SALSA MLPA probemix P463-A1 MRKH Lot A1-0716

Transcription

1 mix P463-A1 MRKH Lot A Mayer-Rokitansky-Küster-Hauser syndrome (MRKH) is characterised by normal physical development of the secondary sexual characteristics and a normal female 46,XX karyotype but with complete aplasia of the uterus, cervix, and superior parts of vagina, leading to failure to menstruate and infertility. This syndrome is distinguishable in type I with normally developed fallopian tubes, ovaries, and urinary tract, and type II with fallopian or ovarian abnormalities, and additional malformations which involve the urinary tract and spine. MRKH has an incidence of approximately 1 in 5,000 new-born girls. Defects in the TBX6, LHX1, HNF1B, and TBX1 genes on chromosomes 16, 17, and 22 are some of the causes for the development of MRKH syndrome. The TBX6 gene (9 s) spans ~6.1 kb of genomic DNA and is located on 16p11.2, 30.1 Mb from the p- telomere. The P463-A1 mix contains one for each of the gene. The LHX1 gene (5 s) spans ~7.1 kb of genomic DNA and is located on 17q12, 36.9 Mb from the p-telomere. This mix contains one for each of the gene. The HNF1B gene (9 s) spans ~58.6 kb of genomic DNA and is located on 17q12, 37.7 Mb from the p-telomere. This mix contains one for each of the gene. TBX1 gene (9 s) spans ~10.6 kb of genomic DNA and is located on 22q11.21, 19.8 Mb from the p-telomere. The P463-A1 mix contains one for each of the gene, two s for 9, and additional two s targeting s present in other transcript variants. Finally, ten reference s are included in this mix, detecting several 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 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 s. Finally, note that most defects in these genes 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). 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 P463 MRKH mix Page 1 of 6

2 Data analysis The P463-A1 MRKH mix contains 45 MLPA s with amplification products between 121 and 496 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 (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 mix can first be normalised 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 normalisation). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalised ratio in a sample by the average intra-normalised ratio of all reference samples. Please note that this type of normalisation 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 s 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 H. Oulhadj 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 included as co-author. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P463 MRKH mix Page 2 of 6

3 Table 1. P463-A1 MRKH mix Chromosomal position reference TBX6 LHX1 HNF1B TBX 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 121 Reference L p Reference L q TBX L28418 Exon HNF1B L27515 Exon TBX L27855 Exon HNF1B L12885 Exon Ж «TBX SP0975- L29167 Exon HNF1B L27838 Exon «TBX L29326 Exon Reference L q «TBX L10879 Exon LHX L27842 Exon TBX L27857 Exon LHX L28951 Exon Ж TBX SP0972- L28952 Exon HNF1B L28900 Exon Reference L q «TBX L24823 Exon TBX L27851 Exon LHX L27844 Exon TBX L27849 Down 283 Reference L q TBX L27852 Exon HNF1B L09335 Exon LHX L27843 Exon «TBX L27848 Exon Reference L q HNF1B L18619 Exon LHX L27845 Exon «TBX L27518 Exon HNF1B L07460 Exon «TBX L27850 Exon Reference L q TBX L27856 Exon TBX L09699 Down 409 «TBX L09694 Exon HNF1B L27839 Exon Reference L q TBX L27858 Exon HNF1B L27840 Exon Reference L q «TBX L27846 Exon TBX L28610 Exon «TBX L27847 Exon Reference L q22 Ж This consists of three parts and has two ligation sites. A low signal of this can be due to depurination of the sample DNA, e.g. due to insufficient buffering capacity or a prolonged denaturation time. «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 P463 MRKH mix Page 3 of 6

4 Note: Exon numbering used here may differ from literature! Please notify us of any mistakes. The identity of the genes detected by the reference s is available on request: info@mlpa.com. A non-specific peak can appear at ~95 nt in the No DNA reaction. Table 2. P463 s arranged according to chromosomal location Table 2a. TBX6 gene TBX6 NM_ start codon (ex 2) L27858 Exon 1 18 nt after 1 GCCTTCGAACTT-TGGAGGCTTGGC 0.7 kb L27855 Exon CAGACGGAACTA-CAACATGTACCA 0.4 kb L28610 Exon TCTGTGGGAACA-GAAATGATCATC 1.8 kb L27852 Exon TCACCAACAGCA-CGCTGGACCCCC 0.2 kb L27851 Exon CACAAGTACCAA-CCCCGCATACAC 0.3 kb 225 Ж SP0972- GAAGATTGCAGC-35 nt spanning Exon & L28952 oligo-gaaactgtaaga 1.8 kb L27857 Exon AGCAGCCACAGA-GGCCTATGGGAG 0.2 kb L28418 Exon GTGAATCAGATC-CAGAACAGGCCC 0.4 kb L27856 Exon reverse GGTGTATGGTAG-AGGGAAGGGGCC stop codon (ex 9) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2b. LHX1 gene LHX1 NM_ start codon (ex 1) L27842 Exon GTGAATGTAAAT-GCAACCTGACCG 2.1 kb L27845 Exon AGGAACTCTACA-TCATCGACGAGA 0.3 kb L28951 Exon CAACGTGTCGGA-CAAGGAAGCGGG 1.5 kb L27844 Exon 4 5 nt before 4 GCCGCCATGTGC-TGCAGGTCTGGT 1.1 kb L27843 Exon CAGCCTCGAGAA-CCATTCTCCTTC stop codon (ex 5) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2c. HNF1B gene HNF1B NM_ start codon (ex 1) L18619 Exon TTGGAAAATGGT-GTCCAAGCTCAC 5.3 kb L12885 Exon TGCAGCAACACA-ACATCCCCCAGA 5.8 kb L27840 Exon ATTCAACCAGAC-AGTCCAGAGTTC 2.0 kb L07460 Exon reverse ACACGGACCTCA-GTGACCAAGTTG 21.2 kb L27839 Exon GCCAGTCGGTTT-TACAGCAAGTCT 5.6 kb L27838 Exon CATAATCCCCAG-CAATCTCAAAAC 3.9 kb L27515 Exon reverse GCTCTGCTGCAT-GAGGGGCTGCTG 2.0 kb L28900 Exon AGCAGCATCAGT-ACACTCACCAAC 12.0 kb L09335 Exon CTCTCCCACGAT-GTCAAGGACTCC stop codon (ex 9) The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. SALSA P463 MRKH mix Page 4 of 6

5 Table 2d. TBX1 gene TBX1 NM_ start codon (ex 2) 166 Ж «09404-SP & 15 nt after AGGAGGAAGGGA-31 nt spanning Exon 1 L oligo-acacttgccgcg 2.8 kb 184 «05408-L29326 Exon CCGGGTGAAGCT-TCGCTGGCTGCC 1.6 kb 472 «20404-L27846 Exon GCCGGTGTGAGC-GTGCAGCTAGAG 2.1 kb 355 «09407-L27518 Exon TCCCACCTTCCA-AGTGAAGCTCTT 1.0 kb 409 «09408-L09694 Exon GCAGTGGATGAA-GCAAATCGTGTC 0.7 kb 318 «20406-L27848 Exon CCACGTGGTCTA-TGTGGACCCACG 0.8 kb 247 «10810-L24823 Exon TCCCTTCGCGAA-AGGCTTCCGGGA 0.2 kb 196 «09411-L10879 Exon CGGCACGGAGAA-AGGTAGGGCCGG 0.6 kb 373 «20408-L27850 Exon GGCCGCCGCCTA-CGACCACTATCT 0.7 kb 490 «20405-L27847 Exon 9 12 nt after 9 AAGCCGCCTGCG-TGTCCATTTATT 12.0 kb L27849 down NM_ ; reverse CTGCGGGGCAGG-GGAGCCCCAGGT 4.0 kb L09699 down NM_ ; AAGTCAGGAGGT-CAAGTGTGCATG stop codon (ex 9) The NM_ sequence represents transcript variant C and is a reference standard in the NCBI RefSeqGene project. Probe L27849 targets an which is only present in NM_ transcript variant A. Probe L09699 targets an which is only present in NM_ transcript variant B. Ж This consists of three parts and has two ligation sites. A low signal of this can be due to depurination of the sample DNA, e.g. due to insufficient buffering capacity or a prolonged denaturation time. «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. 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 P463 MRKH mix Page 5 of 6

6 mix P463-A1 MRKH sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with mix P463-A1 MRKH (lot A1-0716). Note: A non-specific peak can appear at ~95 nt in the No DNA reaction. Implemented Changes compared to the previous product description versions. Version August 2016 (55) - Not applicable, new document. SALSA P463 MRKH mix Page 6 of 6