Certificate of Analysis SALSA MLPA probemix P140 HBA

Transcription

1 COA Version 01; Issued Certificate of Analysis SALSA probemix P140 HBA Catalogue # P R, P140-50R, P R Product name Probemix P140 HBA C , 50 or 100 reactions. Shipping conditions Shipped on dry ice or with cooling elements. Store between -25 C and -15 C. January This product can be frozen / thawed at least 20 times. Use This product has been developed to determine the DNA copy number of 34 sequences in the human HBA1 and HBA2 gene region, as described in Table 1 and 2 of the Product Description. This probemix is designed to be used in combination with SALSA MLPA reagent kit EK1 (100 reactions) or EK5 (500 reactions) as described in the MLPA General Protocol. Quality Test 1 Quality Test 2 Quality Test 3 Quality Test 4 Sufficient distance between peaks, absence of extra or shoulder peaks, complete hybridisation of each individual probe, as tested on Applied Biosystems 3130 and Beckman GeXP sequencers. Variation of each individual probe < 0.10 when tested on 23 different DNA samples of healthy individuals, purified by various extraction methods. Variation of each individual probe meeting reaction-specific criteria when testing a single DNA sample in various experimental conditions. Conditions tested include mimicking pipetting mistakes, evaporation, deviating thermocycler temperatures; presence of salts affecting sample DNA denaturation or impurities affecting polymerase activity in the sample DNA. Reactions without sample DNA resulted in only 5 major peaks shorter than 120 nt: 4 Q fragments at nt and the unused portion of the 19 nt fluorescent PCR primer. No non-specific peaks longer than 120 nt AND with a peak height of more than 25% of the median of the 4 Q fragments observed. Peaks below this 25% threshold are not expected to affect MLPA reactions when sufficient sample DNA is used. FOR RESEARCH USE ONLY. FOR IN VITRO USE ONLY. MSDS: The MLPA probemix sold by does not contain dangerous substances, as defined in European Directive 67/548/EC and its amendments, at concentrations requiring the distribution of Material Safety Data Sheets as specified in European Directives 1999/45/EC and 2001/58/EC. Therefore, a Material Safety Data Sheet is not required for this product. If spills occur, clean with water and follow appropriate site procedures. More information: Phone bv; Willem Schoutenstraat DL, Amsterdam, The Netherlands PASS PASS PASS PASS info@mlpa.com (information & technical questions); order@mlpa.com (orders) (Order information) (Technical support) Certificate of Analysis P140 HBA Page 1 of 2

2 COA Version 01; Issued Certificate of Analysis SALSA probemix P140-C1 HBA sample pictures D y e S i g n a l Size (nt) Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA probemix P140-C1 HBA (lot C1-0114) D y e S i g n a l Size (nt) Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng SD031 binning DNA analysed with SALSA MLPA probemix P140-C1 HBA (lot C1-0114). The location of the mutation-specific probe is indicated. The height of the 105 nt Y-specific probe is similar to that in male samples, indicating the presence of one copy control plasmid / cell. The signal of the 100 nt X chromosome-specific probe is twice as high as in male samples. This lot was certified by on May 1, This certificate is a declaration of analysis at the time of the manufacturing process. All assays were run in compliance with manufacturer s constraints and kit inserts. Certificate of Analysis P140 HBA Page 2 of 2

3 Product Description SALSA probemix P140-C1 HBA To be used with the MLPA General Protocol. Version C1. See the last page for product history. Catalogue numbers: P R: SALSA probemix P140 HBA, 25 reactions. P R: SALSA probemix P140 HBA, 50 reactions. P R: SALSA probemix P140 HBA, 100 reactions. To be used in combination with SALSA MLPA reagent kit EK1 (100 reactions) or EK5 (500 reactions). MLPA reagent kits are available with different fluorescent dyes, e.g. EK1-FAM, EK5-FAM (for Applied Biosystems sequencers) and EK1-Cy5.0, EK5-Cy5.0 (for Beckman sequencers). Intended use: This SALSA MLPA probemix P140 HBA is a research use only (RUO) assay for the detection of copy number changes in the human HBA1 / HBA2 gene region and is not intended to be used as a standalone assay for clinical decisions. Most, but not all, defects in the HBA gene are copy number changes. The majority of defects that are not detected by MLPA are point mutations in the HBA1 and HBA2 genes. It is therefore recommended to use this SALSA MLPA probemix in combination with sequence analysis. Clinical background: The human alpha globin gene cluster spans about 30 kb and is located on chromosome 16p13.3 at a distance of only 0.2 Mb from the p-telomere. The alpha-2 (HBA2) and alpha-1 (HBA1) coding sequences are identical. The two genes differ slightly over the 5' untranslated regions and the introns, while they differ significantly over the 3' untranslated regions. Two alpha chains plus two beta chains constitute HbA, which in normal adult life comprises about 97% of the total hemoglobin. Alpha chains combined with delta chains constitute HbA2, which together with HbF (fetal hemoglobin) makes up the remaining 3% of adult hemoglobin. Most alpha thalassemias result from genomic deletions. In the lethal Hb Bart syndrome, the two HBA1 and the two HBA2 copies are all absent or defect. In HbH disease, one functional HBA copy remains. In α-thalassemia trait, two functional copies remain while in silent α- thalassemia, three functional copies are present. More information is available on This MLPA probemix is expected to detect approximately 90% of all HBA mutations in most populations. See below for the publications on probemix P140 HBA. MLPA technique: The principles of the MLPA technique are described in the MLPA General Protocol. P140-C1 probemix content: This SALSA MLPA probemix P140 HBA contains 45 MLPA probes with amplification products between 130 and 481 nt: 34 probes for the HBA gene region (described in table 2) and 11 reference probes that detect sequences outside this region. One of these 45 probes (136 nt) is specific for the presence of the Constant Spring mutation and will thus not generate a signal in the great majority of samples. HS40 is the major regulatory element of the human alpha-globin locus and is located 40 kb upstream of the zeta-globin gene. Two HS40 specific probes are present. Deletions are known in which only the HS40 region was affected. One probe detects a sequence telomeric of HS40 and 5 probes detect sequences centromeric of HBQ1. These flanking probes are only included to delineate the extent of large HBA region copy number changes. Three probes (172, 214 and 220 nt) detect sequences that are present in both HBA1 and HBA2. The HBA1 and HBA2 genes are almost identical. Pairs of probes were designed for five locations with a very small sequence difference between the two genes: one probe detecting the HBA1 sequence and the other probe detecting the HBA2 sequence. Due to the close proximity of the genes, it is possible that in some healthy individuals the HBA2 sequence at one of these five locations is changed by gene conversion in the HBA1 sequence (or vice versa), without any clinical consequences. SALSA Probemix P140 HBA Page 1 of 17

4 Probe pairs that can be affected this way are the 160 and 165 nt intron 2 probes; the 244 and 250 nt intron 2 probes; the 190 and 391 nt probes; the 226 and 328 nt probes; the 202 and 373 nt probes. When one of the probes of such a probe pair has an ~50% reduced probe signal (0.5 ratio), while the other probe has an ~50% increased probe signal (1.5 ratio), it is likely that this is a benign polymorphism due to sequence exchange between HBA1 and HBA2 rather than a true deletion and duplication (see Table 3). In addition to the 10 different polymorphisms due to gene conversion of these 5 probe pairs, three other frequent (benign) polymorphisms have been described as shown in Table 3. This probemix contains nine quality control fragments generating an amplification product between 64 and 105 nt: four DNA Quantity Fragments (Q-fragments), three DNA Denaturation Fragments (D-fragments), one chromosome X and one chromosome Y-specific fragment (Table 1). More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol. Performance characteristics: The exact product performance characteristics cannot easily be determined due to the difficulty in obtaining DNA samples containing deletions or duplications for each of the probes. Performance of this product can be compromised by, among others: 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. The MLPA General Protocol contains technical guidelines and information on data evaluation / normalization. Internal validation of the MLPA technique in your laboratory is essential and should indicate a standard variation of each probe of 0.10 or lower when testing DNA samples of healthy individuals. Limitations of the procedure: In most populations, around 10% of genetic defects in the HBA gene are small (point) mutations, most of which will not be detected by SALSA MLPA probemix P140 HBA. The combination of a deletion and a similarly sized duplication may result in false negative MLPA results as there is no net change in copy number (example 14 in Table 5). MLPA cannot detect any changes that lie outside the target sequence of the probes and will not detect most 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 (at least up to 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. Not all abnormalities detected by MLPA are pathogenic. In some genes, intragenic deletions are known to result in very mild, or no disease manifestation (Schwartz et al, 2007). Duplications that include the first or last exon of a gene (e.g. exons 1-2) might in some cases not result in inactivation of that gene copy. The complete sequence detected by the probes is available on This product description contains only partial sequences detected by the probes. Required specimens: Purified DNA from human tissues, free from impurities. For more information, see MLPA General Protocol and the website section on DNA sample treatment. RNAse sample treatment (essential for HBA and HBB MLPA probemixes) Since HBA is heavily expressed in red blood cells, an RNAse treatment of samples is essential for wholeblood derived samples (DNA samples purified from white blood cells are less affected). Without RNAse treatment, HBA mrna can bind to the 172, 214 and 220 nt probes that detect a sequence within the HBA1 and HBA2 exons, thereby reducing the effective concentration of probes. Please note that some automatic DNA purification methods (e.g. Roche Magnapure) do not include an RNAse treatment. The following method can be used to treat samples: Mix 4 µl sample and 1 µl 0.5 mg/ml RNAse A. Incubate 30 minutes at 37 C. Continue with the 5 minutes 98 C DNA denaturation step of the MLPA protocol. RNAse A is extremely stable; it can be diluted in TE and stored at -20 C. We recommend RNAse A from Promega (code A7973; 4 mg/ml solution), diluted 8 fold in TE (1 ml of 4 mg/ml RNAse is sufficient for ~8000 samples). Do not use more than the recommended amount. SALSA Probemix P140 HBA Page 2 of 17

5 Reference samples: Reference DNA samples should be derived from the same tissue and should be extracted using the same method as patient samples. Reference samples should be derived from unrelated individuals who are from families without a history of hereditary predisposition to thalassemia. More information on the use of reference samples can be found in the MLPA General Protocol. Please note that approximately 10% of samples from healthy Caucasian individuals did show reproducible copy number changes for one or more of the HBA region probes in our experiments. This % might be higher in other populations. In many cases, these copy number changes were the polymorphisms described in table 3. Positive controls: Inclusion of a positive control DNA sample in your experiment is useful but not essential. has no positive control DNA sample available for this product. Binning DNA sample: A binning DNA sample (SD031) is included with each shipment of the P140 HBA probemix. This DNA sample is a mixture of genomic DNA and a plasmid containing the sequence of the Constant Spring mutation and can be used to determine the amplicon length of the probe for this mutation. In MLPA reactions on SD031, the peak height of the probe detecting the Constant Spring mutation resembles the peak height of this probe on heterozygous samples. Data analysis: For data analysis, Coffalyser.Net software with the appropriate lot specific probe sheet is available free of charge on The analysis method used for P140-C1 is block normalisation. Normalization of results should be performed within one experiment. A more detailed description on how to evaluate runs and how to perform data analysis is given in the MLPA General Protocol. 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 a mutation or polymorphism that prevents ligation of the MLPA probe or that destabilises the binding of a probe oligonucleotide to the sample DNA. Sequence analysis should establish whether any mutations or polymorphisms are present in the probe s target sequence. The finding of a heterozygous mutation or polymorphism would indicate that two different copies of the sequence are present in the sample DNA and that a false positive MLPA result may have been obtained. Copy number changes detected by more than one consecutive probe may still be a false positive result, see below. They should be confirmed by another independent technique such as long range PCR, gap-pcr, qpcr, array CGH or Southern blotting, whenever possible. Deletions / duplications of more than 50 kb in length can often be confirmed by FISH. Incomplete denaturation of the sample DNA, due to the presence of salt in the DNA sample, can result in false positive deletion results showing a decreased probe signal for consecutive probes that are located in a CpG island. The use of an alternative DNA extraction method may resolve such cases. False positive duplication results due to contamination of DNA samples with cdna, or with PCR amplicons of individual exons, have also been described (Varga et al, 2012). Analysis of an independently collected secondary DNA sample can exclude this kind of contamination artifacts. Interpretation of results: Alpha-thalassemia is usually inherited in an autosomal recessive manner. The expected results for most HBA region specific MLPA probes are allele copy numbers of 0 (Hb Bart, homozygous deletion), 1 (heterozygous deletion), 2 (normal), 3 (heterozygous duplication) and occasionally 4 (homozygous duplication), corresponding to probe ratios of respectively 0, 0.5, 1.0, 1.5 or 2.0. Three probes in this product detect a sequence that is present in both HBA1 as well as HBA2 (4 copies / cell). The expected results for these three probes are allele copy numbers of 0, 1, 2, 3, 4 (normal), 5, 6, 7 or 8, corresponding to probe ratios of resp. 0, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75 or 2.0. Examples are in tables 3, 4 and 5. When using MLPA for the first time or whenever changing the DNA extraction method or instruments used, internal validation of the MLPA technique using 16 DNA samples from healthy individuals is strongly recommended. This validation experiment should result in a standard variation of all probes below Arranging probes according to chromosomal location facilitates interpretation of the results and may reveal also more subtle changes such as those observed in mosaic cases. Analysis of parental samples may be SALSA Probemix P140 HBA Page 3 of 17

6 necessary for correct interpretation of complex results, e.g. when one allele contains a deletion while the other allele contains a duplication. Inactivation of a single HBA gene copy is not expected to be pathogenic (α + -thalassemia). Alphaºthalassemia results from deletion or dysfunction of respectively two alleles. Deletion or dysfunction of three alleles results in HbH disease. All four α-globin alleles are deleted or inactivated in Hb Bart syndrome. 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. HBA mutation database: and We strongly encourage users to deposit positive results in these databases. 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 to : info@mlpa.com. 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 in question before drawing any conclusions from findings made with this product. Publications on SALSA Probemix P140 HBA Chen Y.J. et al. (2013). The application of multiplex ligation-dependent probe amplification technology in diagnosis and prenatal diagnosis of α-thalassemia. Zhonghua Xue Ye Xue Za Zhi.34: Coelho A. et al. (2010). Novel large deletions in the human α -globin gene cluster: Clarifying the HS40 long-range regulatory role in the native chromosome environment. Blood Cells Mol.Dis. 45: Colosimo A. et al. (2011). Application of MLPA assay to characterize unsolved alpha-globin gene rearrangements. Blood Cells, Molecules, and Diseases. 46: Gilad O. et al. (2014). Characterization of two unique α-globin gene cluster deletions causing α-thalassemia in Israeli Arabs. Hemoglobin Epub. Sep.15. Harteveld CL. et al. (2013). State of the art and new developments in molecular diagnostics for hemoglobinopathies in multiethnic societies. Int J Lab Hematol. Epub May31. Harteveld CL. et al. (2005). Nine unknown rearrangements in 16p13.3 and 11p15.4 causing alpha- and betathalassemia characterised by high resolution multiplex ligation-dependent probe amplification. J.Med.Genet. 42: Jia X. et al. (2013). Detection of a novel large deletion causing α-thalassemia in South China. Exp.Mol Pathol. 95: Kim SY. et al. (2010). Molecular identification of the novel Gγ-β hybrid hemoglobin: Hb Gγ-β Ulsan (Gγ through 13; β from 19). Blood Cells, Molecules, and Diseases. 45: Li Y. et al. (2014). Molecular diagnosis for a novel deletion mutation of α thalassemia. Zhonghua Xue Ye Xue Za Zhi 35: Liu JZ. et al. (2008). Detection of alpha-thalassemia in China by using multiplex ligation-dependent probe amplification. Hemoglobin. 32: Nezhat N. et al. (2012). Detection of deletions/duplications in α-globin cluster by multiplex ligation-dependent probe amplification. Genet.Test Mol. Biomarkers 16: Origo R. et al. (2013). Complexity of the alpha-globin genotypes identified with thalassemia screening in Sardinia. Blood Cells Mol.Dis. epub. Phylipsen M. et al. (2010). Thalassemia in Western Australia: 11 novel deletions characterized by Multiplex Ligationdependent Probe Amplification. Blood Cells, Molecules, and Diseases 44: Phylipsen M. et al. (2012). A novel α(0)-thalassemia deletion in a Greek patient with HbH disease and β-thalasemia trait. Eur.J.Haematol.88: So CC. et al. (2014). Novel point mutation of the α2-globin gene (HBA2) and a rare 2.4 kb deletion of the α1-globin gene (HBA1), identified in two chinese patients with HbH disease. Hemoglobin 38: Wei XF. et al. (2011). Molecular characterization of a novel 27.6-kb deletion causing α+ thalassemia in a Chinese family. Ann Hematol. 90: Additional References Schouten JP. et al. (2002). Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acid Res.30:e57 (Original description of the MLPA technique). SALSA Probemix P140 HBA Page 4 of 17

7 Schwartz M. et al. (2007). Deletion of exon 16 of the dystrophin gene is not associated with disease. Hum Mutat. 28:205. Varga RE. et al. (2012). MLPA-based evidence for sequence gain: Pitfalls in confirmation and necessity for exclusion of false positives. Anal Biochem. 421: Related SALSA MLPA probemixes P102 HBB Probes for the HBB gene region. SALSA Probemix P140 HBA Page 5 of 17

8 Table 1. SALSA MLPA P140-C1 HBA probemix Length (nt) SALSA MLPA probe Chromosomal position Reference HBA Cluster 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 Ж HBA region, probe S L09493 Generates a signal only on samples containing the Constant Spring mutation! 142 * Reference probe L p * HBA region, probe L kb upstream HBA2 154 HBA region, probe L kb downstream HBA1 160 HBA region, probe L08422 HBA2 intron * HBA region, probe L21607 HBA1 intron * «HBA region, probe L21812 HBA1+HBA2 exon HS-40, probe L04797 HS HBA region, probe L04018 Between HBA2P and HBA1P 190 HBA region, probe L22520 Between HBA2 and HBA1 196 * Reference probe L q * HBA region, probe L24428 Between HBA2 and HBA1 208 * Reference probe L p «HBA region, probe L06288 HBA1+HBA2 exon * «HBA region, probe L22524 HBA1+HBA2 exon * HBA region, probe L22517 Between HBA2 and HBA1 238 * Reference probe L q HBA region, probe L23748 HBA2 intron * HBA region, probe L23600 HBA1 intron HBA region, probe L08417 Between HBA2 and HBA1 262 * DECR2 gene, probe L23601 Centromeric of HBA region 269 Reference probe L p * LUC7L gene, L21960 Centromeric of HBA region 283 HBA region, probe L kb downstream HBA1 292 HBA region, probe L04004 Between HBZ and HBZP 300 * Reference probe L q HBA region, probe L kb downstream HBA1 328 HBA region, probe L kb before HBA2 exon * HBA region, probe L23604 Between HBA2 and HBA1 346 HBA region, probe L kb upstream HBZ 355 Reference probe L q HBA region, probe L kb upstream HBZ 373 HBA region, probe L kb before HBA2 382 HS-40, probe L04175 HS * HBA region, probe L kb before HBA2 400 * HBA region, probe L25313 HBQ1 exon Reference probe L q * AXIN 1 gene, probe L13393 Centromeric of HBA region 436 * Ж HBA region, probe L kb upstream HBZ 445 * ITFG3 gene, probe L20554 Centromeric of HBA region 454 * Reference probe L q * POLR3K gene, probe L25316 Telomeric of HBA region 472 * RGS11 gene, probe L20488 Centromeric of HBA region 481 * Reference probe L q33 * New in version C1, lot «The 172, 214 and 220 nt probes detect sequences that are present in both HBA1 and HBA2. Flanking probe. Included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition tested. The 136 nt probe will only give a signal on samples containing the Constant spring mutation. Ж The 136 and 436 nt probes consist of three oligonucleotides and have two Ligation sites SALSA Probemix P140 HBA Page 6 of 17

9 Table 2. HBA region probes arranged according to chromosomal location Length (nt) SALSA MLPA probe Gene / exon Ligation site Genbank Partial sequence (24 nt adjacent to ligation site) Distance to next probe L kb before HBA2 exon R AGGAAGGGGTGA-GAATGAGAGAAA 0.5 kb L kb before HBA2 exon R ATGTCCAGAAGA-AAAGCGGTGACA 1.6 kb L kb before HBA2 exon R GATAAACAAACT-TGGCTCTGGGTA 0.2 kb L kb before HBA2 exon R CCGGGAAGGAAC-AAACACCAGGAC 0.6 kb 220 «18099-L22524 HBA1 +HBA2 exon R AAGAGTGCCGGG-CCGCGAGCGCGC 0.1 kb 214 «18881-L06288 HBA1 +HBA2 exon R CGCGCCGACCTT-ACCCCAGGCGGC 0.4 kb L08422 HBA2 intron GCGCCTTCCTCT-CAGGGCAGAGGA 0.1 kb L23748 HBA2 intron GGGCCTGGGCCG-CACTGACCCTCT 0.1 kb 172 «15857-L21812 HBA1 +HBA2 exon R GCAGGGGTGAAC-TCGGCGGGGAGG 0.1 kb 136 Ж From p-telomere to POLR3K probe 37 kb L25316 POLR3K gene 66.4 kb AY L04797 HS CTGCCCAAGCCA-AGGGTGGAGGCA 0.2 kb L04175 HS GGTACTGCTGAT-TACAACCTCTGG 30.0 kb NG_ L kb upstream HBZ GGCTGGGGCTCA-AACCAAGGCCCA 5.7 kb L kb upstream HBZ CGCAGTGCTAGA-AGGGAGTTCCTG 3.3 kb 436Ж SP & TTCCTCTCCTGT-37 nt spanning 0.2 kb before HBZ L R oligo-agtctaggagag 6.9 kb 292 ~ L04004 Between HBZ & HBZP GTGGAGTAGGCT-TTGTGGGGAACT 7.8 kb L04018 Between HBA2P & HBA1P AGTGGCCACAAT-TTGGCAGACAGA 2.5 kb S0585-SP0043- L09493 Constant Spring mutation & CCAAATACCGTC-28 nt spanning oligo-tgcccgctgggc SALSA Probemix P140 HBA Page 7 of kb L kb before HBA1 exon 1 (0.4 kb after HBA2 exon 3) TTCTCTCATTCC-CACCCCTTCCTG 0.5 kb L kb before HBA1 exon GTCACTGCTTTC-CTTCTGGACATGG 0.6 kb L kb before HBA1 exon TTCTCTGCCCAA-GGCAGCTTACCC 0.6 kb L kb before HBA1 exon R CTGGAGCATTCA-ACCTCCTCTGGG 0.3 kb L kb before HBA1 exon TCCTGGTGTTTA-TTCCTTCCCGGT 0.6 kb 220 «18099-L22524 HBA1 +HBA2 exon R AAGAGTGCCGGG-CCGCGAGCGCGC 0.1 kb 214 «18881-L06288 HBA1 +HBA2 exon R CGCGCCGACCTT-ACCCCAGGCGGC 0.4 kb L21607 HBA1 intron GCGCCTTCCTCG-CAGGGCAGAGGA 0.1 kb L23600 HBA1 intron GGCCCTCGGCCC-CACTGACCCTCT 0.1 kb 172 «15857-L21812 HBA1 +HBA2 exon R GCAGGGGTGAAC-TCGGCGGGGAGG 0.3 kb L kb after HBA1 exon TGGGACACACAT-GGCTAGAACCTC 0.3 kb L kb after HBA1 exon AAGTCCCACTCC-AGCATGGCTGCA 1.9 kb L kb after HBA1 exon GTTCACTGCCCT-GAAGAAACACCT 1.4 kb L25313 HBQ1 exon TGCTCTCTCGAG-GTCAGGACGCGA 25.1 kb L21960 LUC7L gene ATGTTCCAATGA-AACCAGTGGCAC 33.5 kb L20554 ITFG3 gene GCTGTGATACTT-TTGCCTTTGTCA 31.9 kb L20488 RGS11 gene GAACCTGAGGTT-CCGTGGAATATT 16.3 kb L13393 AXIN1 gene GATCATCGGCAA-AGTGGAGAAGGT kb L23601 DECR2 gene CAGAATCGACAT-TCTCATTAACTG The two sequence blocks between the grey lines are almost identical. Ж The 136 and 436 nt probes consist of three oligonucleotides and have two Ligation sites. R The sequence of these probes is reverse-complement as compared to the NG_ sequence. «The 172 nt L21812, the 214 nt L06288 and 220 nt L22524 probes each detect two sequences: HBA1 + HBA2. Deletion of a single target site results in only 20-25% decrease in signal of these probes. The 136 nt probe S0585-SP0043-L09493 will only give a signal on samples containing the Constant Spring mutation. The probe consists of three synthetic oligonucleotides in order to make it specific for both HBA2 and the Constant Spring mutation. This probe has been tested by several users with good results on mutation-positive samples. has only tested this probe on a plasmid containing the mutation sequence.

10 ~ The 292 probe has been reported to be deleted/duplicated in several samples from Southeast Asia. This is probably a benign deletion polymorphism. + In the sequence detected by this probe, there is only a small difference between the HBA1 and HBA2 gene. Due to their close proximity, it is possible that due to gene conversion the HBA2 sequence at this position in some healthy individuals is changed in a HBA1 sequence (or vice versa), without any consequences. More information can be found in the paragraph below on polymorphisms in the HBA region. The identity of the genes detected by the reference probes is available on request. Please notify us of any mistakes: info@mlpa.com. Exon locations HBA1 and HBA2 in the Genbank NG_ sequence: HBA2: exon 1: ; exon 2: ; exon 3: HBA1: exon 1: ; exon 2: ; exon 3: Figure 1. Schematic representation of the location of the probes in MLPA probemix P140-C1 HBA. The numbers above the arrows represent the amplification size of the respective probe in nt. This picture is not to scale and is only intended to give an impression of the sequential ordering of the probes. * The 172, 214 & 220 nt probes detect sequences that are present in both HBA1 and HBA2. + For more details, see the following page on polymorphisms in the HBA region. Note: the picture becomes unreadable when the view of the document is zoomed out too much! SALSA Probemix P140 HBA Page 8 of 17

11 Polymorphisms in the HBA region When two genomic regions with very high homology are located very close to each other, as is the case for the HBA1 and HBA2 genes, sequence exchange (gene conversion) can easily occur. In Figure 1, 10 probes are marked with +. In the sequence detected by this probe, there is only a small difference between the HBA1 and HBA2 gene. Due to their close proximity, it is possible that in some healthy individuals the HBA2 sequence at this position is changed into a HBA1 sequence (or vice versa) due to gene conversion, without any consequences (Table 3). Probe pairs that can be affected this way are the 160 and 165 nt intron 2 probes; the 244 and 250 nt intron 2 probes; the 190 and 391 nt probes; the 226 and 328 nt probes; and the 202 and 373 nt probes. Please note that for these 5 probe pairs, a single benign nucleotide change can result in a 50% lower signal of one probe and a 50% increased signal of the second probe. Results shown in Table 3 are likely to be due to benign polymorphisms rather than true deletions or duplications. The African polymorphism is a gene conversion between alpha-2 and alpha-1. The African polymorphisms cause the intron that is typical for HBA1 to be located also in HBA2 or vice versa. Alpha-1 and alpha-2 are nearly identical. There are only two spots at which they differ: a point mutation T>G (HBA2: T; HBA1: G) and a 8 nt insertion in intron 2 (where a single G (HBA2) is replaced by CTCGGCCC (HBA1)). Probes at nt and at nt, respectively, detect these intron 2 differences. In the case of the African polymorphism 1, the HBA2 target sites (160 & 244 nt) have disappeared and therefore it looks like a deletion of the alpha-2-gene, while in fact the HBA2 gene is intact, but contains the HBA1 intronic sequence as can be seen by an increased probe signal of the 165 and 250 nt HBA1 probes (vice versa for the African polymorphism 2). Asian polymorphism: The 292 nt probe has been reported to be deleted/duplicated in several samples from Southeast Asia. This is probably a benign deletion polymorphism. Nomenclature: In order to simplify the explanation of the observed polymorphisms, we introduced the terms Polym 1A etc. in Table 3 and elsewhere in this product description. However, when reporting results, we strongly recommend using always only the official HGVS nomenclature as mentioned below. Polym 1A Polym 1B Polym 2A Polym 2B Polym 3A Polym 3B Polym 4A Polym 4B Polym 5A Polym 5B African polym 1 * African polym 2 * Asian polym = NG_000006: g.30717t>c = NG_000006: g.34974c>t = NG_000006: g.31233t>c = NG_000006: g.35489c>t = NG_000006: g.33103g>a = NG_000006: g.36907a>g = NG_000006: g.34247t>g = NG_000006: g.38051g>t = NG_000006: g.34311delginsctcggccc = NG_000006: g.38115_38122delctcggcccinsg = NG_000006: g.[34247t>g; 34311delGinsCTCGGCCC] = NG_000006: g.[38051g>t; 38115_38122delCTCGGCCCinsG] = This seems to be a true deletion covering at least nt of NG_ Exact deletion boundaries are not known by us. This deletion was reported to us in a high proportion of Indonesian samples. *As this MLPA assay can only detect a limited number of specific sequences, we describe the African polymorphisms as two separate small changes, even though they are expected to be usually due to gene conversion events spanning a larger region. SALSA Probemix P140 HBA Page 9 of 17

12 Table 3. Expected probe ratios for predicted non-pathogenic heterozygous HBA Polymorphisms (gene conversion events) Probe number length (nt) Polym 1A Polym 1B Polym 2A Polym 3A Polym 4A Polym 5A African polym 1 African polym 2 Asian polym L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L09493 CS More information on these polymorphisms is on the previous page. These are likely to be benign polymorphisms! Not shown are polymorphisms 2B, 3B, 4B and 5B which are the complement of 2A, 3A, 4A and 5A. Not all of these predicted polymorphisms have been observed in the samples tested at! Asian polym + polym 3B SALSA Probemix P140 HBA Page 10 of 17

13 Table 4A. Expected probe ratios for common heterozygous α 3.7 and α 4.2 HBA deletion types probe length (nt) α 3.7 (A) α 3.7 (B) α 3.7 C) α 3.7 (D) α 3.7 (E) SALSA Probemix P140 HBA Page 11 of 17 α 3.7 (F) α 4.2 (A) α 4.2 (B) α 4.2 (C) ααα (3.7 A) Gene / Probe number L L L L L L L L L L L L L « L « L L L « L L L L L L « L « L L L « L L L L L L L L L ααα (3.7 F) L09493 CS Many similar α 3.7 and α 4.2 deletions, with different breakpoints, exist. The same is true for the α triplication. Homozygote α 3.7 deletion samples are frequently a combination of two different α 3.7 deletions. The combination of a deletion on one allele and a similarly sized duplication on the other allele may resemble a normal sample! As far as we know, distinguishing the various α 3.7 and α 4.2 deletions result has no clinical significance. «The 172 nt, 214 nt and 220 nt probes each detect two sequences: HBA1 and HBA2. Deletion of a single target site results in an ~25% decrease in signal of these probes. Although a single probe, they are mentioned twice in the table! In certain cases such as α 3.7 deletion type A and D, we cannot be certain which copy of these probes is deleted. In the α 3.7 (type E) example, the deletion probably starts and ends within the sequence detected by the 220 nt probe, thereby eliminating two of the four sequence copies detected by this probe. As a result, the probe ratio for the 172 and 214 nt probes is 0.75 (4>3 copies) while the 220 nt probe shows a probe ratio of 0.5 (4>2 copies).

14 Table 4B. Expected probe ratios for common heterozygous HBA deletion types Probe number probe length (nt) SEA MED1 MED2 α 20.5 FIL THAI Dutch1 del HS-40 CS L L L L L L L L L L L L L « L « L L L « L L L L L L « L « L L L « L L L L L L L L L L09493 CS ~60% «The 172 nt, 214 nt and 220 nt probes each detect two sequences: HBA1 and HBA2. Deletion of a single target site results in a ~25% decrease in signal of these probes. Although they are a single probe, they are mentioned twice in the table! When using Coffalyser.Net software for data analysis, the 136 nt probe specific for the Constant Spring mutation will give a 0-3% signal on negative samples and an approximately 60% signal on a heterozygous positive sample. For mutation specific probes, Coffalyser.Net software provides the ratio of the peak as compared to the reference probes. Interpretation Table 4B is courtesy of C.L. Harteveld, M. Phylipsen & R.A. Schaap, LUMC Leiden and is intended as an aid only. MLPA users are responsible for correct interpretation of their results. No rights can be derived from this table. SALSA Probemix P140 HBA Page 12 of 17

15 Table 5. Selected examples of HBA deletions Probe number length L nt L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L09493 CS Deletions are indicated by text boxes (heterozygous) or single borders (bi-allelic). Polymorphisms are in grey. Examples 1-13 are results obtained using probemix P140-C1 on positive samples. These results can be explained as mentioned below, although other explanations are possible in some cases. Examples 1-5 can be due to rearrangement of a single chromosome + presence of one or more polymorphisms. Examples 6-13 are likely to be due to rearrangements in both chromosomes. No further experiments were performed at for confirmation. 1. α 3.7 (D) + polymorphism 3B (328 nt probe: extra copy; 226 nt probe: one copy less). 2. α 3.7 (D) + polymorphism 3B (328 nt probe: extra copy; 226 nt probe: one copy less) + Asian polymorphism. 3. α 3.7 (D) + African polymorphism 1 (165 & 250 nt probes: extra copy; 160 & 244 nt probes: one copy less). 4. α 3.7 (D) + African polymorphism 1 + Polymorphism 2B (165, 250 & 373 nt probes: extra copy; 160, 244 & 202 nt probes: one copy less). 5. Triplication type α 3.7 (A) + polymorphism 4A (165 nt probe: extra copy; 160 nt probe: one copy less). SALSA Probemix P140 HBA Page 13 of 17

16 Biallelic rearrangements: 6. α 3.7 (D) / α3.7 (D) 7. α 4.2 (B) / α 4.2 (C) in combination with polymorphism 3B (328 nt probe: extra copy; 226 nt probe: one copy less). 8. α 3.7 (B) / α3.7 (E) 9. α 3.7 (B) / α 3.7 (E) in combination with polymorphism 4B (160 nt probe: extra copy; 165 nt probe: one copy less). 10. α 3.7 (D) / α3.7 (F) 11. α 3.7 (D) + SEA deletion 12. Triplication type α 3.7 (D) + Sea deletion. 13. ααα + ααα (both type D) 14. Theoretical example: This looks like polymorphism 3B but it could also be a combination of α 3.7 (D) + ααα 3.7 (F)! The combination of a deletion on one allele and a similarly sized duplication on the other allele may resemble a normal sample! Explanation on how to interpret table 5: This is a shortened table, with only a selection of probes. To get the final amount of copies you need to count the number of copies detected by that probe. The final probe ratio obtained by data analysis of MLPA reactions is the copy number in the patient sample divided by the copy number in healthy individuals. Below is an explanation of example 5 of Table 5 describing Triplication type α 3.7 (A) + polymorphism 4A. Similar tables explaining the other examples in table 5 are available upon request. Probe number length Copy number in healthy individuals Polymorphism 4A Triplication α 3.7 type A Copy number in the patient Final probe ratio L L22524 « L06288 « L copy L L21812 « L copy L copy L copy L copy L copy L22524 « copy L06288 « copy L copy +1 copy L copy L21812 « copy L «The 172 nt L21812, the 214 nt L06288 and 220 nt L22524 probes each detect two sequences. SALSA Probemix P140 HBA Page 14 of 17

17 Gene structure: The human alpha globin gene cluster located on chromosome 16 spans about 30 kb and includes seven loci: 5'- zeta - pseudozeta - mu - pseudoalpha-1 - alpha-2 - alpha-1 - theta - 3'. The alpha-2 (HBA2) and alpha-1 (HBA1) coding sequences are identical. These genes differ slightly over the 5' untranslated regions and the introns, but they differ significantly over the 3' untranslated regions: (HBA1) and (HBA2). HBA1 transcript NM_ (576 nt; Coding sequence ) and HBA2 transcript NM_ (622 nt; Coding sequence ) are the reference standards in the NCBI RefSeqGene project. In Table 2, the ligation sites of the P140 HBA MLPA probes are indicated according the Genbank NG_ sequence which contains all genes in the HBA cluster. Product history Version Sold from Modification C probes in the HBA region have been removed and 11 new probes were included. 5 extra flanking probes centromeric of the HBA region have been included. 7 of the 11 reference probes have been replaced. See Table 6 (below) for more information. B The 88 and 96 nt DNA denaturation control fragments replaced (QDX2). B Small change in length of one probe. B Control probes at 100 and 105 nt (X, Y specific) added (QDX1). B new probes in the HBA region included; 1 probe removed. A First release. Implemented changes in the product description Version 33 - Major revision of the text in the product description. - Chromosomal position 400 nt probe L25313 corrected in Table 1. Version 32 - One example removed from Table 4B. - Information added on nomenclature of polymorphisms. Version 31 - Product description completely rewritten. - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new picture included). More information: Manufactured by bv, Amsterdam The Netherlands. info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands SALSA Probemix P140 HBA Page 15 of 17