MRC-Holland MLPA. Description version 11; 20 November 2015

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1 SALSA MLPA probemix P310-B2 TCOF1 Lot B2-0614, B As compared to version B1 (lot B1-0110), the 88 and 96 nt control fragments have been replaced (QDX2). Treacher Collins-Franceschetti 1 syndrome is characterized by abnormal craniofacial development. Defects in the TCOF1 gene on chromosome 5 are the main cause of this disease. This gene encodes a nuclear protein with a LIS1 homology domain. The protein is involved in ribosomal DNA gene transcription through its interaction with upstream binding factor (UBF). The protein encoded by this gene is treacle. This protein is active during early embryonic development in structures that become bones and other tissues in the face. The TCOF1 gene (26 exons) spans ~43 kb of genomic DNA and is located on 5q33, about 150 Mb from the p-telomere. The P310-B2 probemix contains one probe for each exon of the gene with the exception of exons 8, 19 and 20. In addition, the probemix contains a probe for intron 6 and a probe for intron 16. Possible copy number changes of this genomic region in healthy individuals can be found in the database of genome variants ( This probemix furthermore contains 10 reference probes, detecting 10 different autosomal chromosomal locations. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned gene 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 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 P310 TCOF1 probemix Page 1 of 5

2 References Schaefer, E. et al., Autosomal recessive POLR1D mutations with decrease of TCOF1 mrna is responsible for Treacher Collins syndrome. Genetics in Medicine. 16: Vincent, M. et al., Large deletions encompassing the TCOF1 and CAMK2A genes are responsible for Treacher Collins syndrome with intellectual disability. European Journal of Human Genetics. 22: Bowman, M. et al., Gross deletions in TCOF1 are a cause of Treacher-Collins-Franceschetti syndrome. European Journal of Human Genetics. 20: Beygo. J. et al., First report of single exon deletion in TCOF1 causing Treacher Collins syndrome. Molecular Syndromology. 2: Data analysis The P310-B2 TCOF1 probemix contains 35 MLPA probes with amplification products between 135 nt and 436 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 probe s 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 P310 TCOF1 probemix Page 2 of 5

3 Table 1. SALSA MLPA P310-B2 TCOF1 probemix Length Chromosomal position SALSA MLPA probe (nt) reference TCOF 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 92 Ligation-dependent control fragment at 2q X-fragment: Specific for the X chromosome 105 Y-fragment: Specific for the Y chromosome 135 «Reference probe L q TCOF1 probe L12399 Exon TCOF1 probe L17553 Exon TCOF1 probe L17554 Exon TCOF1 probe L17057 Exon TCOF1 probe L17058 Exon TCOF1 probe L17060 Exon TCOF1 probe L12403 Exon Reference probe L p TCOF1 probe L12405 Exon Ж TCOF1 probe SP0128-L17502 Exon Ж TCOF1 probe SP0129-L17501 Exon TCOF1 probe L12410 Exon Reference probe L q TCOF1 probe L15142 Intron Reference probe L q TCOF1 probe L15143 Exon Reference probe L p Ж TCOF1 probe SP0130-L15144 Exon TCOF1 probe L12412 Intron TCOF1 probe L17079 Exon Ж TCOF1 probe SP0131-L17556 Exon TCOF1 probe L17555 Exon Reference probe L p TCOF1 probe L12415 Exon TCOF1 probe L15939 Exon Reference probe L q TCOF1 probe L15940 Exon TCOF1 probe L15146 Exon Reference probe L q Reference probe L q TCOF1 probe L12422 Exon ± TCOF1 probe L15148 Exon TCOF1 probe L16062 Exon Reference probe L q22 ± SNP rs could influence the probe signal. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. A SNP 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. Ж This probes consists of three parts and has two ligation sites. Notes The TCOF1 exon numbering has changed. From description version 11 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequence for this gene (NG_ ). 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 identity of the genes detected by the reference probes is available on request: info@mlpa.com. SALSA P310 TCOF1 probemix Page 3 of 5

4 Table 2. TCOF1 probes arranged according to chromosomal location Length (nt) SALSA MLPA probe TCOF1 Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 1) L17555 Exon TACTTCCCCTGA-TCTACCACCATC 3.3 kb L17553 Exon 2 11 nt before exon 2 GCTTTCTCTTTA-CCTCTCTGCAGA 3.0 kb L15940 Exon CACTGCAAGCTA-AGAAAACCCGTG 3.7 kb L17060 Exon CATCTACCAACT-CCTCAGTCCTGG 0.9 kb L17079 Exon GGGAATTCCATG-CCACACCCTGCC 0.9 kb 418 ± L15148 Exon 6 3 nt after exon 6 ACGTGGAGGTAA-TTGCCACCCATC 2.6 kb L L Ж 265 Ж SP0128- L SP0130- L L L L L L Ж SP0131- L L L L L L L L Ж SP0129- L L17058 Intron 6 (Exon 7) Exon 7 (Exon 8) Exon 9 (Exon 10) Exon 10 (Exon 11) Exon 11 (Exon 12) Exon 12 (Exon 13) Exon 13 (Exon 14) Exon 14 (Exon 15) Exon 15 (Exon 16) Exon 16 (Exon 17) Intron 16 (Exon 18) Exon 17 (Exon 19) Exon 18 (Exon 20) Exon 21 (Exon 23) Exon 22 (Exon 24) Exon 23 (Exon 25) Exon 24 (Exon 26) Exon 25 (Exon 27) Exon 26 (Exon 28) in NM_ (variant 4) GAGGGATCTGAA-AGTGAGGAGGAG 1.9 kb 44 nt before exon 7 CATCACCAGAGA-GTTTTCACAAGC 1.0 kb ; 7 nt after exon 9 46 nt before exon 10; 10 nt before exon 10 GAGGCACTGGCA-26 nt spanning oligo-ggaagccgccct CCTCACTCACAT-36 nt spanning oligo-tgtctcccaggt 0.1 kb 0.4 kb CTCACTCCAGGA-AAAGTCCTTGGG 0.6 kb AACCGTGGGACA-GGTGAGGCCTGT 0.3 kb 12 nt after exon 13 GTGAGGCCTAGA-AGGAGCAGGCCC 2.4 kb CTGCTCAAGCCA-AGCAGAGGTCTC 0.2 kb 19 nt before exon 15 CCTCCAATACTA-TTATCCCCCTGC 0.5 kb 4 nt after exon 16; 29 nt after exon in NM_ (variant 3) CTGCCCAGGTAA-25 nt spanning oligo-acactcactcct TGGTGTGGTCCA-AGCTTCTGTGTG 4.2 kb 4.1 kb CCCAGGCTGCAA-GCACCCCGAGGA 2.0 kb GTCCAGTCGGAT-ATCAGATGGCAA 2.8 kb GACCGCAGCAGA-GTCCAGCGAGGA 0.6 kb 13 nt before exon 22 CCTCTTTCACAA-TGGGCTTCTTCA 3.1 kb GGTCCTGACTGA-GCTGCTGGAACA 2.0 kb 43 nt after exon 24 AAACAGACCCAA-ACCCAAGCCCCC 0.6 kb 16 nt after exon 25; 40 nt after exon 25 GCTTCCCAATCA-24 nt spanning oligo-acagctctggtg GACAGCCAGCTT-CAGGGGTCCCTG stop codon (ex 25) ± SNP rs could influence the probe signal. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. A SNP could influence the probe signal. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. Ж These probes consist of three parts and has two ligation sites. Note: The TCOF1 exon numbering has changed. From description version 11 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequence for this gene (NG_ ). The exon numbering used in previous versions of this product description can be found between brackets in Table 2. Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. 0.8 kb SALSA P310 TCOF1 probemix Page 4 of 5

5 SALSA MLPA probemix P310-B2 TCOF1 sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P310-B2 TCOF1 (lot B2-0614). Implemented Changes compared to the previous product description versions. Version November 2015 (55) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Exon numbering of the TCOF1 gene has been changed on page 3 and 4. Version August 2015 (48) - Electropherogram picture(s) 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) - Various minor textual changes. - Ligation sites of the probes targeting the TCOF1 gene updated according to new version of the NM_reference sequence. Version 07 (46) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, 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. - Warning added in Table 1, 135 nt probe L SALSA P310 TCOF1 probemix Page 5 of 5