In addition, 10 reference probes are included in this probemix, detecting several different autosomal chromosomal locations.

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1 SALSA MLPA probemix P179-B1 Limb Malformations-1 Lot B1-1014, B As compared to the previous lot A2 (lot A2-0311), one GLI3 and three ROR2 probes have been replaced. Four extra GLI3 probes and one extra ROR2 probe have been included. Defects in the GLI3, HOXD13 and ROR2 genes can cause limb malformations. Mutations in GLI3 cause a wide variety of phenotypes, including Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome. Some cases of GCPS are caused by large deletions that include the GLI3 gene. The GLI3 gene (15 exons) spans ~276 kb of genomic DNA and is located on chromosome 7p14.1, ~42 Mb from the p- telomere. The P179-B1 probemix contains probes for each exon of the GLI3 gene. Two probes are present for exon 1, 2, 3, 4, 5 and 10. HOXD13 is a transcription factor involved in distal limb patterning. Four types of HOXD13 mutations are associated with distinct phenotypes. Expansions in the amino-terminal polyalanine tract cause synpolydactyly (SPD), specific missense mutations cause brachydactyly type E, intragenic deletions or other missense mutations cause SPD with an additional foot phenotype, while a splice site mutation has been reported to cause only foot malformation. The HOXD13 gene (2 exons) spans ~3 kb of genomic DNA and is located on chromosome 2q31.1, ~176 Mb from the p-telomere. The two exons are separated by an 808 bp intron. The P179-B1 probemix contains one probe for each exon. The ROR2 gene encodes a transmembrane receptor tyrosine kinase, which is particularly important for the chondrocyte lineage. Mutations in ROR2 have been shown to result in brachydactyly type B, and in autosomal recessive Robinow syndrome. The latter has also been associated with a deletion. The ROR2 gene (9 exons) spans ~228 kb of genomic DNA and is located on chromosome 9q22.31, 92 Mb from the p- telomere. Exon 1 is separated from the rest of the gene by a 174 kb intron. The P179-B1 probemix contains two probes for exon 1 and one probe for each of the other exons. In addition, 10 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. 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 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 1, 1057 DL Amsterdam, the Netherlands SALSA probemix P179 Limb Malformations-1 Page 1 of 7

2 Related SALSA probemixes P180-B1 Limb-2 / Heart: Limb Malformations-2. Genes included: SALL1, SALL4 and TBX5. References for SALSA probemix P179 Limb Malformations-1 Demurger et al., New insights into genotype phenotype correlation for GLI3 mutations. Eur J Hum Genet. 23: Bednarczyk et al., Normal exon copy number of the GLI2 and GLI3 genes in patients with esophageal atresia. Dis Esophagus. 26: Jamsheer et al., Isolated brachydactyly type E caused by a HOXD13 nonsense mutation: a case report. BMC Med Genet. 13:4. Martinez-Garcia M et al., Holt-Oram syndrome: study of 7 cases. Med Clin (Barc) 135: Furniss D et al., Genetic screening of 202 individuals with congenital limb malformations and requiring reconstructive surgery. J Med Genet. 46: Data analysis The P179-B1 Limb-1 probemix contains 43 MLPA probes with amplification products between 130 and 463 nt. In addition, it contains nine 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 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 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. 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 results 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 disease. Users should always verify the latest scientific literature when interpreting their findings. This probemix was developed by D. Furniss at Oxford University and J.P. Schouten at. In case the results obtained with this probemix lead to a scientific publication, it would be very much appreciated if the first probemix designer could be made a co-author. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix P179 Limb Malformations-1 Page 2 of 7

3 Table 1. SALSA MLPA P179-B1 Limb-1 probemix Chromosomal position SALSA MLPA probe reference GLI3 HOXD13 ROR 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 ± GLI3 probe L05451 Exon ± ROR2 probe L05452 Exon ± HOXD13 probe L05005 Exon Reference probe L q GLI3 probe L05453 Exon ROR2 probe L05009 Exon ± HOXD13 probe L05454 Exon * Reference probe L q * ROR2 probe L19859 Exon GLI3 probe L04992 Exon * GLI3 probe L19860 Exon GLI3 probe L20251 Exon GLI3 probe L04995 Exon * ROR2 probe L19861 Exon * GLI3 probe L19862 Exon * Reference probe L q GLI3 probe L04997 Exon ROR2 probe L05012 Exon GLI3 probe L04998 Exon * Reference probe L q * GLI3 probe L19863 Exon GLI3 probe L20252 Exon ROR2 probe L20253 Exon GLI3 probe L05000 Exon Reference probe L q GLI3 probe L05001 Exon ROR2 probe L05014 Exon GLI3 probe L05002 Exon ± Reference probe L p GLI3 probe L05003 Exon ROR2 probe L05015 Exon GLI3 probe L05004 Exon * Reference probe L q ± GLI3 probe L04988 Exon * ROR2 probe L19864 Exon GLI3 probe L04991 Exon Reference probe L p GLI3 probe L04993 Exon ± * ROR2 probe L19865 Exon ± * GLI3 probe L19867 Exon ± * GLI3 probe L20249 Exon * Reference probe L q11 * New in version B1 (from lot B onwards). Changed in version B1 (from lot B onwards). Small change in length, no change in sequence detected. Note: Exon numbering used here may differ from literature! The identity of the genes detected by the reference probes is available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA probemix P179 Limb Malformations-1 Page 3 of 7

4 Table 2. P179-B1 probes arranged according to chromosomal location Table 2a. GLI3 probes SALSA MLPA probe GLI3 Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe Start Codon (exon 2) 453 ± L20249 Exon 1 4 nt after exon 1, reverse TTCGAGCGGGAC-GTACCTGCGGCG 0.2 kb 445 ± L19867 Exon nt after exon 1 GCCCAGATTTAG-AGAGCCGCCGGT 13.6 kb 391 ± L04988 Exon GAGAGCTGAAGT-AATGAGAAGACA 0.1 kb 137 ± L05451 Exon TCCACTCGAACA-GATGTGAGCGAG 74.7 kb L04991 Exon CCTGGACAGACT-TATCACAGAGAG 0.1 kb L05453 Exon GGGGCTCAGCAA-AGTCAGTGAGGA 71.5 kb L04993 Exon reverse GATGAGGAGGGT-CTGAAAAGAAGA 0.1 kb L04992 Exon CTCCTGTACCAA-TTGATGCCAGAC 28.2 kb L20251 Exon CCTGCCCTTCAT-TAGGATCTCCCC 0.1 kb L19860 Exon ACATGGACTATA-TCCGCTCCTTGC 3.1 kb L04995 Exon AGCCCAGCAGAA-TACTATCATCAG 5.4 kb L19862 Exon reverse CTTGCAGATAAG-TGACCATAGGAG 13.8 kb L04997 Exon CTGCCCCAACTT-TTCCAACACAGA 1.0 kb L04998 Exon AGAGGTCCAAGA-TCAAACCCGATG 1.7 kb L20252 Exon CAACCCTTGTCA-AGGAGGAAGGGG 0.1 kb L19863 Exon reverse CTTACGTGCACA-AGCTGCTCTTGG 44.8 kb L05000 Exon CTGGACTGCTCA-AGAGAGCAGAAA 1.1 kb L05001 Exon AAGGCTTTCTCA-AATGCCTCTGAT 5.1 kb L05002 Exon GCAGCCATTCAC-AGTCCAGGTCGC 4.6 kb L05003 Exon GGTCAGTCTTCA-TGCAGCAGCCAA 2.1 kb L05004 Exon TGCAGTATTTAA-ATTCCCAGAACC Stop Codon (exon 15) The NCBI NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2b. HOXD13 probes SALSA MLPA probe HOXD13 Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe Start Codon (exon 1) 148 ± L05005 Exon AGGTATCCTTCT-ACCAGGGCTATA 1.1 kb 172 ± L05454 Exon GAGTATGCCATT-AACAAATTCATT Stop Codon (exon 2) The NCBI NM_ sequence is a reference standard in the NCBI RefSeqGene project. Note: Exon numbering used here may differ from literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA probemix P179 Limb Malformations-1 Page 4 of 7

5 Table 2c. ROR2 probes SALSA MLPA probe ROR2 Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe Start Codon (exon 1) 142 ± L05452 Exon GAGGTCCTCGAA-GTGGACCCGTTT 0.2 kb 433 ± L19865 Exon 1 2 nt after exon 1 CGGACTTCAGGT-AGGATCTGGCGT kb L05009 Exon GAACGACCCTTT-AGGACCCCTTGA 18.5 kb L19859 Exon GGATGAAGACCA-TTACCGCCACTG 1.3 kb L19861 Exon 4 59 nt after exon 4 TTGTTATGTAGG-AATCCGGGGTTT 18.6 kb L05012 Exon AACCGGACCATT-TATGTGGACTCG 4.1 kb L20253 Exon ACCAGTGCTCAC-AGTTCGCCATCC 2.3 kb L05014 Exon CAGGCATGGATT-ACAGAGGAACGG 4.4 kb L05015 Exon TGGGGATTCTGT-ACATCTTGGTCC 2.1 kb L19864 Exon ATGTGCTAGTGT-ACGACAAGCTGA Stop Codon (exon 9) The NCBI NM_ sequence is a reference standard in the NCBI RefSeqGene project. Note: Exon numbering used here may differ from literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA probemix P179 Limb Malformations-1 Page 5 of 7

6 SALSA MLPA probemix P179-B1 Limb-1 sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P179-B1 Limb-1 (lot B1-1014). SALSA probemix P179 Limb Malformations-1 Page 6 of 7

7 Implemented Changes compared to the previous product description version(s). Version 15 (55) 27 November Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Various textual and layout changes. - References updated. - Manufacturer s address adjusted. Version 14 (54) 15 July Figure based on the use of old MLPA buffer (replaced in December 2012) removed. Version 13 (48) - Warning added in Table 1, 346 nt probe L Version 12 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 11 (48) - Product description adapted to standard version 48. Version 10 (46) - 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 minor textual changes on p Remark on RefSeqGene standard added below Table 2. Version 09 (46) - Chromosomal location of reference probe L01642 updated in Table 1. Version 08 (46) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). Version 07 (46) - Exon numbering of the GLI3 gene has been changed in Tables 1 and 2. - Ligation sites of GLI3 and HOXD13 updated according to new version of the NM_reference sequence. - Various minor layout changes. - New reference added on page 1. Version 06 (45) - Various minor textual changes on page 1. - Minor changes in the data analysis section on page 2. Sentence when only small numbers of samples are tested, visual comparison of peak profiles should be sufficient removed from data analysis section. - Various minor layout changes in Table 1 and 2, tables have been numbered. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. SALSA probemix P179 Limb Malformations-1 Page 7 of 7