MRC-Holland MLPA. Related SALSA MLPA probemix P102 HBB: Contains probes for the HBB gene

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1 SALSA MLPA probemix P140-B4 HBA Lot B4-0413, B & B4-0312: Compared to the previous version B3 (lot B3-0510), the 88 and 96 nt control fragments have been replaced (QDX2). The human alpha globin gene cluster is located on chromosome 16 and spans about 30 kb. The alpha-2 (HBA2) and alpha-1 (HBA1) coding sequences are identical. These 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. Alpha thalassemias usually result from deletions of each of the HBA2 and HBA1 genes; some non-deletion alpha thalassemias have also been reported. HS-40 is the major regulatory element of the human alpha-globin locus and is located 40 kb upstream the zeta-globin gene. Deletion of only the HS-40 region has been described. This SALSA probemix is designed to detect copy number changes of 24 different sequences in the HBA region. In addition, the probemix contains one probe that detects the presence of the Constant Spring mutation. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in or near the HBA1 and HBA2 genes. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak height of the amplification product of that probe. The 142 & 166 nt probes detect the exon 3 sequence which is present in both HBA1 and HBA2. Hence, they will show a decrease in signal of only 20-25% in samples of individuals with three instead of the usual four HBA copies. For more information regarding interpretation of probe ratios, please see Table 3. 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. Since HBA is heavily expressed in red blood cells, an RNase treatment of samples is essential when DNA was purified from whole blood. Without RNase treatment, the HBA mrna can bind to the template DNA strand, thus competing with the MLPA probes. This may result in irregular results for probes located within exons. Below, a possible method of RNase treatment is described. SALSA probemixes and reagents are sold by for research purposes and to demonstrate the possibilities of the MLPA technique. They are not CE/FDA certified for use in diagnostic procedures. Purchase of the SALSA test probemixes and reagents includes a limited license to use these products for research purposes. The use of a SALSA probemix and reagents requires a thermocycler with heated lid and sequence type electrophoresis equipment. Different fluorescent PCR primers are available. The MLPA technique has been first described in Nucleic Acid Research 30, e57 (2002). More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands Related SALSA probemix P102 HBB: Contains probes for the HBB gene References for SALSA probemix P140 HBA Wei XF. et al. (2011) Molecular characterization of a novel 27.6-kb deletion causing α+ thalassemia in a Chinese family. Ann Hematol. 90: Colosimo A. et al. (2011) Application of MLPA assay to characterize unsolved alpha-globin gene rearrangements. Blood Cells, Molecules, and Diseases. 46: SALSA probemix P140 HBA Page 1 of 9

2 Description version 31; 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: Liu JZ et al. (2008) Detection of alpha-thalassemia in China by using multiplex ligation-dependent probe amplification. Hemoglobin. 32: 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: Please note that this SALSA MLPA P140 probemix is different from the probemix used by Harteveld et al. in this publication. RNase sample treatment (essential for HBA and HBB MLPA probemixes) The presence of RNA in the sample can result in a higher variability of certain HBA and HBB probes. This is due to the exceptionally large amounts of HBA and HBB mrna present in whole-blood derived samples (DNA samples purified from white blood cells are less affected.) This mrna can compete with MLPA probes when target sites site are located within HBB/HBA exons. 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 is sufficient for ~8000 samples). Do not use more than the recommended amount. Data analysis The P140-B4 HBA probemix contains 38 MLPA probes with amplification products between 130 and 409 nt. nt. Please note that one of these 38 probes, the 135 nt probe, will not generate a signal in the great majority of samples. This 135 nt probe is specific for the presence of the Constant Spring mutation. In addition, the P140-B4 mix 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 More information on how to interpret observations on these control fragments can be found in the MLPA protocol. Please note that the 142 & 166 nt probes detect the exon 3 sequence which is present in both HBA1 and HBA2. Different alpha-thalassemia deletions may generate ratios in these two probes varying from 0.25 to 1.2, as follows: Heterozygous -alpha 3.7 or -alpha 4.2 or -alpha 20.5 deletion: ratio 0.75 Homozygous -alpha 3.7, homozygous -alpha 4.2 or complementary erozygous -alpha 3.7 / 4.2 : ratio 0.5 Compound erozygous -alpha 4.2 AND alpha 0 -thal OR -alpha 3.7 : ratio 0.25 Alpha-triplication: ratio 1.2 Data generated by this probemix can first be intra-sample normalised by dividing the peak height of each probe s amplification product by the total height 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. 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 This probemix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. We would like to thank Marion Phylipsen and Kees Harteveld of Leiden University for sharing the test results of the P140 probemix obtained on various mutation-positive samples. SALSA probemix P140 HBA Page 2 of 9

3 Figure 1. Schematic representation of the location of the probes in the P140-B4 HBA MLPA probemix. 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. *142 nt; # 166 nt: The 142 nt and 166 nt probe both detect a sequence that is present in both HBA1 and HBA2. The African polymorphism is a gene conversion between HBA2 and HBA1; the intron that is typical for HBA1 is located also in HBA2. HBA1 and HBA2 are nearly identical. There are only two spots where they differ: a point mutation T>G (HBA2: T; HBA1: G) and a 7 nt insertion in intron 2 (where a single G (HBA2) is replaced by TCGGCCC (HBA1)). Probes at 160 nt and at 241 nt, respectively, detect these differences, where it should be noted that they both detect the intron 2 sequences that are typical for the HBA2 gene. In case of the African polymorphism these HBA2 target sites have disappeared and it therefore looks like a deletion of the HBA2 gene, while in fact the HBA2 gene is intact but contains the HBA1 intronic sequence. Figure 2. Schematic representation of the location of the probes for HBA2 included in the P140-B4 HBA MLPA probemix. The numbers above the arrows represent the amplification size of the respective probe in nt. Probes at 160 nt and at 241 nt, respectively are specific for HBA2, whereas the remaining ones also detect HBA1. In case of the African polymorphism these HBA2 target sites have disappeared and it therefore looks like a deletion of HBA2, while in fact the HBA2 gene is intact but contains the HBA1 intronic sequence. This picture is not to scale and is only intended to give an impression of the sequential ordering of the probes. SALSA probemix P140 HBA Page 3 of 9

4 Table 1. SALSA MLPA P140-B4 HBA probemix Length (nt) SALSA MLPA probe Chromosomal position Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA D-fragments: Low signal of 88 OR 96 nt fragment indicates incomplete denaturation 100 X-fragment: Specific for the X chromosome 105 Y-fragment: Specific for the Y chromosome 130 Reference probe L q Ж HBA region, probe S0290-SP0043- Signal only on samples containing the Constant Spring L09493 mutation! HBA region, probe L04011 HBA1 +HBA2 exon Reference probe L q HBA region, probe L kb downstream HBA1 160 HBA region, probe L08422 HBA2 intron HBA region, probe L06292 HBA1+HBA2 exon Reference probe L q HS-40, probe L04797 HS HBA region, probe L04018 Between HBA2P and HBA1P 190 HBA region, probe L04740 Between HBA2 and HBA1 196 ± HBA region, probe L08414 End of HBA2 exon HBA region, probe L04007 Between HBA1P and HBA2 208 Reference probe L q HBA region, probe L08415 Between HBA1P and HBA2 220 ~ HBA region, probe L08416 Between HBA2 and HBA1 229 HBA region, probe L04008 Between HBA1P and HBA2 236 POLR3K probe L p13, 60 kb telomeric of HS HBA region, probe L06249 HBA2 intron Reference probe L p HBA region, probe L08417 Between HBA2 and HBA1 265 Reference probe L p Reference probe L q HBA region, probe L kb downstream HBA1 292 ~ HBA region, probe L04004 Between HBZ and HBZP 301 Reference probe L p HBA region, probe L kb downstream HBA1 318 ~ HBA region, probe L04005 Between HBZ and HBZP 325 CREBBP probe L Mb centromeric of HBA1 337 ~ HBA region, probe L08420 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 L08410 Between HBA1P and HBA2 382 HS-40, probe L04175 HS Reference probe L q HBA region, probe L kb downstream HBA1 409 Reference probe L q29 Ж This probe consists of three parts and has two ligation sites. + The 142 & 166 nt probes detect both HBA1 and HBA2. SNP rs influences the 160 nt probe signal. This probe is located at a 1 nt difference between the HBA1 and HBA2 intron sequence. We recommend ignoring apparent deletions of only this probe. ± SNP rs could influence the probe signal of the 196 nt probe. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. ~ An important note on this probe is present in Table 2. 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. Please note that some probes may be variable in samples containing RNA, and that an RNA treatment is essential (p.2). IMPORTANT: Apparent deletions detected by only a single HBA probe frequently prove to be false positives. A decrease in signal of a single MLPA probe in the HBA region is often due to a change of a single nucleotide that affects SALSA probemix P140 HBA Page 4 of 9

5 Description version 31; the ligation reaction or destabilizes the binding of one probe oligonucleotide to the sample DNA, rather than to a deletion. Many SNP s are present in this complicated region. The identity of the genes detected by the reference probes is available on request. Please notify us of any mistakes: info@mlpa.com. Table 2. HBA cluster 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 L01316 POLR3K gene - 43 kb from p-telomere 60.2 kb AY L04797 HS CTGCCCAAGCCA-AGGGTGGAGGCA 0.1 kb L04175 HS GGTACTGCTGAT-TACAACCTCTGG 29.3 kb NG_ L kb upstream HBZ GGCTGGGGCTCA-AACCAAGGCCCA 5.7 kb L kb upstream HBZ CGCAGTGCTAGA-AGGGAGTTCCTG 10.1 kb 292 ~ L04004 Between HBZ & HBZP GTGGAGTAGGCT-TTGTGGGGAACT 3.3 kb 318 ~ L04005 Between HBZ & HBZP TGCTCTAGGTCA-CCCTGTCATCAC 4.6 kb L04018 Between HBA2P & HBA1P AGTGGCCACAAT-TTGGCAGACAGA 2.6 kb 373 * L08410 Between HBA1P & HBA AGAGGGTCACCA-TGACACAGGAAG 0.4 kb L04007 Between HBA1P & HBA TTCCAGCCCAGC-CCCTGCCCCACA 0.1 kb 214 * L08415 Between HBA1P & HBA ATGTCCAGAAGA-AAAGCGGTGACA 1.9 kb 229 * L04008 Between HBA1P & HBA CCGGGAAGGAAC-AAACACCAGGAC 0.7 kb 142 * L04011 HBA1+2 exon CGCGCCGACCTT-ACCCCAGGCGGC 0.4 kb L08422 HBA2 intron GCGCCTTCCTCT-CAGGGCAGAGGA 0.1 kb L06249 HBA2 intron GGGCCTGGGCCG-CACTGACCCTCT 0.1 kb L06292 HBA1+2 exon TCCCCGCCGAGT-TCACCCCTGCGG 0.1 kb 135 S0290-SP0043- CCAAATACCGTC-28 nt spanning Constant Spring mutation L09493 oligo-tgcccgctgggc 0.2 kb 196 ± L08414 End of HBA2 exon ATGTGCCAACAA-TGGAGGTGTTTA 0.4 kb L04740 Between HBA2 & HBA TTCTCTCATTCC-CACCCCTTCCTG 0.6 kb L08416 Between HBA2 & HBA CGCTCTTCCTGC-GCCTCACAGCCC 0.5 kb L08417 Between HBA2 & HBA TTCTCTGCCCAA-GGCAGCTTACCC 0.6 kb L08420 Between HBA2 & HBA GTTTACCTGTTT-AACCAGTATTTA 1.0 kb 142 * L04011 HBA1+2 exon CGCGCCGACCTT-ACCCCAGGCGGC 0.6 kb L06292 HBA1+2 exon TCCCCGCCGAGT-TCACCCCTGCGG 0.3 kb L kb downstream HBA TGGGACACACAT-GGCTAGAACCTC 0.3 kb L kb downstream HBA AAGTCCCACTCC-AGCATGGCTGCA 1.9 kb L kb downstream HBA GTTCACTGCCCT-GAAGAAACACCT 1.4 kb L06294 End of exon 3 of HBQ1, 3.7 kb downstream HBA AGTAAAGCCTCT-CCCAGGAGCAGC ~ The 292 and 318 nt probes have been reported to be deleted/duplicated together in several samples. This is probably a benign polymorphism. One case has been reported to us with these probes duplicated on one allele and a FIL deletion on the other allele, resulting in an apparent erozygous SEA deletion. * These probes detect the reverse sequence. + The 142 nt L04011 and 166 nt L06292 probes each detect two sequences: HBA1 + HBA2. Deletion of a single target site results in only 20-25% decrease in signal of these probes. SNP rs influences the 160 nt probe signal. This probe is located at a 1 nt difference between the HBA1 and HBA2 intron sequence. We recommend ignoring apparent deletions of only this probe. The 135 nt probe S0290-SP0043-L09493 will only give a signal on samples containing the Constant Spring mutation. This probe has been tested with good results on mutation-positive samples by several users. MRC- Holland has only tested this probe on a plasmid containing the mutation sequence. ± SNP rs could influence the probe signal of the 196 nt probe. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. Please disregard apparent copy number changes detected only by the 220 nt probe located between HBA2 and HBA1. In the sequence detected by this probe, there is only a single nucleotide difference between the HBA1 and HBA2 gene. Due to their close proximity, it is likely that the HBA2 sequence at this position in some healthy individuals is changed in a HBA1 sequence (and vice versa) due to gene conversion, without any consequences. SALSA probemix P140 HBA Page 5 of 9

6 Description version 31; The 337 nt probe might be replaced in the future as the probe is not entirely specific. Samples that are homozygous for the alpha-3.7 deletion do not give any signal for 8 out of 9 probes that are deleted as indicated on page 5. The 337 nt probe however does give a (reduced) signal on these samples. Exon locations HBA1 and HBA2 in the Genbank NG_ sequence: HBA2: exon 1: ; exon 2: ; exon 3: HBA1: exon 1: ; exon 2: ; exon 3: SALSA probemix P140 HBA Page 6 of 9

7 Table 3. Interpretation common HBA deletion types and expected probe ratios Probe name length (nt) ratio wt RW hom RW LW alfa-tripl SEA MED1 α 20.5 FIL THAI Dutch1 RW/ SEA african polym del HS-40 POLR3K gene L HS-40 (1) L HS-40 (2) L kb up HBZ L kb up HBZ L HBZ/HBZP(1) L HBZ/HBZP(2) L HBA2P/HBA1P L HBA1P/HBA2(1) L HBA1P/HBA2(2) L HBA1P/HBA2(3) L HBA1P/HBA2(4) L HBA2 int2(1) L or HBA2 int2(2) L or HBA2 ex l HBA2/HBA1(1) L HBA2/HBA1(2) L HBA2/HBA1(3) L HBA2/HBA1(4) L HBA1+2, ex l HBA1+2, ex l HBA region 0.2 kb dw HBA L08423* HBA region 0.5 kb dw HBA L04019* HBA region 2.4 kb dw HBA L04020* HBQ1 3.7kb dw HBA L06294* HBA S00290-SP0043-L09493 Constant- Spring 135 up : upstream; dw : downstream Please note that several similar α 3.7 (RW; rightward) deletions, with different breakpoints, exist. The same is true for the α triplication. Please note: this interpretation table is courtesy of Kees Harteveld, LUMC Leiden. It is meant as an aid only. MLPA users are responsible for a correct interpretation of their results, and no rights can be derived from this table. SALSA probemix P140 HBA Page 7 of 9

8 SALSA MLPA probemix P140-B4 HBA sample picture D ye S ign al Size (nt) Figure 3. Capillary electrophoresis pattern from a control sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P140-B4 HBA (lot B4-0413). SALSA probemix P140 HBA Page 8 of 9

9 Implemented Changes compared to the previous product description version. Version 31 (52) - Product description adapted to a new lot (lot number added, new picture included). Version 30 (52) - Replaced Figure 1 and 2. - Various minor textual changes. Version 29 (52) - Adjusted Figure 1 and 2. - Removed old buffer electropherograms. Version 28 (52) - Product description adapted to new version. Version 27 (48) - Warning added to Table 1 about SNP in 196 nt probe L Version 26 (48) - Corrected information about African Polymorphism and Figure 2 added on page 3. Version 25 (48) - Product description adapted to a new lot (lot number added, new picture included). - Minor changes in text and figure added on page one. - New references added on page two. - Information about African Polymorphism and Figure 2 added on page 3. Version 24 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 23 (48) - Product description adapted to a new version (lot number added, new picture included). Version 22 (48) - Reference probe (325 nt) in Table 1 changed into flanking probe. - Various minor textual changes. Version 21 (46) - Warning added on frequent deletions and duplications of the 292 nt (04624-L04004) and 318 nt (04625-L04005) probes. Version 20 (46) - Product description adapted to a new version (lot number added, new picture included). - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. Version 19 (45) - Product description adapted to a new lot (lot number added, new picture included). Version 18 (44) - Minor changes in the product description on page 1. - Minor changes in the data analysis section on page 2. - Minor changes in table titles. - Changes in layout Table 3. - Warning added to Tables 1 and 2 about SNP in 220 nt probe L Version 17 - Warning added to Table 2 about SNP in 160 nt probe L Minor layout changes. SALSA probemix P140 HBA Page 9 of 9