SALSA MLPA probemix P074-A3 Androgen Receptor (AR) Lot A Compared to previous lot A2-0712, three reference probes have been replaced.

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1 SALSA MLPA probemix P074-A3 Androgen Receptor (AR) Lot A Compared to previous lot A2-0712, three reference probes have been replaced. The androgen insensitivity syndrome (AIS), formerly known as the testicular feminization syndrome (TFM), is a recessive disorder in which affected males have female external genitalia, female breast development, blind vagina and female adnexa, and abdominal or inguinal testes, despite a normal male (2A + XY) karyotype. Partial androgen insensitivity results in hypospadias and micropenis with gynecomastia (Reifenstein syndrome). AIS is caused by mutations in the gene for the androgen receptor (AR). X-linked spinal and bulbar muscular atrophy (SBMA, SMAX1), also known as Kennedy disease, is a recessive form of spinal muscular atrophy. It occurs only in men. Age at onset is usually in the third to fifth decade of life, but earlier involvement has been reported. The disorder is characterized by slowly progressive limb and bulbar muscle weakness with fasciculations, muscle atrophy and gynecomastia. The disorder is clinically similar to, but genetically distinct from, classic forms of autosomal spinal muscular atrophy and is caused by a trinucleotide CAG repeat expansion in exon 1 of the gene encoding the androgen receptor. CAG repeat numbers are usually over 38 in SBMA patients, whereas healthy individuals have 10 to 36 CAG repeats. Note that the length of the trinucleotide repeat cannot be measured by MLPA. The AR gene (8 exons), spans ~187 kb of genomic DNA and is located on chromosome Xq12, 67 Mb from the p-telomere. The P074-A3 probemix contains two probes for each of the 8 exons; although exons 6 and 7 are only represented by one probe each. Furthermore two probes have been included for the first exon of transcript variant 2 (NM_ ) which is located in intron 1 of the reference standard transcript variant 1. In addition, 9 reference probes are included in this probemix, detecting several different locations on the X chromosome. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the AR gene in a DNA sample. 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). More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands SALSA probemix P074 AR Page 1 of 6

2 References Li Y et al. (2012). AR intragenic deletions linked to androgen receptor splice variant expression and activity in models of prostate cancer progression. Oncogene 31: Data analysis The P074-A3 AR probemix contains 25 MLPA probes with amplification products between 178 and 418 nt. In addition, it contains 10 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 two Y-fragments at 105 nt and 118 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 recognised by the reference probes. It is recommended to use reference and patient samples of the same sex to minimize variation, but this is not strictly necessary. Sex determination can also be done by visual examination of the electropherogram. 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 probemix P074 AR Page 2 of 6

3 Table 1. SALSA MLPA P074-A3 AR probemix Length (nt) SALSA MLPA probe Chromosomal position reference AR 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 118 Y-fragment: Specific for the Y chromosome 178 Reference probe L06673 Xp AR probe L13678 Exon 2 (3) 190 AR probe L13679 Exon 6 (7) 202 AR probe L13680 Exon 3 (4) 208 AR probe L13681 Exon 2 (3) 220 Reference probe L25392 Xp AR probe L13682 Exon * Reference probe L09375 Xp AR probe L13683 Exon 5 (6) 260 AR probe L13684 Exon 3 (4) 274 AR probe L13685 Exon 5 (6) 283 Reference probe L12953 Xq AR probe L13686 Exon # AR probe L13687 Exon 1b (2) 310 Reference probe L07954 Xq AR probe L13688 Exon 8 (9) 328 AR probe L13689 Exon 4 (5) 337 Reference probe L07362 Xp AR probe L13690 Exon 7 (8) 364 * Reference probe L03190 Xq # AR probe L13691 Exon 1b (2) 382 AR probe L13692 Exon 8 (9) 399 AR probe L13693 Exon 4 (5) 409 * Reference probe L05288 Xq Reference probe L05999 Xp22 * New in version A3 (from lot A onwards). Changed in version A3 (from lot A onwards). Small change in length, no change in sequence detected. # Probes noted as detecting exon 1b, detect the first exon of transcript variant 2 (NM_ ). Notes: The AR exon numbering has changed. From description version 07 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for this gene. 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 1. The identity of the genes detected by the reference probes is available on request: info@mlpa.com. Please notify us of any mistakes. SALSA probemix P074 AR Page 3 of 6

4 Table 2. AR probes arranged according to chromosomal location Length (nt) SALSA MLPA probe AR exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 1) L13686 Exon CGTGTGTCTTCT-TCTGCACGAGAC 1.1 kb L13682 Exon AGCACTGAAGAT-ACTGCTGAGTAT 22.8 kb 373 # L13691 Exon 1b NM_ ; 12 nt before exon 2 AAGTTGCTCTCT-TCTCCAGTAGGT 0.2 kb 301 # L13687 Exon 1b NM_ ; TGCCTGTCACTT-TTTCCCATGATA 74.3 kb L13681 Exon CAGGGACCATGT-TTTGCCCATTGA 0.1 kb L13678 Exon AGCTTCTGGGTG-TCACTATGGAGC 42.7 kb L13684 Exon CTGTGCGCCAGC-AGAAATGATTGC 0.1 kb L13680 Exon AGCAGGGATGAC-TCTGGGAGGTAA 25.5 kb L13689 Exon GCCAGGTGTAGT-GTGTGCTGGACA 0.1 kb L13693 Exon GAGACAGCTTGT-ACACGTGGTCAA 5.9 kb L13683 Exon ATGGCTGTCATT-CAGTACTCCTGG 0.1 kb L13685 Exon TCAACTCCAGGA-TGCTCTACTTCG 4.3 kb L13679 Exon CACCTCTCTCAA-GAGTTTGGATGG 1.1 kb L13690 Exon 7 3 nt after exon 7 TGCAGCCTGTAA-GCAAACGATGGA 0.7 kb L13688 Exon CATCAGTTCACT-TTTGACCTGCTA 0.1 kb L13692 Exon CCAGTGAAGCAT-TGGAAACCCTAT stop codon (ex 8) The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. # Probes noted as detecting exon 1b, detect the first exon of transcript variant 2 (NM_ ). Notes: The AR exon numbering has changed. From description version 07 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for this gene. 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 1. The identity of the genes detected by the reference probes is available on request: info@mlpa.com. Please notify us of any mistakes. SALSA probemix P074 AR Page 4 of 6

5 SALSA MLPA probemix P074-A3 AR sample pictures Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA probemix P074-A3 AR (lot A3-0814). Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng human female control DNA analysed with SALSA probemix P074-A3 AR (lot A3-0814). SALSA probemix P074 AR Page 5 of 6

6 Implemented Changes compared to the previous product description versions Version 07 (53) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new pictures included). - Adjustment of the exon numbering to the NCBI exon numbering that is present in the NM_ sequences for this gene. - Various minor textual changes. - Updated link for Database of Genomic Variants. Version 06 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 05 (48) - Product description adapted to a new product version (version number changed, lot number added, new pictures included). - Various minor textual changes. Version 04 (48) - Ligation sites of the probes targeting the AR gene 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. Version 03 (46) - Details added on androgen insensitivity syndrome and X-linked spinal and bulbar muscular atrophy. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Data analysis method has been modified. - Various minor textual changes on page 1. - Various minor layout changes. SALSA probemix P074 AR Page 6 of 6