MRC-Holland MLPA. Related SALSA MLPA probemixes P054 FOXL2-TWIST1: Contains probes for the GPR143 gene on Xp22.2.

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1 SALSA MLPA probemix P160-C1 STS Lot C As compared to version B1 (lot B1-0313), two STS probes have been added and one flanking probe and the Y-fragment on 118 nt have been removed. Two STS probes, two flanking probes and one reference probe have been replaced. In addition, several probe lengths have been adjusted. Defects in the Steroid Sulfatase (STS) gene are the cause of X-linked ichthyosis or placental steroid sulfatase deficiency. The protein encoded by this gene catalyses the conversion of sulfated steroid precursors to estrogens during pregnancy. The STS gene (10 exons) spans ~135 kb of genomic DNA and is located on chromosome Xp22.31, 7 Mb from the p-telomere. Most individuals (85-90%) with X-linked ichthyosis have extensive deletions of the STS gene. Point mutations however have also been described. The percentage of gene defects due to deletions in the STS gene is among the highest of all genetic diseases. Most patients have a deletion of the complete gene but partial STS gene deletions have also been described. This P160-C1 STS probemix contains probes for each exon of the STS gene, with the exception of exon 3, as well as two flanking probes targeting the PUDP gene. This probemix furthermore contains several probes for the Xp22 region, including probes for the ANOS1 (KAL1) gene involved in Kallmann syndrome and the NLGN4X gene that might be involved in X-linked autism. In addition, 11 reference probes are included, detecting several different locations on the X-chromosome. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned genes 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 recognizable 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). Related SALSA probemixes P054 FOXL2-TWIST1: Contains probes for the GPR143 gene on Xp22.2. 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 P160 STS probemix Page 1 of 7

2 Data analysis The P160-C1 STS probemix contains 35 MLPA probes with amplification products between 130 and 427 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 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 exon 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 P160 STS probemix Page 2 of 7

3 Table 1. SALSA MLPA P160-C1 STS probemix Length Chromosomal position SALSA MLPA probe (nt) reference STS other 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 probe L13693 Xq STS probe L04565 Exon NLGN4X probe L04580 Exon ANOS1 (KAL1) probe L03817 Exon Reference probe L01615 Xp * STS probe L29265 Exon ANOS1 (KAL1) probe L05579 Exon * Reference probe L06389 Xq STS probe L04566 Exon NLGN4X probe L04578 Intron STS probe L05112 Exon Reference probe L11353 Xq STS probe L04571 Exon * ANOS1 (KAL1) probe L05940 Exon GPR143 probe L02406 Exon * STS probe L29266 Exon «PUDP (HDHD1A) probe L04575 Upstream 247 * STS probe L29267 Exon STS probe L05589 Exon NLGN4X probe L29518 Intron Reference probe L00963 Xq STS probe L04572 Exon NLGN4X probe L04583 Exon * STS probe L29269 Exon Reference probe L04511 Xq STS probe L04568 Exon NLGN4X probe L04579 Exon Reference probe L02108 Xp Reference probe L05990 Xp STS probe L04573 Exon * PUDP (HDHD1A) probe L29270 Upstream 399 Reference probe L07363 Xp Reference probe L00338 Xq STS probe L04569 Exon Reference probe L13935 Xq21 * New in version C1 (from lot C onwards). Changed in version C1 (from lot C onwards). Small change in length, no change in sequence detected. Flanking probe. Included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition tested. «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. Notes Exon numbering used here may differ from literature! Please notify us of any mistakes. The identity of the genes detected by the reference probes is available on request: info@mlpa.com. Gene names of ANOS1 and PUDP have changed from description version 9 onwards. The gene names used in previous versions can be found between brackets in Table 1 and Table 2. Please notify us of any mistakes: info@mlpa.com. SALSA P160 STS probemix Page 3 of 7

4 Table 2. Xp22 probes arranged according to chromosomal location Table 2a. NLGN4X Length (nt) SALSA MLPA probe NLGN4X Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (exon 2) 283 ๑ L29518 Intron 1 (2) 754 nt after exon 1 GACGGCTTGGGT-GATGCACGAAAT 0.8 kb 183 # L04578 Intron 1 (2) 1561 nt after exon 1 AGAGAGAGAGTG-AACTTCAGTCCT 75.5 kb L04579 Exon 2 (3) TTGAACCATGTC-ACGGCCCCAGGG kb L04580 Exon 3 (4) AAGAAGCCCGTC-ATGGTCTATATC kb L04583 Exon 6 (7) CTGACTTAAGAC-AAAAATGCAAAA stop codon (exon 6) ๑ The target sequence of this probe is not present in NM_ but can be found in transcript variant 1 (NM_ ; ligation site in exon 1). # The target sequence of this probe is not present in NM_ but can be found in transcript variant 4 (NM_ ; ligation site in exon 1). Changed in version C1 (from lot C onwards). Small change in length, no change in sequence detected. The NM_ sequence represents transcript variant 2 and is a reference standard in the NCBI RefSeqGene project. Note: The NLGN4X exon numbering has changed. From description version 9 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequence for this gene. The exon numbering used in previous versions of this product description can be found between brackets in Table 2a. Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. Table 2b. STS Length SALSA MLPA (nt) probe 391 * L «05194-L04575 STS Exon PUDP (HDHD1A) Exon 4 PUDP (HDHD1A) Exon 1 Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) GATGGAAACTCC-AATCTCAGGAGG AGGCGGCGGCTG-CACACTACCCAC Distance to next probe 97.9 kb 71.5 kb start codon (exon 1) 160 * L29265 Exon reverse TATTCCCATGAG-AAACCTCTGCTT 0.1 kb L04565 Exon CATCACAGCTCA-GTTCCCCAACAA 33.6 kb L04566 Exon reverse CTTGATGCTGCG-TGGCTCTCGGCT 4.3 kb No probe Exon L04568 Exon CCGCACTGGAGT-TTTCCTCTTCAC 2.1 kb L04569 Exon GGCTACTCCACG-TGCCTCTAGGCG 16.3 kb 326 * L29269 Exon 6 78 nt before exon 6 reverse CATTCTGATTAA-GATCCACCTATT 0.2 kb 247 * L29267 Exon 6 6 nt after exon 6 reverse CACTGAGGAGAA-ACGTACCCACAC 28.9 kb 226 * L29266 Exon 7 96 nt before exon 7 AACGTTTACTTC-CACCTTGAGAAA 0.2 kb L04571 Exon GCACATGTAGAA-GAAGTGTCTTCC 20.3 kb L04572 Exon CAACATGGACAT-ATTTCCTACAGT 8.6 kb L04573 Exon AAAAAGCCAACG-CTCCGATCATGA 17.4 kb L05112 Exon CATGATTTTGTT-TTCATCCCATTT 0.4 kb L05589 Exon TGAAAGTTGGCT-ATAATTTCTCTA stop codon (exon 10) * New in version C1 (from lot C onwards). SALSA P160 STS probemix Page 4 of 7

5 Changed in version C1 (from lot C onwards). Small change in length, no change in sequence detected. Flanking probe. Included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition tested. «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. The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2c. ANOS1 (KAL1) Length (nt) SALSA MLPA probe ANOS1 Exon L02406 GPR143 Exon 9 Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) CTGCAACAAAAA-TGAGGGTGACCC Distance to next probe kb start codon (exon 1) L05579 Exon reverse TACTGCTGACCA-TCCAGCTCCAAA 16.5 kb 209 * L05940 Exon CCCACTCGCCCG-CTGGAAGTCGGA 24.3 kb L03817 Exon ATGATCTTTACT-GAATTTGCCCTT stop codon (exon 14) * New in version C1 (from lot C onwards). Flanking probe. Included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition tested. The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Note: Exon numbering used here may differ from literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA P160 STS probemix Page 5 of 7

6 SALSA MLPA probemix P160-C1 STS sample pictures Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P160-C1 STS (lot C1-0816). Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng human female control DNA analysed with SALSA MLPA probemix P160-C1 STS (lot C1-0816). SALSA P160 STS probemix Page 6 of 7

7 Implemented Changes compared to the previous product description versions. Version September 2016 (55) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new pictures included). - Information on the database of genomic variants added on page 2. - Data analysis method modified. - Updated gene names KAL1 to ANOS1 and HDHDA1 to PUDP. - Exon numbering of the NLGN4X gene has been changed on page 3 and 4. - Salt warning added under Table 1 and Table 2. - Various minor textual changes. - Altered manufacturer s address. Version 08 (50) - 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). Version 07 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. - Various minor textual changes. Version 06 (48) - Warning added to tables about 292 nt probe L Version 05 (47) - Remark on RefSeqGene standard and transcript variant added below Table 2. - Ligation sites of the probes targeting the STS gene updated according to new version of the NM_reference sequence. - Various minor textual changes. - Various minor layout changes. Version 04 (45) - Various minor textual changes on page 1. - Minor changes in the data analysis section on page 2. - Tables have been numbered. - Various minor layout changes. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. SALSA P160 STS probemix Page 7 of 7