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

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1 SALSA MLPA probemix P155-D2 Ehlers-Danlos syndrome III & IV Lot D 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 6p 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

2 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 : 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 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 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 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 probemix P155 EDS Page 2 of 6

3 Table 1. SALSA MLPA P155-D2 EDS type III & IV probemix Length Chromosomal position SALSA MLPA probe (nt) reference TNXB COL3A 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 COL3A1 probe L21415 Exon TNXB probe L04393 Exon COL3A1 probe L04404 Exon TNXB probe L14636 Exon COL3A1 probe L21416 Exon Reference probe L q TNXB probe L15001 Exon COL3A1 probe L04407 Exon CYP21A2 probe L20260 Downstream 190 TNXB probe L22188 Exon COL3A1 probe L04405 Exon Reference probe L q TNXB probe L21386 Exon TNXB probe L21417 Exon COL3A1 probe L21329 Exon COL3A1 probe L28408 Exon CYP21A2 probe L16990 Downstream 238 TNXB probe L04396 Exon Reference probe L q COL3A1 probe L04413 Exon COL3A1 probe L28139 Exon COL3A1 probe L21402 Exon TNXB probe L04397 Exon TNXB probe L01515 Exon Reference probe L q ± TNXB probe L22198 Exon COL3A1 probe L21420 Exon TNXB probe L21389 Exon ATF6B probe L01512 Upstream 349 BAK1 probe L22199 Upstream 358 Reference probe L q COL3A1 probe L04410 Exon COL3A1 probe L21421 Exon ± TNXB probe L14827 Exon TNXB probe L04401 Exon COL3A1 probe L04409 Exon Reference probe L q TNXB probe L15002 Exon COL3A1 probe L21422 Exon TNXB probe L21423 Exon TNXB probe L22201 Exon Reference probe L q TNXB probe L14637 Exon COL3A1 probe L04414 Exon * Reference probe L q31 * New in version D2 (from lot D 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

4 Table 2. P155 probes arranged according to chromosomal location Table 2a. 6p21.33 Length (nt) SALSA MLPA probe TNXB exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe q-telomere L22199 BAK1 GCTTCGTGGTCG-ACTTCATGCTGC kb L01512 ATF6B (CREBL1) GACAACCTGCTT-AGCCCGGAGGAC 18.6 kb start codon (ex 2) L04393 Exon nt before exon 1 CGTGGGAGGTAA-GACCGGGGCTGG 0.3 kb L21389 Exon CCTCCCGGGGTT-GGGGACAGAGCA 11.4 kb L21417 Exon AGGAACAGTGCA-CTGGGGGATGTT 1.1 kb L15001 Exon reverse CCGTGTCGCAAA-TGCATTCGCCGT 7.4 kb L22188 Exon GCCTCAGCCTAT-GACCAGAGAGGA 4.7 kb L04396 Exon CGACTCCTTGCT-CCTGCGCTGGAC 5.5 kb L04397 Exon GTGGGAGGCCTA-GAGCCCGGGCAC 5.2 kb L21386 Exon reverse CCCACAGAGTTG-GGGGTCACATCT 3.6 kb 310 ± L22198 Exon TGTGGTCCAGTT-CAAGGACAAAGA 0.6 kb 382 ± L14827 Exon CAGCCTCAGGGA-GGTCAGCGTGCC 0.8 kb L21423 Exon TGTATGGTTTCA-GTGATGGGAAGC 3.8 kb L22201 Exon GCTCCTCTTGCA-AAGCTGCGCCTA 8.3 kb L04401 Exon GGGAGCTGACCA-TGACAGATGCCA 4.1 kb L15002 Exon reverse GTCACACCCACA-GCGGACACTGGG 6.3 kb L01515 Exon CTCCAGCTCTCT-GCGCCTGTCCTG 2.6 kb L14636 Exon CAGGGGCTGATC-CCAGGCGCTCGC 0.1 kb L14637 Exon reverse GGAAGCCTGTGA-GAGGCTCACTCT 4.0 kb stop codon (ex 44) L16990 CYP21A2 (Exon 6) GGATCACATCGT-GGAGATGCAGCT 0.7 kb 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_ sequence represents transcript variant XB and is a reference standard in the NCBI RefSeqGene project. SALSA probemix P155 EDS Page 4 of 6

5 Table 2b. COL3A1 Length SALSA MLPA COL3A1 Ligation site Partial sequence (24 nt adjacent Distance to (nt) probe exon NM_ to ligation site) next probe start codon (ex 1) L04405 Exon TTGAACTGCTTT-TCTTTTCTCCTT 0.1 kb L04404 Exon CATCCCACTATT-ATTTTGGCACAA 10.3 kb L21329 Exon CGATGACATAAT-ATGTGACGATCA 0.9 kb L21421 Exon CCTGGAATCTGT-GAATCATGCCCT 1.4 kb L04407 Exon CTCGCAGGCTAT-CCTGGACCAGCT 3.0 kb L21420 Exon GTAGACCCGGAC-GACCTGGAGAGC 0.9 kb L21402 Exon reverse GTTTCACCCTTT-TCTCCATTTCGT 1.2 kb L21416 Exon GTGCTCGGGGTA-ATGACGGTGCTC 1.9 kb L04409 Exon 17 4 nt after exon 17 GCGAAATGGTAA-GCTGTCCCCACT 0.7 kb L04410 Exon GGTGTTCCAGGA-GCTAAAGGCGAA 1.4 kb L21415 Exon TACTTCAGGGCA-TGCCCGGAAGTC 2.2 kb L21422 Exon GGTCCTCCAGGA-GAAAATGGAAAA 1.6 kb L28139 Exon GACAGAATGGTG-AACCTGGTGGTA 3.5 kb L04413 Exon 43 4 nt after exon 43 GAGAAAGTGTGA-GTTCCCAAAAGC 1.6 kb L28408 Exon GGTCCCATTGGA-CCACCAGGGCCT 3.6 kb L04414 Exon TGATCAAGAATT-TGGTGTGGACGT stop codon (ex 51) The NM_ 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

6 Implemented Changes compared to the previous product description versions. Version 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 November 2016 (55) - Identity of the last reference probe corrected. - Figure 2 replaced. Version 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 L00945 and 427 nt probe L 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