MRC-Holland MLPA. Description version 07; 11 AUG 2015

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1 mix P347-A2 Hemochromatosis Lot A As compared to version A1 (lot A1-1109), four reference s have been replaced and the control fragments have been adjusted (QDX2). Hereditary hemochromatosis (HH) is an inherited disorder characterised by progressive iron deposition and tissue injury in multiple organs. HH has been demonstrated to result from mutations in several genes involved in the regulation of iron homeostasis such as HFE, TFR2, HFE2, HAMP and SLC40A1. Mutations in these genes act in an autosomal recessive manner except for SLC40A1 that manifest in autosomal dominant phenotypes. The most common form of HH is associated with the homozygous C282Y mutation of the HFE gene (Alexander et al., 2009). The HFE gene comprises 6 exons, spanning about 8 kb of genomic DNA on 6p22.2, 26 Mb from the p- telomere. The SLC40A1 gene contains 8 exons, spanning 20 kb and is located on chromosome 2q32.2, 190 Mb from the p-telomere. The TFR2 gene consists of 18 exons and has a length of approximately 21 kb on chromosome 7q22.1, 100 Mb from the p-telomere. The HFE2 gene comprises 4 exons, spanning about 4 kb of genomic DNA on 1q21.1, 145 Mb from the p- telomere. The HAMP gene contains 3 exons, spanning 3 kb and is located on chromosome 19q13.12, 36 Mb from the p-telomere. The P347-A2 mix contains s for each exon of the HFE, SLC40A1 and HAMP genes. For the HFE2 gene 3 s have been included and for the TFR2 gene 8 s are present. Furthermore, mutationspecific s for the HFE C282Y mutation and HAMP G71D mutation detecting the wild-type sequence are present. Finally, 11 reference s are included, which detect sequences on various autosomal chromosomes. This SALSA mix is designed to detect deletions/duplications of one or more sequences in the aforementioned genes and to detect the presence of the aforementioned mentioned point mutation in a DNA sample. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak area of the amplification product of that. Note that a mutation or polymorphism in the sequence detected by a can also cause a reduction in relative peak area, even when not located exactly on the ligation site! In addition, some signals are more sensitive to sample purity and small changes in experimental conditions. Therefore, deletions and duplications detected by MLPA should always be confirmed by other methods. Not all deletions and duplications detected by MLPA will be pathogenic; users should always verify the latest scientific literature when interpreting their findings. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. Finally, note that most defects in this gene are expected to be small (point) mutations which will not be detected by this SALSA MLPA test. SALSA mixes 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 test mixes and reagents includes a limited license to use these products for research purposes. The use of this SALSA mix 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 SALSA mix P347 Hemochromatosis Page 1 of 7

2 Data analysis The P347-A2 Hemochromatosis mix contains 45 MLPA s with amplification products between 136 and 481 nt. This includes one specific for the HFE C282Y mutation, which will only generate a signal when the mutation is present and one detecting the wild-type sequence of the HAMP G71D mutation. In addition, it contains 9 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at nt, three DNA denaturation control fragments (Dfragments) at nt, one X-fragment at 100 nt and one Y-fragment at 105 nt. More information on how to interpret observations on these control fragments can be found in the MLPA protocol. Data generated by this mix can first be normalised intra-sample by dividing the peak area of each s amplification product by the total area of only the reference s in this mix (block normalisation). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalised ratio in a sample by the average intra-normalised ratio of all reference samples. Please note that this type of normalisation assumes no changes occurred in the genomic regions recognised by the reference s. 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. QFPCR, FISH, Southern blots or long range PCR. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website This mix was developed by R. Vijzelaar at. In case the results obtained with this mix lead to a scientific publication, it would be very much appreciated if the mix designer could be made a co-author. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA mix P347 Hemochromatosis Page 2 of 7

3 Table 1. P347-A2 Hemochromatosis mix Chromosomal position Reference HFE SLC40A1 TFR2 HFE2 HAMP 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 136 Reference L q SLC40A L HFE L b 154 SLC40A L HFE L Reference L q Ж SLC40A SP0151-L HFE L SLC40A L TFR L * Reference L q Ж HFE SP0152-L SLC40A L HFE L TFR L Ж SLC40A SP0153-L Reference L q HFE L a 250 Ж HAMP SP0154-L HFE L TFR L b 274 Ж HFE SP0155-L Reference L q HAMP L / G71D WT 301 HFE L TFR L SLC40A L TFR L Reference L p HFE L Ж HAMP SP0156-L Ж HFE SP0157-L TFR L HFE L * Reference L q * Reference L q Ж ± HFE SP0158-L16030 C282Y mutation 419 HFE L a 427 SLC40A L Ж HAMP SP0159-L * Reference L q TFR L Reference L q TFR L Reference L q26 * New in version A2 (from lot A onwards). Changed from lot A onwards. Small change in length, no change in sequence detected. Ж This consists of three parts and has two ligation sites. ± Mutation-specific. This has been tested on patient samples and will only generate a signal when the C282Y mutation is present. Wild type sequence detected. The presence of the G71D mutation will result in a decreased signal. SALSA mix P347 Hemochromatosis Page 3 of 7

4 Note: numbering might be different as compared to literature! Please notify us of any mistakes. The identity of the genes detected by the reference s is available on request: info@mlpa.com. Table 2. P347 s arranged according to chromosomal location Table 2a. HFE gene HFE NM_ next start codon (ex 1) 208 Ж SP nt before ex 1; GTGTTTCACAAG-33nt spanning oligo 1 L AAGTTCTGAAAG 3.6 kb L a 23 nt before ex 2a CTACACATGGTT-AAGGCCTGTTGC 0.1 kb L a GACCTTGGTCTT-TCCTTGTTTGAA 0.4 kb L nt before ex 3 GGGATTTTTCCA-GAGTCCCACACC 0.1 kb L GACCACCTTGAA-TTCTGCCCTGAC 1.3 kb L a 2 nt before ex 4a TCTTTCCTGTCA-AGTGCCTCCTTT 0.2 kb 409 Ж ± SP0158-4a ; GAGATATACGTA-25nt spanning oligo L16030 C282Y CAGCCCCTCATT 0.2 kb L TTAGAGCCCTCA-CCGTCTGGCACC 0.1 kb L TTGTTCATTGGA-ATTTTGTTCATA 1.2 kb 274 Ж SP ; AACTCCTTGATT-36nt spanning oligo 6 L CCATTTAGGTTT 0.1 kb 364 Ж SP ; TGTCTCTCATGA-28nt spanning oligo 6 L CATTTCCTCCGT stop codon (ex 6) ± Mutation-specific. This has been tested on patient samples and will only generate a signal when the C282Y mutation is present. Ж This consists of three parts and has two ligation sites. The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2b. SLC40A1 gene SLC40A1 NM_ next start codon (ex 1) L TCCTGTTAACAA-GCACCTCAGCGA 0.7 kb L CTTGGCCGACTA-CCTGACCTCTGC 4.6 kb L TGGCACTTTGCG-GTGTCTGTGTTT 2.4 kb L TCGCTGGTGGTA-CAGAATGTTTCA 1.2 kb 172 Ж SP ; ACTGCTACTGCA-36nt spanning oligo 5 L GGAGAAGACAGA 6.2 kb L GATTGACCAGTT-AACCAACATCTT 1.7 kb L ACAGCTTTCCTG-TTTGATCTTGTG 1.8 kb 233 Ж SP ; TGATCTTCTGCA-30nt spanning oligo 8 L TGAAGCTTTTGG stop codon (ex 8) Ж This consists of three parts and has two ligation sites. The NM_ sequence is a reference standard in the NCBI RefSeqGene project. SALSA mix P347 Hemochromatosis Page 4 of 7

5 Table 2c. TFR2 gene TFR2 NM_ next start codon (ex 1) L GGACACAAGCAT-GGAGCGGCTTTG 0.6 kb L GCCAGCCTTCCT-ACTGGGCTACGT 7.4 kb L b CGTGTGGACCGA-CACGCACTACGT 1.3 kb L AGGAGTGCTCAT-ATACCCAGAGCC 2.9 kb L nt after ex 10 TGAGCAACGGTA-AGGTCAGGGCCA 1.5 kb L CTCTCTATGAAC-AGGTGGTGTTCA 0.2 kb L reverse GGACTCCCACAA-AGGCCGTGAAGG 6.8 kb L TCCTCGCTTGAA-TGATTCAGGGTC stop codon (ex 18) The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2d. HFE2 gene HFE2 NM_ next start codon (ex 2) L TGGAGAATTGGA-TAGCAGAGTAAT 1.3 kb L CTTCCGGTCAAA-ATTCACTAGGTA 0.6 kb L b CTCCGCTGCAAT-GCTGAGTACGTA No 4 stop codon (ex 4) The NM_ sequence represents transcript variant a and is a reference standard in the NCBI RefSeqGene project. Table 2e. HAMP gene HAMP NM_ next start codon (ex 1) 355 Ж SP ; CCAGAGCAAGCT-27nt spanning oligo 1 L AGACGGCACGAT 0.1 kb 436 Ж SP ; CAGTGGCTCTGT-27nt spanning oligo 1 L nt after ex 1 -GCCTGGGTCCTT 2.2 kb 250 Ж SP ; AACCCCAGGACA-24nt spanning oligo 2 L TGGTGAGCGCAA 0.2 kb L / G71D WT TTTCTGCTGCGG-CTGCTGTCATCG stop codon (ex 3) Ж This consists of three parts and has two ligation sites. Wild type sequence detected. The presence of the G71D mutation will result in a decreased signal. The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Note: numbering might be different as compared to literature! Complete sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA mix P347 Hemochromatosis Page 5 of 7

6 mix P347-A2 Hemochromatosis sample pictures D ye S ign al Size Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with mix P347-A2 Hemochromatosis (lot A2-0213) D ye S ign al Size Figure 2. Capillary electrophoresis pattern of a male patient sample containing a heterozygous C282Y mutation, analysed with mix P347-A2 Hemochromatosis mix (lot A2-0213). The location of the C282Y specific is indicated. SALSA mix P347 Hemochromatosis Page 6 of 7

7 D ye S ign al Size Figure 3. Capillary electrophoresis pattern of a female patient sample containing a homozygous C282Y mutation, analysed with mix P347-A2 Hemochromatosis mix (lot A2-0213). The location of the C282Y specific is indicated. Implemented Changes compared to the previous product description version(s). Version 07 (49) - Figure(s) based on the use of old MLPA buffer (replaced in December 2012) removed. Version 06 (49) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new picture included). Version 05 (48) - Figures 3 and 4 were replaced with the correct pictures of patient samples showing the C282Y mutation. - Arrows indicating C282Y mutation have been corrected. Version 04 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 03 (47) - New sample pictures of patient samples showing the C282Y mutation included. - Information about NM_ sequences added under Table 2. Version 02 (46) - Warning added for the 292 nt, this detects the wild-type sequence of the HAMP G71D mutation. Version 01 (45) - Not applicable, new document. SALSA mix P347 Hemochromatosis Page 7 of 7