MRC-Holland MLPA. Description version 11; 01 September 2017

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1 SALSA MLPA probemix P262-B1 GHI Lot B As compared to version A2 (lot A2-1114), all reference probes have been replaced and five reference probes have been added. In addition, one STAT5B probe has been removed and several probe lengths have been adjusted. Growth hormone insensitivity (GHI) is characterized by severe short stature, normal to elevated serum levels of growth hormone (GH) and resistance to exogenous GH therapy. The aetiology of GHI is classically associated with mutations in the GH receptor (GHR) gene or with mutations affecting the signalling cascade of the GHR (OMIM ). Intracellular signalling molecules activated by GH belong to the Janus kinasesignal transducer and activator of transcription 5B (JAK2-STAT5B) pathway. Amongst others, this pathway activates the insulin-like growth factor (IGF1), which is implicated in the regulation of protein turnover and exerts potent mitogenic and differentiating effects on most cell types. There are two genomic isoforms of the GHR gene in humans: a full-length isoform (GHRfl) and an isoform lacking exon 3 (GHRd3). The distribution of these genotypes differs among populations, with the frequency for GHRfl/GHRfl ranging from 35 53%, for GHRfl/GHRd3 between 33 58%, and for GHRd3/GHRd3 ranging from 7-26%. Growth hormone is used to increase height in short children who are not deficient in growth hormone receptor, but its efficacy varies widely between individuals. Dos Santos et al. (2004, Nat Genet.) found that the GHRd3 isoform was associated with 1.7 to 2 times more growth acceleration induced by growth hormone than the full-length isoform. The GHR gene (10 exons) spans ~298 kb of genomic DNA and is located on chromosome 5p12, ~42 Mb from the p-telomere. The P262 probemix contains one probe for each GHR exon and an additional one for exon 10. Please note that one GHR probe is for exon 3, which will only be present in the GHRfl isoform. The JAK2 gene (25 exons) spans ~143 kb of genomic DNA and is located on chromosome 9p24.1, ~5 Mb from the p-telomere. The P262 probemix contains 15 JAK2 probes for 15 different exons. The IGF1 gene (5 exons) spans ~63 kb of genomic DNA and is located on chromosome 12q23.2, ~101 Mb from the p-telomere. The P262 probemix contains four IGF1 probes for different exons. The STAT5B (19 exons) spans ~77 kb of genomic DNA and is located on chromosome 17q21.2, ~38 Mb from the p-telomere. The P262 probemix contains 11 STAT5B probes for 11 different exons. 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 these genes 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 Acids 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 P262 GHI probemix Page 1 of 8

2 Related SALSA probemixes P018 SHOX: Contains probes for SHOX (Léri-Weill Dyschondrosteosis). P026 Sotos: Contains probes for NSD1 and FGFR4 (Sotos syndrome). P216 GHD mix 1: Contains probes for genes involved in growth hormone deficiency. P217 IGF1R: Contains probes for IGF1R, IGFBP3 and IGFALS. References Mul D et al. (2012). A mosaic de novo duplication of 17q21-25 is associated with growth hormone insensitivity, disturbed in vitro CD28 mediated signalling and decreased STAT5B, PI3K and NF-kB activation. Eur J Endocrinol. 166: Gorbenko del Blanco D et al. (2012). Growth hormone insensitivity syndrome caused by a heterozygous GHR mutation: phenotypic variability due to moderation by nonsense-mediated decay. Clin Endocrinol. 76: Data analysis The P262-B1 GHI probemix contains 50 MLPA probes with amplification products between 130 and 494 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. Selection of reference samples: The choice of reference samples is important for the correct determination of copy numbers using MLPA. The P262-B1 GHI probemix contains 1 probe that detects exon 3 of the GHR gene, which, apart from the normal two copies, can be either heterozygous and homozygous deleted in a substantial portion of the population. For data analysis, reference samples should be chosen that contain two copies of the sequence detected by the exon 3 probe. is unfortunately not able to supply suitable reference samples. In order to select suitable reference samples for your experiments, we recommend testing DNA from 16 healthy individuals in the first experiment. These should preferably be samples that are derived from the same type of tissue and purified by the same method as your samples to be tested. Analysis of these 16 samples using the average of these 16 samples will allow one to identify the samples that have two copies of GHR exon 3. The correct choice can be confirmed by re-analysis of the data using only these samples as reference sample. 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 P262 GHI probemix Page 2 of 8

3 Table 1. SALSA MLPA P262-B1 GHI probemix Length Chromosomal position SALSA MLPA probe (nt) reference GHR JAK2 IGF1 STAT5B 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 92 Ligation-dependent control fragment at 2q X-fragment: Specific for the X chromosome 105 Y-fragment: Specific for the Y chromosome 130 * Reference probe L p * Reference probe L q JAK2 probe L29395 Exon IGF1 probe L29906 Exon JAK2 probe L30067 Exon GHR probe L29397 Exon JAK2 probe L29398 Exon STAT5B probe L29435 Exon JAK2 probe L29399 Exon * JAK2 probe L29440 Exon * Reference probe L q «GHR probe L29400 Exon JAK2 probe L29649 Exon GHR probe L29403 Exon JAK2 probe L29402 Exon GHR probe L29438 Exon «STAT5B probe L29404 Exon STAT5B probe L09388 Exon * Reference probe L q «JAK2 probe L09389 Exon STAT5B probe L30118 Exon * STAT5B probe L30119 Exon GHR probe L30120 Exon IGF1 probe L29406 Exon IGF1 probe L09967 Exon STAT5B probe L07113 Exon * JAK2 probe L29436 Exon * Reference probe L p STAT5B probe L29407 Exon * GHR probe L30093 Exon JAK2 probe L29409 Exon * GHR probe L29432 Exon * Reference probe L q «JAK2 probe L29410 Exon GHR probe L29411 Exon «JAK2 probe L29412 Exon STAT5B probe L29413 Exon * IGF1 probe L29908 Exon STAT5B probe L29909 Exon JAK2 probe L29910 Exon GHR probe L30117 Exon STAT5B probe L30153 Exon * Reference probe L q * STAT5B probe L30154 Exon GHR probe L07078 Exon JAK2 probe L29414 Exon * «JAK2 probe L29441 Exon * Reference probe L p GHR probe L29415 Exon * Reference probe L q22 * New in version B1 (from lot B onwards). SALSA P262 GHI probemix Page 3 of 8

4 Changed in version B1 (from lot B onwards). Small change in length, no change in sequence detected. This probe is specific for GHR exon 3. It will only give a signal when the GHRfl isoform is present on one or both alleles. Please read the section Data Analysis on page 2 concerning selection of reference samples! «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 The IGF exon numbering has changed. From description version 09 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 2c. The identity of the genes detected by the reference probes is available on request: info@mlpa.com. SALSA P262 GHI probemix Page 4 of 8

5 Table 2. P262 probes arranged according to chromosomal location Table 2a. GHR Length (nt) SALSA MLPA probe GHR exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (exon 2) 209 «07422-L29400 Exon GATCAGAGGCGA-AGCTCGGAGGTA kb L29432 Exon CTGGCAGCTGCT-GTTGACCTTGGC 64.2 kb L30093 Exon nt after exon 3 AACTTTGTCCTT-TACAACATGATA 58.9 kb L29403 Exon ATCATGGTACAA-AGAACCTAGGAC 6.0 kb L30117 Exon , reverse CTTGATACAATA-AGGTATCCAGAT 4.9 kb L29411 Exon ATATCCAAGTGA-GATGGGAAGCAC 11.3 kb L29397 Exon TGTACTCATTGA-AAGTGGATAAGG 2.3 kb L07078 Exon , reverse TCCACACCTACC-TTTGCTGTTTAG 4.6 kb L30120 Exon TCTGCCCCCAGT-TCCAGTTCCAAA 0.8 kb L29438 Exon CAGTGTTATCCA-AGCAGAGAAAAA 0.1 kb L29415 Exon CATCGACTTTTA-TGCCCAGGTGAG stop codon (exon 10) This probe is specific for GHR exon 3 and will only give a signal when the GHRfl isoform is present. «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. Please read the section Data Analysis on page 2 concerning the selection of reference samples! The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2b. JAK2 Length (nt) SALSA MLPA probe JAK2 exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (exon 3) L29649 Exon CTGGTCCTCGCT-GCCGAGGGATGT 0.5 kb L29436 Exon reverse CAGTTCACATCT-TGTTCCTGTTGC 35.7 kb L29398 Exon nt before exon 3 GTGGAGTGCGGA-GGTTTGCTGCAG 8.2 kb L29910 Exon reverse ATTATGCCTGGT-TGACTCATCTAT 14.6 kb L29395 Exon ATTGCAGTGGCA-GCAACAGAGCCT 6.3 kb L29402 Exon reverse CTAACACTGCCA-TCCCAAGACATT 4.0 kb L29399 Exon CTACACAGAGAA-ATTTGAAGTAAA 10.2 kb L29409 Exon ATTACCTCTGTA-AAGAAGTAGCAC 5.0 kb L29414 Exon AACGAATGGTGT-TTCTGATGTACC 3.8 kb 472 «21157-L29441 Exon GAAGCAGCAAGT-ATGATGAGCAAG 4.6 kb 264 «07449-L09389 Exon reverse ATGAAAGGAGGA-TTTCCTGTCTTC 3.5 kb 376 «07450-L29410 Exon TGAAGACCGGGA-TCCTACACAGTT 8.8 kb 391 «07451-L29412 Exon CTCAGATATGCA-AGGTAACTAATA 32.5 kb L30067 Exon GAATCACTGACA-GAGAGCAAGTTT 4.2 kb L29440 Exon AATACCTTGGCA-TCTTGTGTGATG stop codon (exon 25) «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 represents transcript variant 1. SALSA P262 GHI probemix Page 5 of 8

6 Table 2c. IGF1 Length SALSA MLPA Ligation site Partial sequence (24 nt Distance to IGF1 exon (nt) probe NM_ adjacent to ligation site) next probe start codon (exon 1) L09967 exon AGCAATGGGAAA-AATCAGCAGTCT 4.6 kb L29406 exon 2 (3) GTCCTCCTCGCA-TCTCTTCTACCT 56.2 kb L29908 exon 3 (4) GAGGCTGGAGAT-GTATTGCGCACC 23.4 kb L29906 exon 5 (6) ATTTCCCCTGCT-ACTTTGAAACCA stop codon (exon 5) The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Note: The IGF1 exon numbering has changed. From description version 09 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for this gene. The exon numbering used in previous versions of this product description can be found between brackets in Table 2c. Table 2d. STAT5B Length (nt) SALSA MLPA probe STAT5B exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (exon 2) 240 «07454-L29404 exon nt after exon 1 CGCCTCACACCT-TCACGTTGCAAT 43.8 kb L29909 exon CATCAGATGCAA-GCGTTATATGGC 4.4 kb L30154 exon TAATCCACAGGA-GAACATTAAGGC 2.8 kb L29435 exon ATCCGCCATATA-TTGTACAATGAA 4.9 kb L29413 exon 6 95 nt before exon 6 TATCTGAGCCCA-GGAGGGTCTCGC 0.8 kb L30118 exon nt after exon 7 TGCTGTCCTGGA-GATGGGACAGGG 3.1 kb L09388 exon GCAGCCAGGACA-ACAATGCGACGG 3.9 kb L30153 exon GTGCTGTGGCCA-CAGCTGTGTGAG 1.8 kb L29407 exon nt before exon 15 GGTCTTCTCTCT-GGCATCGTAAGT 8.0 kb L30119 exon TCCCCAGGCTCA-CTATAACATGTA 2.3 kb L07113 exon GAGGAGCAGGCT-ACCCGCATCCCA stop codon (exon 19) «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. 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 P262 GHI probemix Page 6 of 8

7 SALSA MLPA probemix P262-B1 GHI sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P262-B1 GHI (lot B1-0717). SALSA P262 GHI probemix Page 7 of 8

8 Implemented Changes compared to the previous product description versions. Version September 2017 (55) - Warning added in Table 1, 209 nt probe L29400, 240 nt probe L29404, 264 nt probe L09389, 376 nt probe L29410, 391 nt probe L29412, and 472 nt probe L Probe numbers and lengths for the exon 18 and exon 19 probes of STAT5B corrected in Table 2d. - Various minor textual changes. Version August 2017 (55) - 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 on pages 1 and 2. Version December 2015 (55) - 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). - Exon numbering of the IGF1 gene has been changed in Table 1 and Table 2c. - Manufacturer s address adjusted. Version March 2015 (54) - Electropherogram pictures using the old MLPA buffer removed. - Warning on GHR exon numbering in table 2 removed. Version 07 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 06 (48) - Product description adapted to a new version (lot number added, small changes in Table 1 and Table 2, new picture included). - Recommended selection of reference samples added in section Data Analysis. - References added on page 2. - Related products added on page 2. - Various minor textual changes. - Various minor layout changes. Version 05 (46) - Warning added below Table 2 that the exon numbering used is different from the NCBI exon numbering in the NM_ reference sequence. - Remark on RefSeqGene standard and transcript variant added below Table 2. - Ligation sites of the probes targeting the GHR gene updated according to new version of the NM_reference sequence. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. SALSA P262 GHI probemix Page 8 of 8