MRC-Holland MLPA. Description version 10; 17 November 2016

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1 SALSA MLPA probemix P058-A3 IGHMBP2 Lot A As compared to the previous lot A2-0412, one reference probe has been removed and one replaced. In addition, one probe length has been adjusted. Spinal muscular atrophy with respiratory distress type 1 (SMARD1), also known as distal spinal muscular atrophy 1 (DSMA1) or distal hereditary motor neuropathies type 6 (dhmn6), is a rare autosomal recessive motor neuron disorder that affects infants and is characterized by diaphragmatic palsy, distal muscular weakness and muscle atrophy. The disease is caused by mutations in the gene encoding immunoglobulin µ- binding protein 2 (IGHMBP2). 'Distal' SMA (DSMA1) is distinguished from 'proximal' autosomal recessive spinal muscular atrophy (SMA) by the primary muscles involved. Like the SMN1 gene, which is mutated in SMA, IGHMBP2 colocalises with the RNA-processing machinery in both the cytoplasm and the nucleus. IGHMBP2 and SMN1 share common functions important to motor neuron maintenance and integrity in mammals. IGHMBP2 is the second gene found to be defective in SMA (Grohmann et al.; Nat Genetics 2001). The IGHMBP2 gene (15 exons) spans ~37 kb of genomic DNA and is located on chromosome 11q13.3, ~69 Mb from the p-telomere. The P058-A3 IGHMBP2 probemix contains one probe for each exon of IGHMBP2 and two probes for exon 1. In addition, nine reference probes are included in this probemix, detecting different autosomal chromosomal locations. This SALSA probemix 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 probe. 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 MLPA 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 1, 1057 DL Amsterdam, the Netherlands Related SALSA probemixes P021 SMA: Spinal Muscular Atrophy (SMA), to determine SMN1 and SMN2 copy number changes (patients). P060 SMA carrier: Spinal Muscular Atrophy (SMA) carrier, to determine SMN1 and SMN2 copy number changes of exon 7 and 8 only. SALSA P058 IGHMBP2 probemix Page 1 of 6

2 Data analysis The P058-A3 IGHMBP2 probemix contains 25 MLPA probes with amplification products between 130 and 375 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 first be normalised intra-sample by dividing the peak height of each probe s amplification product by the total peak height of only the reference probes in this 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 no changes occurred in the genomic regions recognised by the reference probes. 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 P058 IGHMBP2 probemix Page 2 of 6

3 Table 1. SALSA MLPA P058-A3 IGHMBP2 probemix Length (nt) SALSA MLPA probe Chromosomal position Reference IGHMBP 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 L p IGHMBP2 probe L11024 Exon IGHMBP2 probe L11043 Exon Reference probe L q Reference probe L p IGHMBP2 probe L11035 Exon IGHMBP2 probe L11022 Exon IGHMBP2 probe L12572 Exon Reference probe L q IGHMBP2 probe L11026 Exon IGHMBP2 probe L11037 Exon IGHMBP2 probe L11019 Exon Reference probe L q IGHMBP2 probe L11008 Exon * Reference probe L p IGHMBP2 probe L11027 Exon IGHMBP2 probe L11032 Exon IGHMBP2 probe L11011 Exon IGHMBP2 probe L11025 Exon Reference probe L p IGHMBP2 probe L11016 Exon IGHMBP2 probe L11041 Exon Reference probe L q IGHMBP2 probe L11013 Exon Reference probe L q21 * 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. Note: 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. SALSA P058 IGHMBP2 probemix Page 3 of 6

4 Table 2. IGHMBP2 probes arranged according to chromosomal location Length (nt) SALSA MLPA probe IGHMBP2 exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe Start Codon (exon 1) L11008 Exon GTCCGCTGTAAC-ACCGGCCCGGCG 0.1 kb L11011 Exon TGTGGAGAGCTT-CGTGACCAAGCA 2.1 kb L11013 Exon GGCGTGTGTTTG-CTGAAGCTGCAG 2.1 kb L11016 Exon TTGAGCTTGGAC-CGAGAGAATTCC 0.3 kb L11019 Exon CCCAGCCTCCTC-ACTCATAGAAGT 2.9 kb L11022 Exon GCCATCATCCAT-GGACCTCCTGGC 3.3 kb L11024 Exon CGTGGACAATCT-GGTGGAGCGCCT 3.0 kb L11025 Exon CGGCAAACGTGG-TCCTTGCAACAA 11.4 kb L11026 Exon TGCCCGAGAGCT-ACTTCGACGTGG 4.2 kb L11027 Exon TGCACCAGGCTA-TCATGCGCTGGG 0.4 kb L12572 Exon GCCACAGAAGAG-ACGGGTGTGCCC 0.7 kb L11032 Exon GACGCTGGTGTT-CCAGCCCGTGAC 0.9 kb L11035 Exon TCCAAGGCCGAG-AGAAGGAGGCCG 1.0 kb L11037 Exon CCATGCATTTTT-GAAGACCCTGGT 1.9 kb L11041 Exon GCCGCCGTTAAG-GCTGATAACACC 1.8 kb L11043 Exon CAGGGCCTCAAA-CTTGAAGTCACT Stop Codon (exon 15) Changed in version A3 (from lot A onwards). Small change in length, no change in sequence detected. The NCBI 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 P058 IGHMBP2 probemix Page 4 of 6

5 SALSA MLPA probemix P058-A3 IGHMBP2 sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA probemix P058-A3 IGHMBP2 (lot A3-1016). SALSA P058 IGHMBP2 probemix Page 5 of 6

6 Implemented Changes compared to the previous product description versions. Version November 2016 (55) - Product description adapted to a new product version (version number changed, lot number added, new picture included). - Changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Various textual changes throughout the document. Version August 2015 (54) - Various minor textual changes. - Figure(s) based on the use of old MLPA buffer (replaced in December 2012) removed. - Peak area replaced with peak height. Version 08 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 07 (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). Version 06 (48) - Various minor textual changes. Version 05 (48) - Various minor textual changes. - Remark on RefSeqGene standard added below Table 2. - Small correction of chromosomal locations in Table 1. Version 04 (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. SALSA P058 IGHMBP2 probemix Page 6 of 6