Product Description SALSA MLPA probemix P091-D1 CFTR To be used with the MLPA General Protocol.

Transcription

1 Product Description SALSA probemix P091-D1 CFTR To be used with the MLPA General Protocol. Version D1. As compared to version C1, 3 CFTR and 4 reference probes have been replaced. In addition, the 88 and 96 nt control fragments have been replaced (QDX2). For complete product history see page 8. Catalogue numbers: P R: SALSA probemix P091 CFTR, 25 reactions. P R: SALSA probemix P091 CFTR, 50 reactions. P R: SALSA probemix P091 CFTR, 100 reactions. To be used in combination with a SALSA 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. SALSA probemix P091 CFTR should not be used for initial diagnosis of cystic fibrosis (CF) or congenital absence of the vas deferens (CAVD). Intended use: SALSA probemix P091 CFTR is an in vitro diagnostic (IVD) 1 or research use only (RUO) assay for the detection of the F508 point mutation and deletions and/or duplications of one or more exons in the human CFTR gene of patients diagnosed with cystic fibrosis (CF) or congenital absence of the vas deferens (CAVD). When an aberration has been identified in patient DNA, this product can also be used for carrier screening of family members. This assay is optimised for use with peripheral blood derived genomic DNA. Deletions or duplications obtained with the P091 CFTR probemix must be verified by another technique. In particular, copy number changes detected by only a single probe always require validation by another method. This SALSA probemix is not intended to be used as a standalone assay for clinical decisions. Most defects in the CFTR gene are point mutations. Except F508, the majority of point mutations will not be detected by MLPA. It is therefore recommended to use this SALSA probemix in combination with sequence analysis. The results of this test should be interpreted by a clinical molecular geneticist or equivalent. 1 Please note that this probemix is for In Vitro Diagnostic use (IVD) in the countries specified at the end of this product description. In all other countries, the product is for Research Use Only (RUO). Clinical background: Cystic fibrosis (CF) and congenital absence of the vas deferens (CAVD), which are both recessive genetic diseases, are caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR functions as a chloride channel regulating water and ion transport across membranes in the lungs, liver, pancreas, intestine, reproductive tracts, and sweat glands. Azoospermia can result in men with CAVD. CF related pathologies include thickened mucus in the lungs with frequent respiratory infection and inflammation, and malnutrition and diabetes due to pancreatic insufficiency. More information is available on Deletions and duplications of complete exons in the CFTR gene account for ~2% of mutations found in patients and are usually missed by standard sequence analysis. Most of these deletions and duplications can be detected by the MLPA technique and hence MLPA complements sequence analysis of the CFTR gene. SALSA Probemix P091 CFTR Page 1 of 8

2 Additionally, one of the most common mutations found in patients is F508, for which the wild type allele can be detected with this probemix. Gene structure and transcript variants: The CFTR gene spans ~190 kilobases (kb) on chromosome 7q31.2 and has 27 exons. Only one transcript variant has been defined: NM_ (6132 nt; coding sequence , The LRG sequence ( for CFTR is pending approval. The GenBank chromosomal DNA sequence is NG_ Exon numbering: We adopted the exon numbering used by NCBI. Exon numbering might be different as compared to literature! P091-D1 probemix content: This SALSA probemix P091 CFTR contains 44 MLPA probes with amplification products between 130 and 481 nt (Table 1) including 34 probes for the CFTR gene region (Table 2) and 10 reference probes that detect sequences outside this region. The identity of the genes detected by the reference probes is available online ( The probemix contains probes for each of the 27 CFTR exons and a second probe for exon 1, 11, 13 and 27. One of the exon 11 (330 nt) probes detects the wild type allele of the F508 mutation. This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity Fragments (Q-fragments), three DNA Denaturation Fragments (D-fragments), and one chromosome X and one chromosome Y-specific fragment (Table 1). The Q-fragments are only visible when less than 100 ng sample DNA is used. Low signal of the 88 or 96 nt fragment indicates incomplete DNA denaturation. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol. MLPA technique: The principles of the MLPA technique (Nygren et al. 2005, Schouten et al. 2002) are described in the MLPA General Protocol ( MLPA technique validation: Internal validation of the MLPA technique using 16 DNA samples from healthy individuals is required, in particular when using MLPA for the first time, or when changing the sample handling procedure, DNA extraction method or instruments used. This validation experiment should result in a standard deviation <0.10 for all probes over the experiment. Required specimens: Purified DNA from human peripheral blood, 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: Reference DNA samples should be derived from the same tissue type, handled using the same procedure, and prepared using the same DNA extraction method as the patient samples. Reference samples should be derived from unrelated individuals who are from families without a history of CF and CAVD. 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. Inclusion of a positive sample in each experiment is recommended. Performance characteristics: The expected number of CFTR deletions/duplications which can be detected with this MLPA probemix is ~2% of all CFTR mutations (Tang et al. 2013). This probemix will also be able to detect the wild type allele of the F508 mutation, which accounts for ~66% of all CFTR mutations (Bobadilla et al. 2002). The analytical sensitivity and specificity for the detection of deletions/duplications in the CFTR gene (based on a literature review) is very high and can be considered >99%. Analytical performance can be compromised by: SNPs or other polymorphisms (e.g. indels) in the DNA target sequence, impurities in the DNA sample, incomplete DNA denaturation, the use of insufficient or too much sample DNA, the use of insufficient or unsuitable reference samples, problems with capillary electrophoresis or a poor data normalisation procedure and other technical errors. The MLPA General Protocol contains technical guidelines and information on data evaluation/normalisation. SALSA Probemix P091 CFTR Page 2 of 8

3 Data analysis: Coffalyser.Net software must be used for data analysis in combination with the appropriate lot-specific MLPA Coffalyser sheet. For both, the latest version should be used which are 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, see the Coffalyser.Net Manual. Interpretation of results: The expected results for CFTR region specific MLPA probes are allele copy numbers of 2 (normal), 0 (homozygous deletion), 1 (heterozygous deletion), 3 (heterozygous duplication) and occasionally 4 (homozygous duplication or heterozygous triplication). The standard deviation of all 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 When these criteria are fulfilled, the following cut-off values for the DQ of the probes can be used to interpret MLPA results: Copy Number status Dosage quotient Normal 0.80 < DQ < 1.20 Homozygous deletion DQ = 0 Heterozygous deletion 0.40 < DQ < 0.65 Heterozygous duplication 1.30 < DQ < 1.65 Heterozygous triplication/ Homozygous duplication 1.75 < DQ < 2.15 Ambiguous copy number All other values - Arranging probes according to chromosomal location facilitates interpretation of the results and may reveal more subtle changes such as those observed in mosaic cases. Analysis of parental samples may be necessary for correct interpretation of complex results. - False positive results: Please note that abnormalities detected by a single probe (or multiple consecutive probes) still have a considerable 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 within a CpG island in or near the CFTR gene. The use of an additional purification step or an alternative DNA extraction method may resolve such cases. - False positive duplication results: Contamination of DNA samples with cdna or PCR amplicons of individual exons can lead to false positive duplication results (Varga et al. 2012). Analysis of an independently collected secondary DNA sample can exclude these kinds of contamination artefacts. - Normal copy number variation in healthy individuals is described in the database of genomic variants: Users should always verify the latest updates of the database and scientific literature when interpreting their findings. - Copy number changes detected by reference probes are unlikely to have any relation to the condition tested for. Notes CFTR results: - CF and CAVD are autosomal recessive disorders caused by defects in the CFTR gene. Inactivation of both copies of the CFTR gene is expected to result in disease. Inactivation of a single copy of the CFTR gene is typically seen in carriers. - Deletion or duplication of the flanking probes for ASZ1 and CTTNBP2 are not expected to be the cause of CF or CAVD. These probes have only been included to delineate the extent of large deletions and duplications nt probe detects the wild type allele of the F508 mutation. The presence of the F508 mutation results in a lower signal. Limitations of the procedure: - In most populations, the major cause of genetic defects in the CFTR gene are small (point) mutations, most of which, except F508, will not be detected by using SALSA probemix P091 CFTR. - MLPA cannot detect any changes that lie outside the target sequence of the probes and will not detect copy number neutral inversions or translocations. Even when MLPA did not detect any aberrations, the possibility remains that biological changes in that gene or chromosomal region do exist but remain undetected. - 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 SALSA Probemix P091 CFTR Page 3 of 8

4 reduce the probe signal by preventing ligation of the probe oligonucleotides or by destabilising the binding of a probe oligonucleotide to the sample DNA. Confirmation of results: Copy number changes detected by only a single probe always require confirmation by another method. An apparent deletion detected by a single probe can be due to e.g. a mutation/polymorphism that prevents ligation or destabilises the binding of probe oligonucleotides to the DNA sample. Sequence analysis can establish whether mutations or polymorphisms are present in the probe target sequence. The finding of a heterozygous mutation or polymorphism indicates that two different alleles of the sequence are present in the sample DNA and that a false positive MLPA result was obtained. Copy number changes detected by one or more than one consecutive probe should be confirmed by another independent technique such as long range PCR, qpcr, array CGH or Southern blotting, whenever possible. Deletions/duplications of more than 50 kb in length can often be confirmed by FISH. CFTR mutation database: We strongly encourage users to deposit positive results in the Clinical and Functional Translation of CFTR (CFTR2) database. Recommendations for the nomenclature to describe deletions/duplications of one or more exons can be found on Please report copy number changes detected by the reference probes, false positive results due to SNPs, and unusual results (e.g., a duplication of CFTR exons 6 and 8 but not exon 7) to : info@mlpa.com. SALSA Probemix P091 CFTR Page 4 of 8

5 Table 1. SALSA P091-D1 CFTR probemix Length Chromosomal position (a) SALSA MLPA probe (nt) reference CFTR Q-fragments (Only visible with <100 ng sample DNA) D-fragments (Low signal of 88 or 96 fragment indicates incomplete denaturation) 100 X-fragment (X chromosome specific) 105 Y-fragment (Y chromosome specific) 130 Reference probe L q ASZ1 probe L kb before CFTR 142 CFTR probe L13079 Exon CFTR probe L03315 Exon CFTR probe L02376 Exon ± CFTR probe L02389 Exon * Reference probe L q CFTR probe L02939 Exon CFTR probe L02390 Exon * Reference probe L p CFTR probe L02400 Exon CFTR probe L13077 Exon CFTR probe L13651 Exon Reference probe L q CFTR probe L02379 Exon * CFTR probe L22807 Exon CFTR probe L03312 Exon CFTR probe L02380 Exon CFTR probe L02393 Exon Reference probe L p CFTR probe L15981 Exon CFTR probe L02394 Exon CFTR probe L03314 Exon CFTR probe L15081 Exon CFTR probe L13076 Exon * Reference probe L p CFTR probe L14978 Exon 11; F508 WT 337 CFTR probe L13653 Exon CFTR probe L03313 Exon CFTR probe L15083 Exon * CFTR probe L22808 Exon CFTR probe L23339 Exon CFTR probe L02397 Exon CFTR probe L02385 Exon * CFTR probe L22809 Exon CFTR probe L02398 Exon ± CFTR probe L02387 Exon * Reference probe L q CFTR probe L02399 Exon CTTNBP2 probe L Kb after CFTR 454 Reference probe L q CFTR probe L23460 Exon Reference probe L p * Reference probe L q11 (a) Exon numbering has changed from version C1 (lots 0809 & 1109) onwards. We adopted the exon numbering used by NCBI. Exon numbering might be different as compared to literature! * New in version D1 (from lot 0712 onwards). Changed in version D1 (from lot 0712 onwards). Small change in length, not in sequence detected. This probe detects the wild type allele of the F508 mutation. The presence of the F508 mutation results in a lower signal. We have no information on the effect of polymorphisms very close to the F508 site. ± SNP and/or (point) mutations close to the ligation site can influence the probe signal: exon 12 (418 nt) probe signal is likely to be influenced by the G542X mutation; exon 15 (160 nt) probe signal is influenced by the W846X mutation (D. Bunyan, Salisbury). SALSA Probemix P091 CFTR Page 5 of 8

6 Flanking probe. Included to help determine the extent of a deletion/duplication. Copy number alterations of only the flanking or reference probes are unlikely to be related to the condition tested. Table 2. CFTR probes arranged according to chromosomal location Length (nt) SALSA MLPA probe L03264 CFTR exon (a) ASZ1 gene (GASZ) Ligation site (b) NM_ Partial sequence (c) (24 nt adjacent to ligation site) TATGCTGCTAGT-GTTGCCAATGCA Distance to next probe 57.1 kb CFTR start codon (ex 1) L23339 Upstream 747 nt before ATG of CFTR startcodon AGCGCTAAGGTA-AATGCATCAGAC 0.5 kb L02376 Exon nt before ATG startcodon GCTAGAGCAAAT-TTGGGGCCGGAC 0.1 kb L03312 Exon AGAAAAAGGGTT-GAGCGGCAGGCA 24.3 kb L13077 Exon , reverse GGATTTGGTATA-TGTCTGACAATT 4.8 kb L02379 Exon TGGCTTCAAAGA-AAAATCCTAAAC 21.9 kb L02380 Exon GGGAAGAATCAT-AGCTTCCTATGA 3.4 kb L03313 Exon GATAAAATAAGT-ATTGGACAACTT 1.0 kb L15981 Exon CTGGGAGTTGTT-ACAGGCGTCTGC 1.3 kb L15081 Exon , reverse CAGTATGCCTTA-ACAGATTGGATA 3.5 kb L13653 Exon AGATACTTCAAT-AGCTCAGCCTTC 1.9 kb 400 * L22809 Exon ATAAGACATTGG-AATATAACTTAA 6.1 kb L02385 Exon nt before exon 10, reverse CTACTCCATCAC-ACTGGTAGCACC 11.4 kb L23460 Exon , reverse TTCTTCCACTGT-GCTTAATTTTAC 0.1 kb L14978 Exon 11 F508 WT AGAAAATATCAT-CTTTGGTGTTTC 28.2 kb 418 ± L02387 Exon TTTGCAGAGAAA-GACAATATAGTT 2.6 kb 226 * L22807 Exon TTGTATTTATTA-GACTCTCCTTTT 0.2 kb L03314 Exon nt after exon 13 AGATTGCATTTT-ACCTCTTGAGAA 1.9 kb L13079 Exon GTACCAGATTCT-GAGCAGGGAGAG 2.6 kb 160 ± L02389 Exon , reverse GAAGGTATGTGT-TCCATGTAGTCA 7.9 kb L02390 Exon GTGGCTGCTTCT-TTGGTTGTGCTG 0.8 kb L13651 Exon CTTTGCTTGCTA-TGGGATTCTTCA 3.0 kb 364 * L22808 Exon TCCAAAGATATA-GCAATTTTGGAT 3.9 kb L02393 Exon , reverse CTGTTGCAACAA-AGATGTAGGGTT 1.1 kb L02394 Exon , reverse TTGGCAGTATGT-AAATTCAGAGCT 3.0 kb L13076 Exon TTAGCCATGAAT-ATCATGAGTACA 12.9 kb L15083 Exon ACAGAAGGTAAA-CCTACCAAGTCA 15.0 kb L02397 Exon GTGTCTTGGGAT-TCAATAACTTTG 10.4 kb L02398 Exon , reverse CATCTGCAACTT-TCCATATTTCTT 11.8 kb L02399 Exon GATAGAACAGTT-TCCTGGGAAGCT 0.8 kb L03315 Exon , reverse CATTCCAGCATT-GCTTCTATCCTG 1.4 kb L02400 Exon TTCCATCCAGAA-ACTGCTGAACGA 0.1 kb L02939 Exon , reverse CTGTCTCCTCTT-TCAGAGCAGCAA 58.1 kb CFTR stop codon (ex 27) CTTNBP2 gene L03267 CAAGAAGCAATA-TTGTCAAGAGCC (CORTBP2) (a) Exon numbering has changed from version C1 (lots 0809 & 1109) onwards. We adopted the exon numbering used by NCBI. Exon numbering might be different as compared to literature! (b) Ligation sites of the P091 CFTR MLPA probes are indicated according to RefSeq sequence NM_ containing 27 exons. (c) Only partial probe sequences are shown. Complete probe sequences are available at Please notify us of any mistakes: info@mlpa.com. * New in version D1 (from lot 0712 onwards). Changed in version D1 (from lot 0712 onwards). Small change in length, not in sequence detected. This probe detects the wild type allele of the F508 mutation. The presence of the F508 mutation results in a lower signal. We have no information on the effect of polymorphisms very close to the F508 site. SALSA Probemix P091 CFTR Page 6 of 8

7 ± SNP and/or (point) mutations close to the ligation site can influence the probe signal: exon 12 (418 nt) probe signal is likely to be influenced by the G542X mutations; exon 15 (160 nt) probe signal is influenced by the W846X mutation (D. Bunyan, Salisbury). Flanking probe. Included to help determine the extent of a deletion/duplication. Copy number alterations of only the flanking or reference probes are unlikely to be related to the condition tested. References Bobadilla JL et al. (2002). Cystic fibrosis: a worldwide analysis of CFTR mutations--correlation with incidence data and application to screening. Hum Mutat. 19: Schouten JP et al. (2002). Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. 30:e57. Nygren AO et al. (2005). Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences. Nucleic Acids Res. 33:e128. Tang S et al. (2013). Characterization of a recurrent 3.8kb deletion involving exons 17a and 17b within the CFTR gene. J Cyst Fibros. 12: 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 Probemix P091 CFTR Alibakhshi R et al. (2008). Analysis of the CFTR gene in Iranian cystic fibrosis patients: identification of eight novel mutations. J Cyst Fibros. 7: Bonadia LC et al. (2014). CFTR genotype and clinical outcomes of adult patients carried as cystic fibrosis disease. Gene. 540: Cordovado SK et al. (2012). CFTR mutation analysis and haplotype associations in CF patients. Mol Genet Metab. 105: Costantino L et al. (2011). A wide methodological approach to identify a large duplication in CFTR gene in a CF patient uncharacterised by sequencing analysis. J Cyst Fibros. 10: Diana A et al. (2012). A large deletion causes apparent homozygosity for the D1152H mutation in the cystic fibrosis transmembrane regulator (CFTR) gene. Gene. 497: Farjadian S et al. (2014). Rare large homozygous CFTR gene deletion in an Iranian patient with cystic fibrosis. World J Clin Cases. 2: Giordano S et al. (2013). Molecular and functional analysis of the large 5' promoter region of CFTR gene revealed pathogenic mutations in CF and CFTR-related disorders. J Mol Diagn. 15: Giuliani R et al. (2010). Identification of the second CFTR mutation in patients with congenital bilateral absence of vas deferens undergoing ART protocols. Asian J Androl. 12: Ivady G et al. (2011). Distribution of< i> CFTR</i> mutations in Eastern Hungarians: Relevance to genetic testing and to the introduction of newborn screening for cystic fibrosis. Journal of Cystic Fibrosis. 10: Jung H et al. (2011). Heterogeneous spectrum of CFTR gene mutations in Korean patients with cystic fibrosis. Korean J Lab Med. 31: Krenkova P et al. (2013). Distribution of CFTR mutations in the Czech population: positive impact of integrated clinical and laboratory expertise, detection of novel/de novo alleles and relevance for related/derived populations. J Cyst Fibros. 12: Marson FA et al. (2014). Polymorphisms in the glutathione pathway modulate cystic fibrosis severity: a crosssectional study. BMC medical genetics. 15:27. Nakakuki M et al. (2012). Detection of a large heterozygous deletion and a splicing defect in the CFTR transcripts from nasal swab of a Japanese case of cystic fibrosis. J Hum Genet. 57: Nectoux J et al. (2006). A frequent large rearrangement in the CFTR gene in cystic fibrosis patients from Reunion Island. Genet Test. 10: Neocleous V et al. (2014). Apparent Homozygosity of p.phe508del in CFTR due to a Large Gene Deletion of Exons Case Rep Genet. 2014: Paracchini V et al. (2008). Molecular and clinical features associated with CFTR gene rearrangements in Italian population: identification of a new duplication and recurrent deletions. Clin Genet. 73: Ramos MD et al. (2010). CFTR rearrangements in Spanish cystic fibrosis patients: first new duplication (35kb) characterised in the Mediterranean countries. Ann Hum Genet. 74: Schneider M et al. (2007). Large deletions in the CFTR gene: clinics and genetics in Swiss patients with CF. Clin Genet. 72: Schneider M et al. (2009). Ligation dependent allele specific quantification (LASQ) of CFTR cdna on the LightCycler using MLPA hybridization probes. Clin Chim Acta. 402: Svensson AM et al. (2010). Detection of large rearrangements in the cystic fibrosis transmembrane conductance regulator gene by multiplex ligation-dependent probe amplification assay when sequencing fails to detect two disease-causing mutations. Genet Test Mol Biomarkers. 14: SALSA Probemix P091 CFTR Page 7 of 8

8 Tomaiuolo AC et al. (2011). Characterization of a novel isolated deletion of the exon 3 within the CFTR gene: Relevance for phenotypic expression and genetic counseling. Clin Biochem. 44: Ziętkiewicz E et al. (2014). CFTR Mutations Spectrum and the Efficiency of Molecular Diagnostics in Polish Cystic Fibrosis Patients. PloS one. 9:e P091 Product history Version Modification D1 Three CFTR and four reference probes have been replaced. In addition, the 88 and 96 nt control fragments have been replaced (QDX2). C1 Four CFTR and one reference probe have been replaced. B1 The wildtype-specific probe for F508 and control fragments at 88, 96, 100, and 105 nt have been included. A1 First release. Implemented changes in the product description Version D May 2017 (03) - Product description restructured and adapted to a new template. Version D August 2015 (02) - Minor textual changes. Version D July 2015 (02) - Product description completely rewritten and adapted to a new template. Version January 2013 (48) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new pictures included). Version December 2012 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 07 (48) 11 January Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Minor textual changes on page 1 - Remark on RefSeqGene standard added below Table 2. - Small changes in ligation sites of the probes targeting the CFTR gene updated according to new version of the NM_reference sequence - Small correction of chromosomal locations in Table 1. Version 06 (45) 12 December Product description adapted to a new lot (lot number added, new picture included). - Minor textual changes on page 1 - Data analysis section modified - Tables have been numbered - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. More information: bv; Willem Schoutenstraat DL, Amsterdam, The Netherlands info@mlpa.com (information & technical questions); order@mlpa.com (orders) Phone SALSA Probemix P091 CFTR Page 8 of 8