SALSA MLPA mix P075-B1 TCF4-FOXG1 Lot B1-0614: As compared to version A1 (lot A1-0510), seven target specific s have been replaced and eight new s have been included. Furthermore, three reference s have been removed, seven reference s have been replaced and the control fragments have been replaced (QDX2). Pitt Hopkins Syndrome (PTHS) is a rare disorder characterized by severe intellectual disability, pervasive developmental delay, atypical autistic characteristics, and hyperventilation. PTHS is caused by heterozygous hypomorphic or null mutation or deletion of the transcription factor 4 (TCF4; E2-2; ITF2) gene on human chromosome 18. The TCF4 gene is also a risk factor with highly significant linkage to schizophrenia, presumably via overexpression of the TCF4 gene product in the central nervous system. Another disorder that has some phenotypical overlap with Pitt-Hopkins syndrome is the congenital variant of Rett syndrome. This syndrome occurs almost exclusively in females and is characterized by hypotonia and mental retardation from the very first months of life. It has been reported that the main cause of the congenital variant of Rett syndrome is mutations in the FOXG1 gene, which encodes a transcription factor of the forkhead family on chromosome 14. The TCF4 gene (20 exons) spans ~366.3 kb of genomic DNA and is located on 18q21.2, 53 Mb from the p-telomere. The FOXG1 gene (1 exon) spans ~3.2 kb of genomic DNA and is located on 14q12, 29 Mb from the p-telomere. The P075-B1 mix contains one for each exon of the TCF4 gene. Also, additional s for exons 1, 3, 4, 5, 6, 9, 11 and 20 are included in P075-B1. Moreover, based on the main transcript of TCF4, extra s for intron 5, 6 and 8 are included in this mix. In most cases these s detect exonic sequences present in other transcripts. To date 12 different transcript variants are known while multiple other transcripts are predicted. This mix contains 4 additional s located upstream of the main transcript variant 1 (NM_001083962.1), of which 3 are located in transcript variant 3 (NM_001243226.3). In addition, the P075-B1 mix contains three s for exon 1 of the FOXG1 gene and two s upstream of exon 1. Finally, 10 reference s are included in this mix, detecting different autosomal chromosomal locations. This SALSA mix 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. Note that a mutation or polymorphism (e.g. SNP) in the sequence detected by a can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some 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 mixes 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 mixes and reagents includes a limited license to use these products for research purposes. The use of a SALSA mix 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). SALSA mix P075 TCF4-FOXG1 Page 1 of 6
More information Website : www.mlpa.com E-mail : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands Related SALSA MLPA mixes ME028 PWS/AS: Prader-Willi / Angelman syndrome P015 MECP2: RETT syndrome P169-Hirschsprung: Contains s for ZEB2 (Mowat-Wilson) and other genes. P189 RETT-like: RETT-like syndrome P336 UBE3A: UBE3A / Angelman P379 NRXN1: Pitt-Hopkins-like syndrome 2 Data analysis The P075-B1 TCF4-FOXG1 mix contains 50 MLPA s with amplification products between 130 nt and 483 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 (D-fragments) 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 mix can first be normalized intra-sample by dividing the peak height of each s amplification product by the total peak height of only the reference s in this mix (block normalization). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalized ratio in a sample by the average intra-normalized ratio of all reference samples. Please note that this type of normalization assumes no changes occurred in the genomic regions recognised by the reference s. 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 s are unlikely to be the cause of the condition tested for. Users should always verify the latest scientific literature when interpreting their findings. This mix was developed by K. Tuin at. In case the results obtained with this mix lead to a scientific publication, it would be very much appreciated if the mix designer could be made a co-author. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA mix P075 TCF4-FOXG1 Page 2 of 6
Table 1. SALSA MLPA P075-B1 TCF4-FOXG1 mix Length (nt) SALSA MLPA Chromosomal position reference TCF4 FOXG1 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 00797-L13645 5q31 138 TCF4 13335-L14761 Exon 4 143 TCF4 13334-L26942 Exon 14 148 TCF4 13337-L14763 Exon 18 154 * Reference 08375-L08229 15q24 160 TCF4 13336-L14762 Exon 20 166 * TCF4 17729-L21843 Upstream 172 * Reference 11007-L11678 4q22 178 * TCF4 16847-L19641 Exon 5 184 * TCF4 17345-L20919 Exon 15 190 «FOXG1 13756-L15243 Upstream 196 TCF4 13333-L14759 Exon 6 202 TCF4 13339-L14765 Exon 8 208 «TCF4 12506-L13556 Exon 1 214 TCF4 13338-L15889 Exon 4 220 TCF4 13342-L15879 Exon 16 226 * «TCF4 17730-L21844 Upstream 232 * Reference 09641-L26943 17q25 238 TCF4 13326-L14752 Exon 10 247 * «FOXG1 17346-L20920 Exon 1 256 TCF4 13324-L14750 Exon 6 264 Reference 08773-L08842 19q13 274 «FOXG1 13755-L15242 Exon 1 283 TCF4 13346-L14772 Exon 7 292 TCF4 13325-L14751 Exon 11 301 * TCF4 17731-L21845 Upstream 310 «TCF4 13341-L14767 Exon 1 316 * Ж «TCF4 19601-SP0845-L26944 Exon 3 322 TCF4 13345-L26945 Exon 17 328 * Reference 08881-L08937 7q32 335 * «FOXG1 16850-L27389 Upstream 342 * TCF4 19602-L27390 Intron 6 350 * «TCF4 16851-L27392 Exon 3 358 TCF4 13327-L27395 Exon 11 364 * Reference 12656-L19936 16q22 372 * TCF4 16852-L19646 Exon 19 379 * TCF4 19603-L26947 Intron 5 386 TCF4 13329-L26946 Exon 12 391 TCF4 12522-L13572 Exon 20 401 TCF4 13343-L14769 Exon 5 409 TCF4 13344-L14770 Exon 9 418 Reference 06876-L05967 3p12 427 * «TCF4 16853-L19647 Exon 2 436 TCF4 13340-L14766 Exon 9 445 * Reference 10667-L11249 6p12 454 * TCF4 19604-L26951 Intron 8 461 TCF4 13348-L26950 Exon 13 468 «FOXG1 13754-L26949 Exon 1 476 * Ж TCF4 17732-SP0544-L26948 Upstream 483 * Reference 10038-L11452 1q44 * New in version B1 (from lot B1-0614 onwards). Changed in version B1 (from lot B1-0614 onwards). Small change in length, no change in sequence detected. Ж This consists of three parts and has two ligation sites. «This is located within, or close to, a very strong CpG island. A low signal of this can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. SALSA mix P075 TCF4-FOXG1 Page 3 of 6
Table 2. P075-B1 s arranged according to chromosomal location Table 2a. TCF4 gene Length (nt) SALSA MLPA TCF4 exon Ligation site NM_001083962.1 Partial sequence (24 nt adjacent to ligation site) Distance to next Start Codon 613-615 (ex 2) 476 * Ж 17732-SP0544- NM_001243226.3; TAGCACAGGTGC-33 nt spanning Upstream L26948 133-134; 166-167 oligo-caaattactcaa 0.1 kb 301 * 17731-L21845 Upstream NM_001243226.3; 271-272 GCTACTCGAGCT-TCTCCAGGAGGC 4.0 kb 166 * 17729-L21843 Upstream NM_001243226.3; 4250 nt after ATG CTAAGGCACTCA-CCTACTCTCAGA 41.9 kb 226 * «17730-L21844 Upstream NM_001243227.1; 49-48 reverse TCCCTGAAAGAT-ACATTGTAATCC 1.2 kb 208 «12506-L13556 Exon 1 99-100 CCGAGGGATGCA-ACGGGCAAAAAC 0.3 kb 310 «13341-L14767 Exon 1 403-402 reverse CTCTTAACACCA-ACTCTCTTCTCC 1.2 kb 427 * «16853-L19647 Exon 2 663-662 reverse TCCAGTAAATCA-CTCAGCTCTTTG 1.1 kb 316 * Ж «19601-SP0845- XM_005266746.1; CCCTAGGCAGGC-30 nt spanning Exon 3 L26944 55-56; 85-86 oligo-ctttctccattc 0.7 kb 350 * «16851-L27392 Exon 3 742-743 TGGCAAGTGGAC-ATTTTACTGGCT 121.0 kb 214 13338-L15889 Exon 4 148 nt before exon 4 AGTGGCTTCTGA-CCCATCTACTTA 0.2 kb 138 13335-L14761 Exon 4 14 nt after exon 4 reverse TGGGAGAAAAGA-TTAGATATACTT 2.8 kb 401 13343-L14769 Exon 5 130 nt before 5 GATTCCTTCTAG-TGAAGTTCCAGG 0.2 kb 178 * 16847-L19641 Exon 5 883-884 CACATGACAATC-TCTCTCCACCTT 38.8 kb 379 * 19603-L26947 Intron 5 NM_001243233.1; 215-216 GCCACAACAGTT-TATTCATCCACA 18.7 kb 196 13333-L14759 Exon 6 100 nt before exon 6 CCTACTTTACGT-ATGTAAACATCG 0.1 kb 256 13324-L14750 Exon 6 949-950 ACTCATCTTATG-GGAGAGAATCAA 1.3 kb 342 * 19602-L27390 Intron 6 XM_005266759.1; 78-79 ACAGTGCTTGGT-TAAGAGCTCCTG 51.2 kb 283 13346-L14772 Exon 7 1073-1072 reverse TTCGGGGATTAT-TGCTAGAATACT 0.5 kb 202 13339-L14765 Exon 8 1140-1139 reverse AAACCTGGAGGA-ACTTTTCGAACT 28.7 kb 454 * 19604-L26951 Intron 8 NM_001243234.1; 137-138 ACTGCGCATACA-CAATCCCGGGCA 42.1 kb 436 13340-L14766 Exon 9 1177-1178 ATGCTCCATCAG-CAAGCACTGCCG 0.1 kb 409 13344-L14770 Exon 9 8 nt after exon 9 CAAGGTAAGATG-CTGCTGCTTCTG 3.9 kb 238 13326-L14752 Exon 10 1356-1357 TCTTCTCATATT-CCACAGTCCAGC 5.8 kb 292 13325-L14751 Exon 11 1457-1458 TCCGATGTCCAC-TTTCCATCGTAG 0.1 kb 358 13327-L27395 Exon 11 18 nt after exon 11 CACAGAAATGCC-AATTCTGATACC 8.3 kb 386 13329-L26946 Exon 12 1599-1598 reverse TTCCTCACCGAA-GCAAGTGCTTTC 1.6 kb 461 13348-L26950 Exon 13 17 nt after exon 13 GTATTTCAAATC-CCATTTCATCAT 2.5 kb 143 13334-L26942 Exon 14 1684-1685 TAATATCAGCAG-GCACAGCTGTTT 2.8 kb 184 * 17345-L20919 Exon 15 1857-1856 reverse TGCATGTCCCCA-TGACCACCAGGC 20.0 kb 220 13342-L15879 Exon 16 2046-2047 CCACAGCTTCCT-GTCCAGTCTGCG 1.9 kb 322 13345-L26945 Exon 17 60 nt before exon 17 GCAGCCTTGCAA-TCTGGTGTGCAG 3.7 kb 148 13337-L14763 Exon 18 2264-2265 GTCCCACAGCAA-TAATGACGATGA 0.8 kb 372 * 16852-L19646 Exon 19 2593-2594 CACACCCTGGAA-TGGGAGACGCAT 0.5 kb 160 13336-L14762 Exon 20 2904-2905 ACAGGCTGAGAC-ACAGCCCAGAGA 0.6 kb 391 12522-L13572 Exon 20 3465-3466 CCTGTAGTGCCA-ACTCTGCTTCCA Stop Codon 2626-2628 (ex 19) * New in version B1 (from lot B1-0614 onwards). Changed in version B1 (from lot B1-0614 onwards). Small change in length, no change in sequence detected. Ж This consists of three parts and has two ligation sites. «This is located within, or close to, a very strong CpG island. A low signal of this can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. The NCBI NM_001083962.1 sequence is a reference standard in the NCBI RefSeqGene project. SALSA mix P075 TCF4-FOXG1 Page 4 of 6
Note: Exon numbering used here may differ from literature! Complete sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. Table 2b. FOXG1 gene Length (nt) SALSA MLPA FOXG1 Exon Ligation site NM_005249.4 Partial sequence (24 nt adjacent to ligation site) Distance to next 335 * «16850-L27389 Upstream 844 nt before exon 1 AAATGCCAGACA-CTGGCCTGCAAG 0.2 kb 190 «13756-L15243 Upstream 634 nt before exon 1 GAGGAAGCCGGA-AATGTGAGCTAT 1.9 kb Start Codon 209-211 (ex 1) 247 * «17346-L20920 Exon 1 1223-1224 CCAGCCACCCCA-TGCCCTACAGCT 0.4 kb 274 «13755-L15242 Exon 1 1622-1621 reverse GAAATAATCAGA-CAGTCCCCCAGA 0.2 kb 468 «13754-L26949 Exon 1 1816-1817 TCTAGGGTTGTT-TATTATTCTAAC Stop Codon 1676-1678 (ex 1) * New in version B1 (from lot B1-0614 onwards). Changed in version B1 (from lot B1-0614 onwards). Small change in length, no change in sequence detected. «This is located within, or close to, a very strong CpG island. A low signal of this can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. The NCBI NM_005249.4 sequence is a reference standard in the NCBI RefSeqGene project. Note: Exon numbering used here may differ from literature! Complete sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA MLPA mix P075-B1 TCF4-FOXG1 sample picture 15000 12500 10000 7500 85.31 96.06 90.81 105.30 127.08 218.94 152.29 141.32 183.51 164.63 145.95 232.08 273.44 176.39 189.63 246.47 171.12 327.67 135.12 195.35 237.76 321.77 341.53 308.16 213.26 201.37 301.95 263.00 158.79 206.64 226.15 255.65 282.85 334.11 291.45 315.54 348.98 357.22 364.66 370.61 378.51 385.25 401.06 427.79 417.23 391.62 409.88 436.81 446.75 467.93 483.37 461.30 453.94 5000 100.43 475.81 2500 D ye S ign al 0 100 150 200 250 300 350 400 450 500 Size (nt) Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA mix P075-B1 TCF4-FOXG1 (lot B1-0614). SALSA mix P075 TCF4-FOXG1 Page 5 of 6
Implemented Changes compared to the previous product description versions. Version 08 (53) - For the TCF4 and FOXG1 gene exon numbers and ligation sites in table 1 and 2 have been adjusted according to NCBI Map Viewer. - 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. - Changes of lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. Version 07 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 06 (48) - Exon numbering of the TCF4 gene has been changed due to the identification of new exons - Various textual changes on page 1 related to the exon numbering change. Version 05 (48) - Remark on RefSeqGene standard and transcript variant added below Table 2. - Various minor textual changes on page 1. - Ligation sites of the s targeting the TCF4 and FOXG1 genes updated according to new version of the NM_reference sequence. - Small correction of chromosomal locations in Table 1 and 2. Version 04 (46) - Warning changed on page 2. Use new PCR primer mix to prevent primer-dimer formation. Version 03 (46) - Warning added on page 2 regarding high formation of primer-dimers. Version 02 (46) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Table 2 for more than one gene/region has been named Table 2a and Table 2b. - Warning added in Table 1 for salt sensitive s. Version 01 (44) - Not applicable, new document. SALSA mix P075 TCF4-FOXG1 Page 6 of 6