SALSA MLPA probemix P155-D2 Ehlers-Danlos syndrome III & IV

SALSA MLPA probemix P155-D2 Ehlers-Danlos syndrome III & IV Lot D2-0617. As compared to previous version D1 (lot D1-1114), one reference probe has been replaced. The Ehlers-Danlos syndrome type III (EDS III) is a benign hypermobility syndrome. Marked joint hyperextensibility without skeletal deformity dominates the clinical picture. Skin manifestations are relatively inconspicuous. Differentiation from familial joint laxity is often uncertain. This phenotype can by caused by mutations in either the Collagen type III (COL3A1) gene located on chromosome 2q32.2 or the Tenascin XB (TNXB) gene located on chromosome 6p21.33. Deficiency of Tenascin-X, the product of TNXB, leads to a clinically distinct autosomal recessive form of Ehlers-Danlos syndrome, a connective tissue disorder characterised by hyperextensible skin, hypermobile joints, and tissue fragility. TNXB is closely linked to the HLA locus. Other genes near TNXB include CYP21A2, the complement C4A and C4B genes, CYP21P and the pseudogene TNXA. The sequences of CYP21A2 and CYP21A1P, and those of C4A and C4B, are almost identical. Orientation of genes from the 6p telomere to the centromere is as follows: C4A-CYP21A1P-TNXA-C4B-CYP21A2-TNXB-ATF6B (see Figure 1). TNXA is a small duplicated part of the TNXB gene. Ehlers-Danlos syndrome type IV (EDS IV) is an autosomal dominant disorder characterised by the joint and dermal manifestations as in other forms of the syndrome but also by proneness to spontaneous rupture of bowel and large arteries. The cause of EDS IV is mutations in the COL3A1 gene. Figure 1. A schematic representation of the chromosome 6p21.3 region. Arrows indicate direction of transcription. The TNXB gene (44 exons) spans ~68 kb of genomic DNA and is located on 6p21.33, 32 Mb from the p- telomere. The P155-D2 EDS probemix contains 17 probes for the TNXB gene. Also, 2 probes are included for the region upstream of TNXB, located in the ATF6B (previously called CREBL1) and BAK1 genes. Furthermore, 2 CYP21A2 probes downstream of TNXB are included. The COL3A1 gene (51 exons) spans ~38 kb of genomic DNA and is located on 2q32.2, 190 Mb from the p- telomere. This P155-D2 EDS probemix contains 16 probes for the COL3A1 gene. In addition, 9 reference probes are included in this P155-D2 EDS 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. Note that a mutation or polymorphism (e.g. SNP) 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 SALSA probemix P155 EDS Page 1 of 6

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 Acids Research 30, e57 (2002). More information Website : www.mlpa.com E-mail : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands Related SALSA probemixes P050 CAH: Contains probes for the CYP21A2, CYP21A1P, TNXB, C4A, C4B, and ATF6B (CREBL1) genes. P272 COL1A2: Contains probes for the COL1A2 gene (EDS type VIIA&B). P331/P332 COL5A1: Contain probes for the COL5A1 gene (EDS type I&II). P359 PLOD1: Contains probes for the PLOD1 gene (EDS type VI, Kyphoscoliosis). Data analysis The P155-D2 EDS probemix contains 46 MLPA probes with amplification products between 130 nt and 490 nt. In addition, it contains 9 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at 64-70-76-82 nt, three DNA Denaturation control fragments (Dfragments) at 88-92-96 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 probemix can first be normalised intra-sample by dividing the peak height of each probes amplification product by the total peak 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. Please note that 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 www.mlpa.com. Many copy number alterations in healthy individuals are described in the database of genomic variants: http://dgv.tcag.ca/dgv/app/home. 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 probemix P155 EDS Page 2 of 6

Table 1. SALSA MLPA P155-D2 EDS type III & IV probemix Length Chromosomal position SALSA MLPA probe (nt) reference TNXB COL3A1 64-70-76-82 Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA 88-92-96 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 00797-L00463 5q31 135 COL3A1 probe 17516-L21415 Exon 23 142 TNXB probe 05007-L04393 Exon 1 148 COL3A1 probe 05018-L04404 Exon 1 154 TNXB probe 13291-L14636 Exon 35 161 COL3A1 probe 17517-L21416 Exon 14 166 Reference probe 04111-L03471 9q34 172 TNXB probe 05008-L15001 Exon 3 178 COL3A1 probe 05021-L04407 Exon 5 185 CYP21A2 probe 15221-L20260 Downstream 190 TNXB probe 05009-L22188 Exon 5 196 COL3A1 probe 05019-L04405 Exon 1 202 Reference probe 08187-L08081 11q12 208 TNXB probe 05012-L21386 Exon 12 215 TNXB probe 17518-L21417 Exon 2 221 COL3A1 probe 05020-L21329 Exon 2 226 COL3A1 probe 20544-L28408 Exon 47 232 CYP21A2 probe 15216-L16990 Downstream 238 TNXB probe 05010-L04396 Exon 8 244 Reference probe 11039-L11707 14q31 252 COL3A1 probe 05223-L04413 Exon 43 257 COL3A1 probe 20545-L28139 Exon 36 274 COL3A1 probe 05022-L21402 Exon 11 283 TNXB probe 05011-L04397 Exon 11 292 TNXB probe 01982-L01515 Exon 31 301 Reference probe 10074-L10498 8q22 310 ± TNXB probe 03033-L22198 Exon 14 319 COL3A1 probe 17521-L21420 Exon 9 326 TNXB probe 01980-L21389 Exon 1 337 ATF6B probe 01979-L01512 Upstream 349 BAK1 probe 01994-L22199 Upstream 358 Reference probe 16442-L18895 18q21 364 COL3A1 probe 05024-L04410 Exon 20 372 COL3A1 probe 17522-L21421 Exon 4 382 ± TNXB probe 05013-L14827 Exon 15 391 TNXB probe 05015-L04401 Exon 23 400 COL3A1 probe 05023-L04409 Exon 17 409 Reference probe 12866-L19707 13q14 418 TNXB probe 05016-L15002 Exon 26 427 COL3A1 probe 17523-L21422 Exon 28 436 TNXB probe 17524-L21423 Exon 16 456 TNXB probe 05014-L22201 Exon 19 463 Reference probe 09705-L10085 17q11 472 TNXB probe 13292-L14637 Exon 35 481 COL3A1 probe 05028-L04414 Exon 51 490 * Reference probe 15129-L16900 1q31 * New in version D2 (from lot D2-0617 onwards). ± 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. The 472 nt and 154 nt probes are within the 121 nt sequence that is absent in the TNXA pseudogene. The identity of the genes detected by the reference probes is available on request: info@mlpa.com. SALSA probemix P155 EDS Page 3 of 6

Table 2. P155 probes arranged according to chromosomal location Table 2a. 6p21.33 Length (nt) SALSA MLPA probe TNXB exon Ligation site NM_019105.6 Partial sequence (24 nt adjacent to ligation site) Distance to next probe q-telomere 349 01994-L22199 BAK1 GCTTCGTGGTCG-ACTTCATGCTGC 1445.9 kb 337 01979-L01512 ATF6B (CREBL1) GACAACCTGCTT-AGCCCGGAGGAC 18.6 kb start codon 203-205 (ex 2) 142 05007-L04393 Exon 1 165 nt before exon 1 CGTGGGAGGTAA-GACCGGGGCTGG 0.3 kb 326 01980-L21389 Exon 1 148-149 CCTCCCGGGGTT-GGGGACAGAGCA 11.4 kb 215 17518-L21417 Exon 2 566-567 AGGAACAGTGCA-CTGGGGGATGTT 1.1 kb 172 05008-L15001 Exon 3 1368-1367 reverse CCGTGTCGCAAA-TGCATTCGCCGT 7.4 kb 190 05009-L22188 Exon 5 2617-2618 GCCTCAGCCTAT-GACCAGAGAGGA 4.7 kb 238 05010-L04396 Exon 8 3438-3439 CGACTCCTTGCT-CCTGCGCTGGAC 5.5 kb 283 05011-L04397 Exon 11 4492-4493 GTGGGAGGCCTA-GAGCCCGGGCAC 5.2 kb 208 05012-L21386 Exon 12 4663-4662 reverse CCCACAGAGTTG-GGGGTCACATCT 3.6 kb 310 ± 03033-L22198 Exon 14 5325-5326 TGTGGTCCAGTT-CAAGGACAAAGA 0.6 kb 382 ± 05013-L14827 Exon 15 5685-5686 CAGCCTCAGGGA-GGTCAGCGTGCC 0.8 kb 436 17524-L21423 Exon 16 6059-6060 TGTATGGTTTCA-GTGATGGGAAGC 3.8 kb 456 05014-L22201 Exon 19 6781-6782 GCTCCTCTTGCA-AAGCTGCGCCTA 8.3 kb 391 05015-L04401 Exon 23 8108-8109 GGGAGCTGACCA-TGACAGATGCCA 4.1 kb 418 05016-L15002 Exon 26 9298-9297 reverse GTCACACCCACA-GCGGACACTGGG 6.3 kb 292 01982-L01515 Exon 31 10590-10591 CTCCAGCTCTCT-GCGCCTGTCCTG 2.6 kb 154 13291-L14636 Exon 35 11644-11645 CAGGGGCTGATC-CCAGGCGCTCGC 0.1 kb 472 13292-L14637 Exon 35 11706-11705 reverse GGAAGCCTGTGA-GAGGCTCACTCT 4.0 kb stop codon 12929-12931 (ex 44) 232 15216-L16990 CYP21A2 (Exon 6) GGATCACATCGT-GGAGATGCAGCT 0.7 kb 185 15221-L20260 CYP21A2 (Exon 3) GGAGACTACTCC-CTGCTCTGGAAA p-telomere ± 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. The 472 nt and 154 nt probes are within the 121 nt sequence that is absent in the TNXA pseudogene. The NM_019105.6 sequence represents transcript variant XB and is a reference standard in the NCBI RefSeqGene project. SALSA probemix P155 EDS Page 4 of 6

Table 2b. COL3A1 Length SALSA MLPA COL3A1 Ligation site Partial sequence (24 nt adjacent Distance to (nt) probe exon NM_000090.3 to ligation site) next probe start codon 118-120 (ex 1) 196 05019-L04405 Exon 1 71-72 TTGAACTGCTTT-TCTTTTCTCCTT 0.1 kb 148 05018-L04404 Exon 1 177-178 CATCCCACTATT-ATTTTGGCACAA 10.3 kb 221 05020-L21329 Exon 2 314-315 CGATGACATAAT-ATGTGACGATCA 0.9 kb 372 17522-L21421 Exon 4 540-541 CCTGGAATCTGT-GAATCATGCCCT 1.4 kb 178 05021-L04407 Exon 5 633-634 CTCGCAGGCTAT-CCTGGACCAGCT 3.0 kb 319 17521-L21420 Exon 9 829-830 GTAGACCCGGAC-GACCTGGAGAGC 0.9 kb 274 05022-L21402 Exon 11 939-938 reverse GTTTCACCCTTT-TCTCCATTTCGT 1.2 kb 161 17517-L21416 Exon 14 1081-1082 GTGCTCGGGGTA-ATGACGGTGCTC 1.9 kb 400 05023-L04409 Exon 17 4 nt after exon 17 GCGAAATGGTAA-GCTGTCCCCACT 0.7 kb 364 05024-L04410 Exon 20 1494-1495 GGTGTTCCAGGA-GCTAAAGGCGAA 1.4 kb 135 17516-L21415 Exon 23 1729-1730 TACTTCAGGGCA-TGCCCGGAAGTC 2.2 kb 427 17523-L21422 Exon 28 2070-2071 GGTCCTCCAGGA-GAAAATGGAAAA 1.6 kb 257 20545-L28139 Exon 36 2575-2576 GACAGAATGGTG-AACCTGGTGGTA 3.5 kb 252 05223-L04413 Exon 43 4 nt after exon 43 GAGAAAGTGTGA-GTTCCCAAAAGC 1.6 kb 226 20544-L28408 Exon 47 3600-3601 GGTCCCATTGGA-CCACCAGGGCCT 3.6 kb 481 05028-L04414 Exon 51 4484-4485 TGATCAAGAATT-TGGTGTGGACGT stop codon 4516-4518 (ex 51) The NM_000090.3 sequence is a reference standard in the NCBI RefSeqGene project. Note: Exon numbering may differ compared to literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA MLPA probemix P155-D2 EDS sample picture Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P155-D2 EDS (lot D2-0617). SALSA probemix P155 EDS Page 5 of 6

Implemented Changes compared to the previous product description versions. Version 14 28 August 2017 (55) - Product description adapted to a new product version (version number changed, 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. - Minor textual changes. Version 13-04 November 2016 (55) - Identity of the last reference probe corrected. - Figure 2 replaced. Version 12-20 April 2015 (54) - Product description adapted to a new product version (version number changed, lot number added, new picture included). - Figure 1 replaced. Version 11 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 10 (48) - Product description adapted to a new product version (version number changed, lot number added, new picture included). - Various textual changes on page 1 and 2. - Various layout changes. - Changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. Version 09 (47) - Ligation sites of the probes targeting the TNXB and COL3A1 gene updated according to new version of the NM_reference sequence. - Remark on RefSeqGene standard and transcript variant added below Table 2. Version 08 (46) - Various minor textual changes on page 1. - Various minor layout changes. - Warnings added in Table 1 and 2, 184 nt probe 01435-L00945 and 427 nt probe 05224-L14828. - Warning added about gene name change: CREBL1 to ATF6B. - Data analysis method has been modified. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. SALSA probemix P155 EDS Page 6 of 6