Product Description SALSA MLPA Probemix P438-D2 HLA

Transcription

1 Product Description SALSA Probemix P438-D2 HLA To be used with the MLPA General Protocol. Version D2. Catalogue numbers: P R: SALSA MLPA Probemix P438 HLA, 25 reactions. P R: SALSA MLPA Probemix P438 HLA, 50 reactions. P R: SALSA MLPA Probemix P438 HLA, 100 reactions. To be used in combination with a SALSA MLPA reagent kit, available for various number of reactions. MLPA reagent kits are either provided with FAM or Cy5.0 dye-labelled PCR primer, suitable for Applied Biosystems and Beckman capillary sequencers, respectively (see Certificate of Analysis: Information regarding storage conditions, quality tests, and a sample electropherogram from the current sales lot is available at Precautions and warnings: For professional use only. Always consult the most recent product description AND the MLPA General Protocol before use: It is the responsibility of the user to be aware of the latest scientific knowledge of the application before drawing any conclusions from findings generated with this product. General Information: The SALSA MLPA Probemix P438 HLA is a research use only (RUO) assay for the detection of HLA class II molecule variants associated with celiac disease. Celiac disease is a chronic, small intestinal enteropathy, which is triggered by gluten proteins from wheat, barley and rye. Celiac disease is characterized by an autoimmune response in genetically susceptible individuals resulting in small intestine mucosal injury. Celiac disease is a multigenic disorder, in which the most dominant genetic risk factors are the genotypes encoding the HLA class II molecules. The HLA-DQ2.5 variant is present in 90-95% of patients with celiac disease and is encoded by HLA-DQA1*05 / HLA- DQB1*02 alleles. The HLA-DQ8 variant found in most of the remaining patients is encoded by HLA-DQA1*03 / HLA- alleles (Tack et al., 2010). The HLA-DQ2.2 variant, encoded by HLA-DQA1*02 / HLA- DQB1*02 alleles, was also linked to celiac disease (Mubarak et al., 2013). All aforementioned alleles can be detected by this MLPA probemix. This SALSA MLPA Probemix is not CE/FDA registered for use in diagnostic procedures. Purchase of this product includes a limited license for research purposes. Gene structure and Transcript variants: Entrez Gene shows transcript variants of each gene: For NM_ mrna reference sequences: Locus Reference Genomic (LRG) database: Probemix content: The SALSA MLPA Probemix P438-D2 HLA contains 20 MLPA probes with amplification products between 119 and 344 nt. This includes 11 probes specific for detecting HLA-DQA1 and HLA-DQB1 alleles for the subtypes mentioned in Table 3, which will only generate a signal when a certain allele is present. In addition, nine reference probes are included and detect nine different autosomal chromosomal locations. Complete probe sequences and the identity of the genes detected by the reference probes is available online ( This probemix contains nine quality control fragments generating amplification products between 64 and 121 nt: four DNA Quantity Fragments (Q-fragments), two DNA Denaturation Fragments (D-fragments), one benchmark fragment, and one chromosome X and one chromosome Y-specific fragment (see table below). More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at SALSA MLPA Probemix P438-HLA Page 1 of 5

2 Name Q-fragments (Only visible with <100 ng sample DNA) D-fragments (Low signal of 88 or 96 fragment indicates incomplete denaturation) 92 Benchmark fragment 100 X-fragment (X chromosome specific) 105 Y-fragment (Y chromosome specific) No DNA controls result in only five major peaks shorter than 121 nucleotides : four Q-fragments at 64, 70, 76 and 82 nt, and one 19 nt peak corresponding to the unused portion of the fluorescent PCR primer. Non-specific peaks longer than 121 nt AND with a height >25% of the median of the four Q-fragments should not be observed. Note: peaks below this 25% threshold are not expected to affect MLPA reactions when sufficient amount of sample DNA ( ng) is used. MLPA technique: The principles of the MLPA technique (Schouten et al. 2002) are described in the MLPA General Protocol ( Required specimens: Extracted DNA free from impurities known to affect MLPA reactions. For more information please refer to the section on DNA sample treatment found in the MLPA General Protocol. Reference samples: All samples tested, including reference DNA samples, should be derived from the same tissue type, handled using the same procedure, and prepared using the same DNA extraction method when possible. Reference samples should be derived from unrelated individuals who are from families without a history of celiac disease. More information regarding the selection and use of reference samples can be found in the MLPA General Protocol. Positive control DNA samples: cannot provide positive DNA samples. SALSA Binning DNA SD058 and SD059: The SD058 and SD059 Binning DNAs provided with this probemix can be used for binning of the allele-specific probes. SD059 and SD059 both contain human genomic DNA purified from a selected cell line. The SD058 contains one copy/cell for the HLA-DQ2.2 variant. The SD059 contains one copy/cell for the HLA-DQ2.5 and the HLA DQ8 variants. Furthermore, both contain two copies/cell for each sequence detected by the reference probes in this probemix. Inclusion of one reaction with 5 µl SD058 or SD059 sample DNA in MLPA experiments is essential it can be used to aid in data binning of the peak pattern using Coffalyser.Net software. Furthermore, Binning DNA should be included in the experiment whenever changes have been applied to the set-up of the capillary electrophoresis device (e.g. when capillaries have been renewed). Binning DNA should never be used as a reference sample in the MLPA data analysis, neither should it be used in quantification of mutation signals. It is strongly advised to use DNA sample and reference DNA samples extracted with the same method and derived from the same source of tissue. For further details, please consult the SD058 and SD059 Binning DNA product description provided. This product is for research use only (RUO). Data analysis: Coffalyser.Net software should be used for data analysis in combination with the appropriate lot-specific MLPA Coffalyser sheet. For both, the latest version should be used. Coffalyser.Net software is freely downloadable at Use of other non-proprietary software may lead to inconclusive or false results. For more details on MLPA quality control and data analysis, including normalisation, see the Coffalyser.Net Reference Manual. Interpretation of results: The standard deviation of all reference probes in the reference samples should be <0.10 and the dosage quotient (DQ) of the reference probes in the patient samples should be between 0.80 and Table 1 and 2 can be used or HLA variant annotation, describing single and combined variants within one sample and specific HLA-DQB1 variants, respectively. - False positive results: Please note that abnormalities detected by a single probe (or multiple consecutive probes) still have a chance of being a false positive result. Incomplete DNA denaturation (e.g. due to salt contamination) can lead to a decreased probe signal, in particular for probes located in or near a GC-rich region. The use of an additional purification step or an alternative DNA extraction method may resolve such cases. Additionally, contamination of DNA samples with cdna or PCR amplicons of SALSA MLPA Probemix P438-HLA Page 2 of 5

3 individual exons can lead to an increased probe signal (Varga et al. 2012). Analysis of an independently collected secondary DNA sample can exclude these kinds of contamination artefacts. - Copy number changes detected by reference probes are unlikely to have any relation to the condition tested for. Table 1. Expected probe signals in different HLA variants and combinations of HLA variants DQ2.5/ DQ2.5/ DQ2.2/ Probe HLA allele DQ2.5 DQ2.2 DQ8 DQ2.2 DQ8 DQ L12018 DQA1* L12019 DQA1* L12022 DQB1* L12021 DQB1* L25062 DQA1* SP0747- L25064 DQA1* L12015 DQA1* S0371-SP0074- L12959 S0482-SP0849- L26302 DQA1* DQB1* *0305 * Table 2. Expected probe signals in specific HLA-DQB1-03 variants Probe HLA allele B1*0301 B1*0302 B1*0303 B1* S0482-SP0849- L26302 DQB1* *0305 * Warnings: In our quality tests we have encountered a sample which had two copies of the target site of the 229 nt probe and one each of the target sites of the 136 nt and 149 nt probes. Two possible explanations could be: one HLA-DQB1*0301 and one allele or one HLA-DQB1*0303 and one HLA-DQB1*0305 allele. A small peak can appear at 150 nt, in height clearly distinguishable from a positive peak of the DQB1 probe detecting the *0302 or *0303 allele. We suspect this signal may be due to a certain HLA subtype. Any information on the peak is very much appreciated. Please note that in theory the probes at 136 nt and 149 nt, detecting *0302 *0305 and *0302 *0303, respectively, could also detect very rare variants. We have not encountered this in the validation studies. Limitations of the procedure: - Sequence changes (e.g. SNPs, point mutations, small indels) in the target sequence detected by a probe can cause false positive results. Mutations/SNPs (even when >20 nt from the probe ligation site) can reduce the probe signal by preventing ligation of the probe oligonucleotides or by destabilising the binding of a probe oligonucleotide to the sample DNA. SALSA MLPA Probemix P438-HLA Page 3 of 5

4 Please report copy number changes detected by the reference probes, false positive results due to SNPs and unusual results to : info@mlpa.com. Table 3. SALSA MLPA Probemix P438-D2 HLA Chromosomal position (hg18) SALSA MLPA probe Reference HLA-DQA1 HLA-DQB Control fragments see table in probemix content section for more information 119 Reference probe S0698-L q Reference probe L q Ж HLA-DQB1 probe *0302 * Ж HLA-DQA1 probe S0371-SP0074-L12959 * Ж «HLA-DQB1 probe S0482-SP0849-L26302 *0302 * Reference probe L q HLA-DQA1 probe L12015 * «HLA-DQB1 probe L12022 * Reference probe L p «HLA-DQA1 probe L12018 * Reference probe L q «HLA-DQA1 probe L25062 * Ж HLA-DQB1 probe * Reference probe L p Reference probe L p Ж «HLA-DQA1 probe SP0747-L25064 * Reference probe L q «HLA-DQB1 probe L12021 * «HLA-DQA1 probe L12019 * Reference probe L p11 Ж This probe consists of three parts and has two ligation sites. A low signal of this probe can be due to depurination of the sample DNA, e.g. due to insufficient buffering capacity or a prolonged denaturation time. «Probe located in or near a GC-rich region. A low signal can be caused by salt contamination in the DNA sample leading to incomplete DNA denaturation, especially of GC-rich regions. Note: The exon numbering used in this P438-D2 HLA product description is the exon numbering from the RefSeq transcripts NM_ and NM The exon numbering and NM sequence used is from 01/2018, but can be changed (e.g. by NCBI) after the release of the product description. Please notify us of any mistakes: info@mlpa.com. Table 4. HLA probes arranged according to chromosomal location SALSA MLPA probe Exon Ligation site Partial sequence (24 nt adjacent to ligation site) Distance to next probe HLA-DQA1 NM_ start codon (exon 1) L12015 Exon GGTCCCTCTGGG-CAGTACAGCCAT 0.05 kb 222 «19115-L25062 Exon AGACTGTCTGGA-AGTTGCCTCTGT 0.05 kb 142 Ж S0371-SP0074- ATTTGCACTGAC-35 nt spanning Exon , L12959 oligo-tgattaaacgct 0.3 kb 202 «11292-L12018 Exon nt after exon 2 TAAATCCTTCTC-GGAGAGGTCTCA 0.5 kb 332 «11293-L12019 Exon TGCTGAGGAGAG-TTATGACTGCAA 0.3 kb 289 Ж «19117-SP , AACCCCAGGGCA-33 nt spanning Exon 4 L25064 reverse oligo-gctggaatctca 16.0 kb stop codon (ex 4) HLA-DQB1 NM_ stop codon (exon 5) 184 «11296-L12022 Exon 4 37 nt before exon 4 AACTATGGGGTA-TGGGGACAAACA 3.4 kb 229 Ж GACCCGGGCGGA-32 nt spanning Exon , oligo -TGGAGCTCCGCA 0.0 kb 136 Ж , CAGTACTCGGCG-30 nt spanning Exon 2 reverse oligo -TACACCCCCACG 0.1 kb SALSA MLPA Probemix P438-HLA Page 4 of 5

5 SALSA MLPA Partial sequence (24 nt Distance to Exon Ligation site probe adjacent to ligation site) next probe 319 «11295-L12021 Exon TATAACCGAGAA-GAGATCGTGCGC 0.1 kb S0482-SP nt before exon 2, GGGCCGGGGCCT-40 nt spanning 149 Ж «Exon 2 L oligo -CCAGTTTAAGGG start codon (exon 1) Ж This probe consists of three parts and has two ligation sites. A low signal of this probe can be due to depurination of the sample DNA, e.g. due to insufficient buffering capacity or a prolonged denaturation time. «Probe located in or near a GC-rich region. A low signal can be caused by salt contamination in the DNA sample leading to incomplete DNA denaturation, especially of GC-rich regions. Note: The exon numbering used in this P438-D2 HLA product description is the exon numbering from the RefSeq transcripts NM_ and NM The exon numbering and NM sequence used is from 01/2018, but can be changed (e.g. by NCBI) after the release of the product description. Please notify us of any mistakes: info@mlpa.com. References Schouten JP et al. (2002). Relative quantification of 40 nucleic acid sequences by multiplex ligationdependent probe amplification. Nucleic Acids Res. 30:e57. Varga RE et al. (2012). MLPA-based evidence for sequence gain: pitfalls in confirmation and necessity for exclusion of false positives. Anal Biochem. 421: Selected publications using SALSA MLPA Probemix P438 HLA Vijzelaar R et al. (2016). Rapid Detection of the Three Celiac Disease Risk Genotypes HLA-DQ2.2, HLA- DQ2.5, and HLA-DQ8 by Multiplex Ligation-Dependent Probe Amplification. Genet Test Mol Biomarkers. 20(3): Van Beek EM et al. (2013). A multiplex assay to rapidly exclude HLA-DQ2.5 and HLA-DQ8 expression in patients at risk for celiac disease. Clin Chem Lab Med. 51(6): P438 Product history Version Modification D2 First unrestricted release. Implemented changes in the product description Version D May 2018 (01P) - Minor textual change to the table 3 title. Version D March 2018 (01P) - Product description restructured and adapted to a new template. Version May 2016 (55) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new pictures included). - Added information on SD058 and SD059. Version 02 (53) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new picture included). - Warning added below Table 2 - Various minor textual changes on page 1. Version 01 (52) - Not applicable, new document. More information: bv; Willem Schoutenstraat DL, Amsterdam, The Netherlands info@mlpa.com (information & technical questions); order@mlpa.com (orders) Phone SALSA MLPA Probemix P438-HLA Page 5 of 5