MRC-Holland MLPA. Description version 11; 07 December 2015

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1 SALSA MLPA probemix P049-C1 SLC6A8 ABCD1 Lot C1-1015, C Compared to the previous version (B2-0511), one probe for both GDI1 and FLNA have been replaced and one probe for exon 6 of ABCD1 has been added. Two reference probes have been replaced and three removed. Defects in the SLC6A8 gene encoding the X-linked creatine transporter are a relatively frequent cause of mental retardation in males. Several other genes located at a short distance from SLC6A8 have also been implicated in neurological syndromes, including L1CAM gene (X-linked hydrocephalus, this probemix), MECP2 (RETT syndrome; P015 probemix), FLNA (lissencephaly; P061 probemix) and ABCD1 (X-linked adrenoleukodystrophy, this probemix). Defects in the ABCD1 gene result in adrenoleukodystrophy due to defective peroxisomal beta oxidation and the accumulation of the saturated very long chain fatty acids (VLCFA) in all tissues of the body. The manifestations of the disorder occur primarily in the adrenal cortex, in the myelin of the central nervous system, and in the Leydig cells of the testes. The SLC6A8 gene (14 exons) spans ~8.3 kb of genomic DNA and is located approximately 30 kb centromeric of the ABCD1 gene. Exon 1 of SLC6A8 is located within a CpG island; the gene is transcribed towards the telomeric end. The ABCD1 gene contains 10 exons and spans about 20 kb of genomic DNA. SLC6A8 and ABCD1 are located approximately 153 Mb from the p-telomere. This P049-C1 SLC6A8 ABCD1 probemix contains 9 probes for SLC6A8 and 9 probes for 10 ABCD1 exons (no probe is present for exon 9). In addition, it contains 16 other probes in the Xq28 region, including probes for PNCK, BCAP31, IDH3G, L1CAM, IRAK1, MECP2, FLNA and GDI1. Finally, 9 probes detecting sequences elsewhere on the X chromosome are included as reference probes. 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 P178 F8: Probes for the FVIII gene also located at Xq28. P015 MECP2: Additional probes for the MECP2 gene and one probe for the VAMP7 (SYBL1) gene. P061 Lissencephaly: Additional probes for the FLNA gene. 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 probemix P049 SLC6A8 - ABCD1 Page 1 of 6

2 Data analysis The P049-C1 SLC6A8 ABCD1 probemix contains 43 MLPA probes with amplification products between 130 and 472 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 (D-fragments) 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 P049 SLC6A8 - ABCD1 Page 2 of 6

3 Table 1. SALSA MLPA P049-C1 SLC6A8 ABCD1 probemix Length Chromosomal position SALSA MLPA probe (nt) reference SLC6A8 ABCD1 Xq 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 130 * Reference probe L02104 Xp * ± GDI1 probe L19669 Xq Reference probe L08942 Xq BCAP31 probe L05470 Xq ± PNCK probe L03677 Xq ± SLC6A8 probe L01438 Exon SLC6A8 probe L01448 Exon Reference probe L07370 Xp BCAP31 probe L14008 Xq * Reference probe L15467 Xq ± FLNA probe L03492 Xq ± L1CAM probe L06657 Xq ± SLC6A8 probe L01440 Exon ABCD1 probe L11868 Exon ABCD1 probe L11870 Exon SLC6A8 probe L05472 Exon L1CAM probe L08340 Xq L1CAM probe L06660 Xq ABCD1 probe L13913 Exon Reference probe L01054 Xp ± FLNA probe L03493 Xq * ABCD1 probe L20813 Exon ± MECP2 probe L21151 Xq ± SLC6A8 probe L21152 Exon ± SLC6A8 probe L01445 Exon ABCD1 probe L02870 Exon ± L1CAM probe L00337 Xq ± SLC6A8 probe L01444 Exon Reference probe L02293 Xq ± MECP2 probe L00895 Xq ± SLC6A8 probe L01446 Exon ABCD1 probe L14009 Exon ABCD1 probe L13912 Exon IDH3G probe L21148 Xq * FLNA probe L21149 Xq ± SLC6A8 probe L01447 Exon Reference probe L00338 Xq ABCD1 probe L02868 Exon ABCD1 probe L11873 Exon ± PNCK probe L04286 Xq ± PNCK probe L08341 Xq Reference probe L07689 Xp Reference probe L02309 Xq12 * New in version C1 (from lot C onwards). Changed in version C1 (from lot C onwards): Small change in length or peak height, but no change in the sequence detected. ± 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 signal of the 222 nt probe might be influenced by a rare polymorphism (SNP; rs ). Complete probe sequences and the identity of the genes detected by the reference probes are available on request: info@mlpa.com. Note that exon numbering of the SLC6A8 and BCAP31 has changed! See details below Table 2. SALSA probemix P049 SLC6A8 - ABCD1 Page 3 of 6

4 Table 2. Xq28 probes arranged according to chromosomal location Length (nt) SALSA MLPA probe Gene exon Ligation site Partial sequence (24 nt adjacent to ligation site) Distance to next probe 154 ± L03677 PNCK Exon 13 NM_ GCCAAGTGGACT-GACCCCCAGATT 2.0 kb 454 ± L08341 PNCK Exon 5 NM_ ATGGAACTGTGA-GGAGGGCCTGGG 0.9 kb 445 ± L04286 PNCK Exon 3 NM_ AGAAACACACGG-AGGACATCAGCA 15.6 kb SLC6A8 NM_ start codon (exon 1) 160 ± L01438 Exon TGTCCGGCGACG-AGAAGAAGGGCC 2.9 kb 202 ± L01440 Exon 3 (4) AGACTGTGCCAA-TGCCAGCCTGGC 1.6 kb 292 ± L21152 Exon 5 (6) GGCGCCCTGGAT-GGCATCATTTAC 0.4 kb 328 ± L01444 Exon 7 (8a) CAGAGCAGGGCG-TGCACATCTCCA 0.4 kb 301 ± L01445 Exon 8 (9) ATCGCCTACCCG-CGGGCTGTCACG 0.2 kb 364 ± L01446 Exon 9 (10) CCGGCCTCCTCG-ACCTCCTCCCGG 0.3 kb 400 ± L01447 Exon 10 (11) GCCTGGGTGTAC-GGTAGGTCATGG 0.2 kb L01448 Exon 11 (12) ACCCCGCTGGTC-TGCATGGTAAGG 0.6 kb L05472 Exon 13 (14) AGAGTGTCATGT-GACAACTCAGCT 20.4 kb stop codon (exon 13) BCAP31 NM_ stop codon (exon 8) L14008 Exon 4 (5) CTTCCACATGAA-GCTTTTCCGTGC 7.6 kb L05470 Exon 2 (3) CAGTTGCCACCT-TCCTCTATGCGG 2.3 kb start codon (exon 2) ABCD1 NM_ start codon (exon 1) L11868 Exon TGAACCGGGTAT-TCCTGCAGCGGC 3.8 kb L13913 Exon CTCATCCTTCTG-GAACGCCTGTGG 6.9 kb L11870 Exon GAGGAGCTGGTG-AGCGAGCGCACA 0.3 kb L14009 Exon CCAGGGAGCTAG-AGGACGCTCAGG 0.7 kb L02868 Exon GTGGAACAGGGG-ATCATCTGCGAG 3.1 kb L20813 Exon 6 1 nt after exon 6 TCCCGCAGAGGT-AAGGAAGCCCGT 0.5 kb L11873 Exon CCATCCTGGACG-TCGTGCACCTGC 2.3 kb L13912 Exon reverse CTCGCCACCCGA-CAGGACGTCCTT 0.5 kb L02870 Exon CTGGCCAGGAAA-TACCACACACAC 50.9 kb stop codon (exon 10) L21148 IDH3G Exon 1 NM_ TCCCCGAAACTT-CGCACCCCGTCG 70.3 kb L06660 L1CAM Exon 23 NM_ CAGCGGGTGAAA-ACTACAGTGTCG 2.1 kb L08340 L1CAM Exon18 NM_ TATGAGATCAAA-GTCCAGGCCGTC 5.5 kb L00337 L1CAM Exon 4 NM_ AACAGCAACTTT-GCTCAGAGGTTC 3.6 kb L06657 L1CAM Exon 1 NM_ CTCTCCTCCTCT-GCAGCCCCTGCC kb L21151 MECP2 Exon 4b NM_ TTTCATCCTCCA-TGCCAAGGCCAA 2.0 kb L00895 MECP2 Exon 3 NM_ GCCCACCACTCT-GCTGAGCCCGCA kb L21149 FLNA Exon 38 NM_ GCCTGCAGAGTT-TATCATTGATAC 12.2 kb L03493 FLNA Exon 11 NM_ GGCTTCGAGTAT-TACCCCATGGTC 2.5 kb L03492 FLNA Exon 4 NM_ AGCAAGCCCGTT-ACCAATGCGCGA 69.6 kb 136 ± L19669 GDI1 Exon 1 NM_ CCTGACCATGGA-CGAGGAATACGA ± 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 signal of the 222 nt probe might be influenced by a rare polymorphism (SNP; rs ). The mentioned NM_sequences are reference standards in the NCBI RefSeqGene project. Notes! The SCL6A8 and BCAP31 exon numbering has changed. From description version 11 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 2. Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA probemix P049 SLC6A8 - ABCD1 Page 4 of 6

5 SALSA MLPA probemix P049-C1 SLC6A8 ABCD1 sample pictures Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P049-C1 SLC6A8 ABCD1 (lot C1-1015). Figure 2. Capillary electrophoresis pattern from a sample of approximately 50 ng human female control DNA analysed with SALSA MLPA probemix P049-C1 SLC6A8 ABCD1 (lot C1-1015). SALSA probemix P049 SLC6A8 - ABCD1 Page 5 of 6

6 Implemented Changes compared to the previous product description version(s). Version 11 (55) 07 December Product description adapted to a new lot (lot number added, new picture included). - Exon numbering of the SLC6A8 and BCAP31 genes changed. - Various textual and lay-out changes. Version 10 (53) - Various textual and lay-out changes. - Updated link for Database of genomic variants. - Peak area replaced with peak height. Version 09 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 08 (48) - 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). - Various minor textual changes. Version 07 (48) - Warnings about salt sensitivity of the 154, 190, 196, 257, 319, 355, 418, 445 and 454 nt probes added. - Various minor textual changes. Version 06 (46) - 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). - Warning added below Table 2 that the exon numbering of the FLNA and PNCK genes has changed. - Warning added to tables about shoulder peak of 154 nt probe and salt sensitivity of 129 and 136 nt probes. - Remarks on RefSeqGene standard and transcript variants added below Table 2. - Various minor textual changes on page 1. Version 05 (46) - Various minor textual changes on page 1. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Sentence when only small numbers of samples are tested, visual comparison of peak profiles should be sufficient removed from data analysis section - Tables have been numbered. - Various minor layout changes. - Warning added below Table 1 and 2 that the exon numbering of the SLC6A8 and BCAP31 gene has changed. - Ligation sites updated according to new version of the NM_reference sequence. SALSA probemix P049 SLC6A8 - ABCD1 Page 6 of 6