SALSA MLPA probemix P207-C3 F9 Lot C As compared to version C three reference probes have been removed.

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1 SALSA MLPA probemix P207-C3 F9 Lot C As compared to version C three reference probes have been removed. Factor IX deficiency (also called Hemophilia B or Christmas disease) is characterized by a recessive X-linked bleeding disorder. Defects in the F9 gene on chromosome X are one of the main causes of Factor IX deficiency. This gene encodes a vitamin K-dependent serine protease that plays an important function in the blood coagulation cascade. The F9 gene (8 exons) spans ~33 kb of genomic DNA and is located on Xq27.1, ~139Mb from the p- telomere. The P207-C3 probemix contains one probe for each exon of the F9 gene and two probes for exon 8. Furthermore, this probemix contains several probes for the F7 gene and the F8 gene. The F7 gene (9 exons) spans ~15 kb of genomic DNA and is located on 13q34, ~114 Mb from the p-telomere. This probemix contains one or more probes for each exon of the F7 gene with the exception of exon 3. The F8 gene (26 exons) spans ~187 kb of genomic DNA and is located on Xq28, ~154 Mb from the p-telomere. There are 7 probes for F8 included in the probemix. In addition, 13 reference probes are included in this probemix, detecting several different autosomal chromosomal locations. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned genes in a DNA sample. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak height of the amplification product of that probe. Deletions of a probe s recognition sequence on the X-chromosome will lead to a complete absence of the corresponding probe amplification product in males, whereas female heterozygotes are recognisable by a 35-50% reduction in relative peak height. 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. 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 test. 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). Related SALSA probemixes P178 F8: Contains probes for the F8 gene, involved in Hemophilia A. P011 VWF-1 and P012 VWF-2: Contain probes for the vwf gene, involved in von Willibrand factor deficiency. P440 F10 + F11: Contains probes for the F10 and the F11 gene, involved in bleeding disorders. More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : Bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands SALSA P207 F9 probemix Page 1 of 7

2 References Pavlova A. et al. (2015) Congenital combined deficiency of coagulation factors VII and X Different genetics mechanism. Haemophilia. DOI: /hae Payne A.B. et al. (2012) Utility of multiplex ligation-dependent probe amplification (MLPA) for haemophilia mutation screening. Journal of Thrombosis and Haemostasis, 10: Casaña, P. et al. (2009) Identification of deletion carriers in hemophilia B: quantitative real-time polymerase chain reaction or multiple ligation probe amplification. Transl Res. 153(3): Kwon, M.J. (2008) Identification of mutations in the F9 gene including exon deletion by multiplex ligationdependent probe amplification in 33 unrelated Korean patients with haemophilia B. Haemophilia. 14(5): Data analysis The P207-C3 F9 probemix contains 39 MLPA probes with amplification products between 130 and 454 nt. In addition, it contains 10 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 two Y-fragments at 105 nt and 118 nt. More information on how to interpret observations on these control fragments can be found in the MLPA protocol. Data generated by this probemix can be normalised intra-sample by dividing the peak height of each amplification product by the total peak height of only the reference probes in the 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. Please note that this type of normalisation assumes that no changes occurred in the genomic regions targeted by the reference probes. It is strongly recommended to use reference and patient samples of the same sex to minimize variation, as intersex comparison makes analysis more difficult. Sex determination can also be done by visual examination of the electropherogram. 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 Many copy number alterations in healthy individuals are described in the database of genomic variants: For example, a duplication of a complete gene might not be pathogenic, while a partial duplication or a deletion may result in disease. For some genes, certain in-frame deletions may result in a very mild, or no disease. Copy number changes of reference probes are unlikely to be the cause of the condition tested for. Users should always verify the latest scientific literature when interpreting their findings. This probemix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P207 F9 probemix Page 2 of 7

3 Table 1. SALSA MLPA P207-C3 F9 probemix Length (nt) SALSA MLPA probe Chromosomal position reference F7 F8 F 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 118 Y-fragment: Specific for the Y chromosome 130 Reference probe L p F9 probe L06388 Exon F8 probe L05060 Exon F8 probe L05068 Exon F9 probe L06904 Exon Reference probe L q F9 probe L06389 Exon F8 probe L06057 Exon Reference probe L p Reference probe L p F9 probe L28016 Exon F8 probe L06927 Exon F9 probe L06383 Exon Reference probe L q F9 probe L06381 Exon Reference probe L q ٨ F8 probe L05067 Exon Reference probe L q F9 probe L15258 Exon Reference probe L q F9 probe L06385 Exon Reference probe L q F9 probe L06384 Exon F8 probe L15887 Exon F8 probe L15888 Exon Reference probe L q F7 probe L14965 Exon 4 (4a) 355 F7 probe L14966 Exon 9 (8) 364 F7 probe L14967 Exon F7 probe L14968 Exon 8 (7) 391 F7 probe L14969 Exon 5 (4c) 402 F7 probe L01297 Exon 6 (5) 409 Reference probe L q F7 probe L14970 Exon Reference probe L p ± F7 probe L14971 Exon 7 (6) 445 F7 probe L14972 Exon 8 (7) 454 Reference probe L q22 ٨ SNP rs could influence the probe signal. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. ± This probe is located within, or close to, a very strong CpG island. A low signal of this probe can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. Changed in version C3 (from lot C onwards). Small change in length, no change in sequence detected. Note I: The exon numbering has changed for the F7 gene. From description version 10 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequence for this gene. The exon numbering used in previous versions of this product description can be found between brackets in Table 1 and 2. Note II: Exon numbering used here may differ from literature! Please notify us of any mistakes. The identity of the genes detected by the reference probes is available on request: info@mlpa.com. SALSA P207 F9 probemix Page 3 of 7

4 Table 2. P207 probes arranged according to chromosomal location Table 2a. F7 Length (nt) SALSA MLPA probe F7 exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 1) L14967 exon nt after exon 1 GCTCCTGTTCAA-TTTCTTTCCTTC 0.8 kb L14970 exon TAAGGCCTCAGG-AGGAGAAACACG 6.9 kb no probe exon L14965 exon 4 (4a) TTCTGGATTTCT-TACAGTGGTGAG 0.2 kb L14969 exon 5 (4c) 12 nt after exon 5 TAAGGCCCCACT-TTGGGTCCCATA 1.7 kb L01297 exon 6 (5) ATCTGTGTGAAC-GAGAACGGCGGC 1.2 kb 436 ± L14971 exon 7 (6) 2 nt after exon 7 CCATGGCAGGTA-AGGCTTCCCCTG 0.6 kb L14968 exon 8 (7) TTGGTGAATGGA-GCTCAGTTGTGT 0.1 kb L14972 exon 8 (7) TTCGACAAAATC-AAGAACTGGAGG 1.4 kb L14966 exon 9 (8) CCACGCCCAGGA-GTCCTCCTGCGA stop codon (ex 9) ± This probe is located within, or close to, a very strong CpG island. A low signal of this probe can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. Note: The exon numbering has changed for the F7 gene. From description version 10 (C3-1214) onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequence for this gene. The exon numbering used in previous versions of this product description can be found between brackets in Table 1 and 2. NM_ represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2b. F8 Length (nt) SALSA MLPA probe F8 exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 1) L05060 exon GCACATCCAGTG-GGTAAAGTTCCT 25.7 kb L15887 exon reverse TTTCCAGTAGGA-TACACCAACAGC 27.5 kb 247 ٨ L05067 exon CACAGGAAATCA-GTCTATTGGCAT 0.2 kb L05068 exon CACTCTTGATGG-ACCTTGGACAGT 3.4 kb L06927 exon CAGCATGAATCA-GGAATCTTGGGA 35.6 kb L06057 exon ATGGAAAGAACT-CTCTGAACTCTG 67.2 kb L15888 exon 23 (24) CTGGGATAAAAC-ACAATATTTTTA stop codon (ex 26) ٨ SNP rs could influence the probe signal. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. Notes: The F8 exon numbering has changed. From description version 11 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for this gene. This exon numbering used here may differ from literature! The exon numbering used in previous versions of this product description can be found between brackets in Table 2. The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA P207 F9 probemix Page 4 of 7

5 Table 2c. F9 Length (nt) SALSA MLPA probe F9 exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 1) L06381 exon nt after exon 1 AGGTTGGTAAGT-ACTGGTTCTTTG 6.1 kb L15258 exon TAAATTGGAAGA-GTTTGTTCAAGG 0.3 kb L06383 exon 3 3 nt after exon 3 GTATGTTGGTAA-GCAATTCATTTT 3.7 kb L06384 exon ATCAGTGTGAGT-CCAATCCATGTT 7.3 kb L06385 exon AGTTTTGTAAAA-ATAGTGCTGATA 2.8 kb L28016 exon GGTGGAGAAGAT-GCCAAACCAGGT 9.5 kb L06904 exon AGTTGATGCATT-CTGTGGAGGCTC 0.8 kb L06388 exon TGCAGCTATTAA-TAAGTACAACCA 0.3 kb L06389 exon GGAGGTAGAGAT-TCATGTCAAGGA stop codon (ex 8) Changed in version C3 (from lot C onwards). Small change in length, no change in sequence detected. Note: The NM_ sequence is a reference standard in the NCBI RefSeqGene project. The exon numbering used here may differ from literature! Complete probe sequences are available on request: info@mlpa.com. SALSA P207 F9 probemix Page 5 of 7

6 SALSA MLPA probemix P207-C3 F9 sample pictures Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P207-C3 F9 (lot C3-1214). Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng human female control DNA analysed with SALSA MLPA probemix P207-C3 F9 (lot C3-1214). SALSA P207 F9 probemix Page 6 of 7

7 Implemented Changes compared to the previous product description versions. Version February 2016 (55) - F8 exon numbering adjusted. - Sample pictures adjusted; probe labels changed. Version March 2015 (54) - Product description adapted to a new product version (version number changed, lot number changed, changes in table 1). - Electropherogram pictures using the old MLPA buffer (replaced in December 2012) removed. Version 09 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 08 (48) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - New references added on page 1. - Exon numbering of the F7 gene has been changed in Table 1 and Table 2. - Ligation sites of the probes targeting the F7 gene updated according to new version of the NM_reference sequence. Version 07 (46) - Remark on RefSeqGene standard and transcript variant added below Table 2. - Small correction of chromosomal locations in Table 1 and 2. - Tables have been numbered. Version 06 (46) - Various minor textual changes on page 1. - Various minor layout changes. - Data analysis method has been modified. - Sentence when only small numbers of samples are tested, visual comparison of peak profiles should be sufficient removed from data analysis section. Version 05 - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new picture included). - Various textual changes on page 1. - New related SALSA MLPA kits added on page 1. - Tables have been numbered. - Table of chromosomal location for F7 and F8 gene have been added. - Ligation sites of the F9 probes adjusted to the latest NM_sequence. Version 04 - A new paragraph added containing information about product changes compared to the previous lot. - Product description adapted to a new product version (version number changed, lot number added, change in Table 1 and Table 2, new picture included). - Various textual changes on page 1. - Minor textual change in tables. - Use of symbols in first table indicating presence of the new probes. Version 03 - Change in lay-out of page 1. - Various textual changes on page 1. - Related SALSA MLPA-paragraph has been added on page 1. - Control Fragments- paragraph has been deleted on page 2. - Data analysis paragraph When only a small.. has been changed. - Change lay-out first table. - Minor textual change in first table. Version 02 - Data analysis- Coffalyser paragraph has been added. Version 01 - Not applicable, new document. SALSA P207 F9 probemix Page 7 of 7