MRC-Holland MLPA. Description version 17;

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1 SALSA MLPA probemix P080-C1 CRANIOFACIAL Lot C As compared to version B1 (lot B1-0710, B1-0909) one of two ALX4 exon 2 probes is removed, the two FGFR2 and FGFR3 probes at the location of the APERT mutation have been redesigned and several reference probes have been replaced. Furthermore, control fragments have been adjusted (QDX2). CRANIOFACIAL DISORDERS can be caused by alterations in many different genes. This P080-C1 probemix contains probes for the FGFR1, FGFR2, FGFR3, TWIST1, MSX2, ALX1, ALX3, ALX4, EFNB1 and RUNX2 genes. The FGFR1 (8p11.23; 18 exons), FGFR2 (10q26.13; 21 exons) and FGFR3 (4p16.3; 19 exons) genes encode fibroblast growth factor receptors and cause a diverse group of skeletal disorders. In general, mutations in FGFR1 and FGFR2 cause most craniosynostosis. The dwarfing syndromes are often associated with FGFR3 mutations. Deletion of the TWIST1 gene (7p21.1; 2 exons) is the cause of disease in an estimated 11 % of Saethre- Chotzen syndrome patients. Included is also a probe for the TWISTNB (TWIST nearby) gene located at a distance of ~500 kb from TWIST. Large deletions of the TWIST region often result in mental retardation. Dosage of the MSX2 gene (5q35.2; 2 exons) is critical for human skull development. Enlarged parietal foramina and craniosynostosis can result, respectively, from loss and gain of activity in an MSX2 pathway of calvarial osteogenic differentiation. Mutations in ALX4 (11p11.2; 4 exons) can result in parietal foramina as well as craniosynostosis (premature fusion of the cranial sutures). Potocki-Shaffer syndrome, also known as the proximal 11p deletion syndrome, is a contiguous gene syndrome caused by deletion of this 11p13-p11 region. Mutations in the ALX3 gene (1p13.3; 4 exons) can result in frontonasal dysplasia. The ALX1 gene (CART1; 12q21.31; 4 exons) is known to be essential for normal skull bone development, null mice are born with severe craniofacial defects such as a lacking cranium. Defects in the RUNX2 gene (6p21.1; 9 exons) cause the dominant disorder cleidocranial dysplasia. Loss-of-function mutations in the EFNB1 gene (Xq13.1; 5 exons) cause craniofrontonasal syndrome. In addition, nine reference probes are included, 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 on autosomal chromosomes 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 P310 TCOF1: Contains probes for TCOF1, involved in Treacher Collins-Franceschetti 1. 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 P080 Craniofacial Page 1 of 8

2 References of SALSA probemix P080 Jehee, F.S. et al. (2008). High frequency of submicroscopic chromosomal imbalances in patients with syndromic craniosynostosis detected by a combined approach of microsatellite segregation analysis, multiplex ligation-dependent probe amplification and array-based comparative genome hybridisation. J Med Genet. 45: Babbs, C. et al. (2011). Duplication of the EFNB1 Gene in Familial Hypertelorism: Impalance in Ephrin-B1 Expression and Abnormal Phenotypes in Humans and Mice. Hum Mutat. 32: Data analysis The P080-C1 Craniofacial probemix contains 48 MLPA probes with amplification products between 122 and 503 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 (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 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. 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 blots or long range PCR. 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 by R. Vijzelaar at. In case the results obtained with this probemix lead to a scientific publication, it would be very much appreciated if the probemix designer could be made a co-author. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix P080 Craniofacial Page 2 of 8

3 Table 1. SALSA MLPA P080-C1 Craniofacial probemix Length (nt) SALSA MLPA probe Chromosomal position reference ALX1 ALX3 ALX4 EFNB1 RUNX2 TWIST1 Other 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 122 Reference probe L q EFNB1 probe L ALX3 probe L RUNX2 probe L * Reference probe L q RUNX2 probe L ± RUNX2 probe L a 166 ALX3 probe L MSX2 probe L16117 MSX2 ex FGFR1 probe L13122 FGFR1 ex FGFR2 probe L16118 FGFR2 ex 11b 193 ± TWIST1 probe L ALX4 probe L FGFR3 probe L16417 FGFR3 ex ALX1 probe L RUNX2 probe L ALX4 probe L Reference probe L p TWISTNB probe L16629 TWISTNB ex RUNX2 probe L * FGFR2 probe L24455 FGFR2 ex ALX3 probe L Reference probe L p RUNX2 probe L EFNB1 probe L ALX1 probe L RUNX2 probe L ± TWIST1 probe L RUNX2 probe L EFNB1 probe L ± TWIST1 probe L * FGFR3 probe L24464 FGFR3 ex * Reference probe L q ± TWIST1 probe L ALX3 probe L ALX4 probe L ALX1 probe L FGFR1 probe L24470 FGFR1 ex Reference probe L q ALX1 probe L Reference probe L q MSX2 probe L16131 MSX2 ex ALX1 probe L * Reference probe L q EFNB1 probe L ± RUNX2 probe L a 478 EFNB1 probe L * Reference probe L p12 * New in version C1 (from lot C onwards). Changed in version C1 (from lot C onwards). Small change in length, no change in sequence detected. ± 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: numbering might be different as compared to 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 probemix P080 Craniofacial Page 3 of 8

4 Table 2. P080 probes arranged according to chromosomal location Table 2a. ALX3 gene NM_ adjacent to ligation site) next probe start codon (ex 1) L ACTGCGCGCCTT-TCCGCGTGGGGC 5.7 kb L AGCTGAGGAGAA-GACCTCCAAAGC 3.4 kb L AGCGTTATGGGA-AGATCCAGGAGG 0.7 kb L GATGGTGACTAT-AAGTCTCCAAGC stop codon (ex 4) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2b. FGFR3 gene NM_ adjacent to ligation site) next probe start codon (ex 2) L CTGGTCATGGAA-AGCGTGGTGCCC 0.2 kb 334 * L AGAGCGCTCCCC-GCACCGGCCCAT stop codon (ex 19) The 334 nt probe detects the wild type sequence at the site of the C749>G mutation. A 50% reduced signal is expected when this exon is deleted or when samples contain one allele of this mutation. The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2c. MSX2 gene NM_ adjacent to ligation site) next probe start codon (ex 1) L nt after exon 1 CGCCGCCGCCAA-GTGAGTGCGCGC 4.0 kb L nt before exon 2 GGGAGGCCCGAA-AGGAAAAAACCT stop codon (ex 2) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2d. RUNX2 gene NM_ adjacent to ligation site) next probe start codon (ex 2) L CAAACAGCCTCT-TCAGCACAGTGA 93.6 kb ; 3a 470 ± L24474 NM_ GTGTTCCAAAGA-CTCCGGCAAAGA 0.3 kb ; 3a 160 ± L16738 NM_ GTTGTGATGCGT-ATTCCTGTAGAT 9.3 kb L ATGTAGGTGGTA-GCCCTCGGAGAG 6.1 kb L CACCTTGACCAT-AACCGTCTTCAC 54.1 kb L GTCCCGCCTCAG-AACCCACGGCCC 20.3 kb L reverse GACGGGGACGTC-ATCTGGCTCAGG 33.0 kb L reverse CTGGCTCTTCTT-ACTGAGAGTGGA 1.9 kb L TCCAGAATGCTT-CCGCCATGCACC stop codon (ex 9) 3a is not present in the NM_ transcript variant. The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. SALSA probemix P080 Craniofacial Page 4 of 8

5 Table 2e. TWIST1 gene Length (nt) SALSA MLPA probe L16629 TWISTNB 4 Ligation site NM_ NM_ Partial sequence (24 nt adjacent to ligation site) AGCTAGCAGATG-ATGCAGATGACA Distance to next probe kb start codon (ex 1) 353 ± L reverse CTTGCCGCGCTT-GCCCTGGGCCGG 0.3 kb 319 ± L ACGAGCTGGACT-CCAAGATGGCAA 0.7 kb 295 ± L ATTGTTTCCAGA-GAAGGAGAAAAT 0.3 kb 193 ± L TCGTGCCAATCA-GCCACTGAAAGG stop codon (ex 1) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2f. FGFR1 gene NM_ adjacent to ligation site) next probe start codon (ex 4) L (2) CAACCTCTAACT-GCAGAACTGGGA 29.5 kb L (5) CAAATGCCCTTC-CAGTGGGACCCC stop codon (ex 21) The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2g. FGFR2 gene NM_ adjacent to ligation site) next probe start codon (exon 3b) 249 * L ACCAGAGCGATC-GCCTCACCGGCC 4.9 kb L b TGTATGGTGGTA-ACAGTCATCCTG stop codon (ex 21a) The 249 nt probe detects the wildtype sequence at the site of the C755>G mutation. A 50% reduced signal is expected when this exon is deleted or when samples contain one allele of this mutation. The exon number was erroneously mentioned as exon 7 in previous product descriptions. The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2h. ALX4 gene NM_ adjacent to ligation site) next probe start codon (ex 1) L nt after exon 2 AGTGAGGCTGGT-AAAGCAGAGCCT 7.7 kb L GCGGGAGCGTTT-TGGGCAGATGCA 2.6 kb L GCCGGACCGCAA-GACCTCGAGCAT stop codon (ex 4) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. SALSA probemix P080 Craniofacial Page 5 of 8

6 Table 2i. ALX1 gene NM_ adjacent to ligation site) next probe start codon 5-7 (ex 1) L GTTTGCCCTCAA-GAGCCCTCCGAG 0.1 kb L GTCTGCAGGCAA-ATGCGTGCAGGC 3.2 kb L ACTATGGGATCA-CTAAAGTAGAAG 3.3 kb L AAATGGAGAAAA-AGGGAACGTTAT 14.4 kb L ATGACACCTTAT-TCTCACTCGCCT stop codon (ex 4) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2j. EFNB1 gene NM_ adjacent to ligation site) next probe start codon (ex 1) L CCGAAGTGCAGT-CTGCCCCCGGGA 9.1 kb L AGCACCATGATT-ACTACATTACCT 0.8 kb L TTCCTGCAGCAA-CATCCAATGGAA 0.3 kb L AGCTGACTACCA-GCAGGCCCAGCA 0.4 kb L CCTACTACTGAA-GCTACGCAAGCG stop codon (ex 5) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. For all Tables 2: * New in version C1 (from lot C onwards). Changed in version C1 (from lot C onwards). Small change in length, no change in sequence detected. ± 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 FGFR1 exon numbering has changed. From description version 13 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for the FGFR1 gene. The exon numbering used in previous versions of this product description can be found between brackets in Table 2. numbering might be different as compared to literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA probemix P080 Craniofacial Page 6 of 8

7 SALSA MLPA probemix P080-C1 Craniofacial sample pictures D ye S ign al Size (nt) Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA probemix P080-C1 Craniofacial (lot C1-1213) D ye S ign al Size (nt) Figure 2. Capillary electrophoresis pattern from a sample of approximately 50 ng human female control DNA analysed with SALSA probemix P080-C1 Craniofacial (lot C1-1213). SALSA probemix P080 Craniofacial Page 7 of 8

8 Implemented Changes compared to the previous product description version(s). Version 17 (53) - This product description has been changed to incorporate a new product version (lot number added, new pictures included). - Table 2 has been arranged according to chromosomal location. Version 16 (49) - Warning added in Table 1, 335 nt probe L Version 15 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 14 (48) - References adapted. Version 13 (48) - Ligation sites of the probes targeting the MSX2 and RUNX2 genes updated according to new version of the NM_reference sequence. - Various minor textual changes. - Remark on RefSeqGene standard and transcript variant added below Table 2. - Warning added below Table 2 that the exon numbering used is different from the NCBI exon numbering in the NM_ reference sequence. Version 12 (45) - This product description has been changed to incorporate a new lot (lot number added, new pictures included). - number of FGFR2 probe L16630 corrected from 7 to 8. Version 11 (45) - Table 2 for more than one gene/region has been named Table 2a and Table 2b etc. - This product description has been changed to incorporate a new lot (lot number added, small changes in Table 1 and Table 2, new pictures included). Various textual changes on page 1. SALSA probemix P080 Craniofacial Page 8 of 8