MRC-Holland MLPA. Description version 08;

Transcription

1 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_ ), of which 3 are located in transcript variant 3 (NM_ ). 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

2 More information Website : 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 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 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 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 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

3 Table 1. SALSA MLPA P075-B1 TCF4-FOXG1 mix Length (nt) SALSA MLPA Chromosomal position reference TCF4 FOXG 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 L q TCF L14761 Exon TCF L26942 Exon TCF L14763 Exon * Reference L q TCF L14762 Exon * TCF L21843 Upstream 172 * Reference L q * TCF L19641 Exon * TCF L20919 Exon «FOXG L15243 Upstream 196 TCF L14759 Exon TCF L14765 Exon «TCF L13556 Exon TCF L15889 Exon TCF L15879 Exon * «TCF L21844 Upstream 232 * Reference L q TCF L14752 Exon * «FOXG L20920 Exon TCF L14750 Exon Reference L q «FOXG L15242 Exon TCF L14772 Exon TCF L14751 Exon * TCF L21845 Upstream 310 «TCF L14767 Exon * Ж «TCF SP0845-L26944 Exon TCF L26945 Exon * Reference L q * «FOXG L27389 Upstream 342 * TCF L27390 Intron * «TCF L27392 Exon TCF L27395 Exon * Reference L q * TCF L19646 Exon * TCF L26947 Intron TCF L26946 Exon TCF L13572 Exon TCF L14769 Exon TCF L14770 Exon Reference L p * «TCF L19647 Exon TCF L14766 Exon * Reference L p * TCF L26951 Intron TCF L26950 Exon «FOXG L26949 Exon * Ж TCF SP0544-L26948 Upstream 483 * Reference L q44 * 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. 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

4 Table 2. P075-B1 s arranged according to chromosomal location Table 2a. TCF4 gene Length (nt) SALSA MLPA TCF4 exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next Start Codon (ex 2) 476 * Ж SP0544- NM_ ; TAGCACAGGTGC-33 nt spanning Upstream L ; oligo-caaattactcaa 0.1 kb 301 * L21845 Upstream NM_ ; GCTACTCGAGCT-TCTCCAGGAGGC 4.0 kb 166 * L21843 Upstream NM_ ; 4250 nt after ATG CTAAGGCACTCA-CCTACTCTCAGA 41.9 kb 226 * «17730-L21844 Upstream NM_ ; reverse TCCCTGAAAGAT-ACATTGTAATCC 1.2 kb 208 «12506-L13556 Exon CCGAGGGATGCA-ACGGGCAAAAAC 0.3 kb 310 «13341-L14767 Exon reverse CTCTTAACACCA-ACTCTCTTCTCC 1.2 kb 427 * «16853-L19647 Exon reverse TCCAGTAAATCA-CTCAGCTCTTTG 1.1 kb 316 * Ж «19601-SP0845- XM_ ; CCCTAGGCAGGC-30 nt spanning Exon 3 L ; oligo-ctttctccattc 0.7 kb 350 * «16851-L27392 Exon TGGCAAGTGGAC-ATTTTACTGGCT kb L15889 Exon nt before exon 4 AGTGGCTTCTGA-CCCATCTACTTA 0.2 kb L14761 Exon 4 14 nt after exon 4 reverse TGGGAGAAAAGA-TTAGATATACTT 2.8 kb L14769 Exon nt before 5 GATTCCTTCTAG-TGAAGTTCCAGG 0.2 kb 178 * L19641 Exon CACATGACAATC-TCTCTCCACCTT 38.8 kb 379 * L26947 Intron 5 NM_ ; GCCACAACAGTT-TATTCATCCACA 18.7 kb L14759 Exon nt before exon 6 CCTACTTTACGT-ATGTAAACATCG 0.1 kb L14750 Exon ACTCATCTTATG-GGAGAGAATCAA 1.3 kb 342 * L27390 Intron 6 XM_ ; ACAGTGCTTGGT-TAAGAGCTCCTG 51.2 kb L14772 Exon reverse TTCGGGGATTAT-TGCTAGAATACT 0.5 kb L14765 Exon reverse AAACCTGGAGGA-ACTTTTCGAACT 28.7 kb 454 * L26951 Intron 8 NM_ ; ACTGCGCATACA-CAATCCCGGGCA 42.1 kb L14766 Exon ATGCTCCATCAG-CAAGCACTGCCG 0.1 kb L14770 Exon 9 8 nt after exon 9 CAAGGTAAGATG-CTGCTGCTTCTG 3.9 kb L14752 Exon TCTTCTCATATT-CCACAGTCCAGC 5.8 kb L14751 Exon TCCGATGTCCAC-TTTCCATCGTAG 0.1 kb L27395 Exon nt after exon 11 CACAGAAATGCC-AATTCTGATACC 8.3 kb L26946 Exon reverse TTCCTCACCGAA-GCAAGTGCTTTC 1.6 kb L26950 Exon nt after exon 13 GTATTTCAAATC-CCATTTCATCAT 2.5 kb L26942 Exon TAATATCAGCAG-GCACAGCTGTTT 2.8 kb 184 * L20919 Exon reverse TGCATGTCCCCA-TGACCACCAGGC 20.0 kb L15879 Exon CCACAGCTTCCT-GTCCAGTCTGCG 1.9 kb L26945 Exon nt before exon 17 GCAGCCTTGCAA-TCTGGTGTGCAG 3.7 kb L14763 Exon GTCCCACAGCAA-TAATGACGATGA 0.8 kb 372 * L19646 Exon CACACCCTGGAA-TGGGAGACGCAT 0.5 kb L14762 Exon ACAGGCTGAGAC-ACAGCCCAGAGA 0.6 kb L13572 Exon CCTGTAGTGCCA-ACTCTGCTTCCA Stop Codon (ex 19) * 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. 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_ sequence is a reference standard in the NCBI RefSeqGene project. SALSA mix P075 TCF4-FOXG1 Page 4 of 6

5 Note: Exon numbering used here may differ from literature! Complete sequences are available on request: Please notify us of any mistakes: Table 2b. FOXG1 gene Length (nt) SALSA MLPA FOXG1 Exon Ligation site NM_ 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 (ex 1) 247 * «17346-L20920 Exon CCAGCCACCCCA-TGCCCTACAGCT 0.4 kb 274 «13755-L15242 Exon reverse GAAATAATCAGA-CAGTCCCCCAGA 0.2 kb 468 «13754-L26949 Exon TCTAGGGTTGTT-TATTATTCTAAC Stop Codon (ex 1) * 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. «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_ 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 D ye S ign al 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

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