probe L * Reference probe L p RHO probe L PRPF31 probe L PRPF31 probe L ± IMPDH1 probe L RPE65 probe L p RHO probe L RP1 probe L Reference probe L p Reference probe L q RHO probe L RP1 probe L PRPF31 probe L PRPF31 probe L PRPF31 probe L PRPF31 probe L ± IMPDH1 probe L PRPF31 probe L PRPF31 probe L PRPF31 probe L PRPF31 probe L ± IMPDH1 probe L ± IMPDH1 probe L * Reference probe L p ± IMPDH1 probe L * Reference probe L q25 * New in version B2 (from lot B onwards). Changed in version B2 (from lot B onwards). ± 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. Note: 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. probemix P235 Retinitis Pigmentosa Page 3 of 6

4 Table 2. P235 probes arranged according to chromosomal location Table 2a. RHO gene RHO NM_ start codon (ex 1) L CCTGCTCAACCT-AGCCGTGGCTGA 2.0 kb L GAGAACCATGCC-ATCATGGGCGTT 1.3 kb L CAAGCCGGAGGT-CAACAACGAGTC 0.3 kb L TCAGCCACCACA-CAGAAGGCAGAG 1.1 kb L TCACCACCATCT-GCTGCGGCAAGA stop codon (ex 5) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2b. IMPDH1 gene IMPDH1 NM_ start codon (ex 1) 229 ± L nt after ex 1 CTAACTTCCCGA-AACGCCACGCTC 0.1 kb 172 ± L GACCTCGCTACA-CACCCGACGACA 0.1 kb 427 ± L CCTCCTAGAACT-ATCTTCAGTGGT 5.6 kb 445 ± L CTTCATAGCTGA-TGAGGTGGTGAG 2.7 kb 373 ± L nt after ex 6 TGCAGGTGTGTA-ACTCACAGCCAT 0.8 kb 421 ± L reverse GCCACCACCAGT-TCAATCCTTGGC 3.7 kb 270 ± L nt after ex 12 GCCAGGCCCTGT-TCTGCCATGGTA 1.5 kb 189 ± L reverse GGGGCCTCCGTA-GTGGCGGCCAGC 1.9 kb 166 ± L nt before ex 17 CCGTACGGCTGA-GATCTCAGGGCT stop codon (ex 17) ± 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. The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2c. RP1 gene RP1 NM_ start codon (ex 2) L nt before exon 1 TTCTGGCTGTTG-TCTCCTTAGGGT 0.1 kb L CATACTGAGAAT-AAATCCAAAGAC 4.9 kb L ACCCCTTCTACT-GGTTTTTCCATC 1.2 kb L ATCCTGAGCTCT-GGAGCTGTGGTG 2.9 kb L AGCAGTAATCAA-GAGGGCAGTTTG stop codon (ex 4) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. probemix P235 Retinitis Pigmentosa Page 4 of 6

5 Table 2d. PRPF31 gene PRPF31 NM_ start codon (ex 2) L GCTTAAAGGCCT-TGCTTTCTTGTC 0.2 kb L reverse TCTCGCGCCGTT-ATAGAGGCAAAG 2.7 kb L GGATTCAGTCAA-GACCATCGCCAA 0.4 kb L nt after ex 3 TGAGCTTACTGA-TCATGATAGGAC 3.1 kb L GTGGAGATCGAA-AACGAGCTGAGT 0.6 kb L AATGCACTGGAT-TACATCCGCACG 0.9 kb L ACAAGTGCAAGA-ACAATGAGAACC 0.4 kb L ATCTACGAGTAT-GTGGAGTCCCGG 0.8 kb L GTCTACCTCAGT-GCTGCCCCACAC 1.9 kb L nt before ex 9 TTACCTCTGTCT-GTCTGTCTCACA 1.6 kb L GATCGAGCGCAA-ATTCGACAAGTG 0.3 kb L GGCCAACCGTAT-GAGCTTCGGAGA 0.8 kb L CAGACACAGGTA-AACGAGGCCACC 0.2 kb L ATATGGCGGGAA-GTCCACCATCCG 2.3 kb L GGATCGGGTTCT-GGCAGGGAGAAC stop codon (ex 14) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2e. RPE65 gene RPE65 NM_ start codon (ex 1) L GTCAGAGATCGT- TGTACAATTCCC stop codon (ex 14) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Note: 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. probemix P235 Retinitis Pigmentosa Page 5 of 6

6 mix P235-B2 Retinitis Pigmentosa sample picture D ye S ign al Size Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human control DNA analysed with mix P235-B2 Retinitis Pigmentosa (lot B2-1013). Implemented Changes compared to the previous product description version(s). Version 05 (53) - 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 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 03 (46) - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - s of the probes targeting the PRPF31 gene updated according to new version of the NM_reference sequence. - Small correction of chromosomal locations in Table 1 and 2. - Remark on transcript variant used and RefSeqGene standard added below Table 2. - Various minor textual changes throughout the document. Version 02 (45) - Various minor textual changes on page 1. - Minor changes in the data analysis section on page 2. - Sentence when only small numbers of samples are tested, visual comparison of peak profiles should be sufficient removed from data analysis section. - Tables have been numbered. - Various minor layout changes in Table 1 and 2. probemix P235 Retinitis Pigmentosa Page 6 of 6