MRC-Holland MLPA. Description version 14;

Transcription

1 SALSA MLPA KIT P018-E1 SHOX Lot As compared to version D1 (lots 0409, 0408), six probes located on chromosome X and six reference probes have been replaced. Léri-Weill Dyschondrosteosis (LWD) is a dominant skeletal disorder characterized by short stature and distinct bone anomalies. Deletions of the SHOX gene and/or regulatory elements downstream of SHOX, are detected in approximately 60% of LWD patients (Benito-Sanz et al, 2006; Huber et al, 2006; Fukami et al, 2006; Chen et al, 2009). The SHOX gene and the regulatory elements downstream of SHOX are located within the PAR1 region (Pseudo Autosomal region 1) which covers the 3000 kb of the X and Y chromosomes next to the p-telomere. This P018-E1 SHOX probemix contains probes for each exon of the human SHOX gene, as well as a probe just before the SHOX promoter region. In addition, several probes are present detecting sequences in a region downstream of SHOX which has been implicated in regulation of SHOX transcription. Furthermore, several probes on the X chromosome are included in this probemix that can be used to characterise larger deletions and to distinguish SHOX deletions from a Turner syndrome karyotype. Finally, ten autosomal reference probes are included. This SALSA kit is designed to detect copy number changes of one or more sequences in the aforementioned gene and chromosomal regions in a DNA sample. For sequences within the PAR regions and the autosomal reference probes, heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak area of the amplification product of that probe. Deletions of a probe s recognition sequence on the X-chromosome, outside the PAR regions, 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 area. Note that a mutation or polymorphism in the sequence detected by a probe can also cause a reduction in relative peak area, 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 many defects in the SHOX gene are expected to be small (point) mutations which will not be detected by this SALSA MLPA test. SALSA kits are sold by for research purposes and to demonstrate the possibilities of the MLPA technique. This kit is not CE/FDA certified for use in diagnostic procedures. SALSA MLPA kits are supplied with all necessary buffers and enzymes. Purchase of the SALSA MLPA test kits includes a limited license to use these products for research purposes. The use of this SALSA kit 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 MLPA kits P329 CRLF2-CSF2RA-IL3RA: contains probes for these PAR1 region genes. Deletions of the IL3RA and CSF2RA genes, resulting in CRLF2 overexpression, are found in Acute Lymphoblastic Leukaemia samples. P216 GHD: contains probes for GH1, PROP1, POU1F1, GHRHR, HESX1, LHX3 and LHX4. P217 IGF1R: contains probes for IGF1R and IGFBP3. P262 GHI: contains probes for IGF1, GHR, JAK2 and STAT5B. 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 kit P018 SHOX Page 1 of 6

2 References for SALSA MLPA kit P018 SHOX Chen, J. et al (2009). Enhancer mutations of the SHOX gene as a frequent cause of short stature - the essential role of a 250 kb downstream regulatory domain. J.Med.Genet. on line Benito-Sanz S. et al. (2006). PAR1 deletions downstream of SHOX are the most frequent defect in a Spanish cohort of Leri-Weill dyschondrosteosis (LWD) probands. Hum Mutat Oct;27(10):1062. Gatta V. et al. (2006). Identification and characterization of different SHOX gene deletions in patients with Leri-Weill dyschondrosteosys by MLPA assay. J Hum Genet Nov 8. Fukami, M. et al (2006). Transactivation function of an approximately 800-bp evolutionarily conserved sequence at the SHOX 3' region: implication for the downstream enhancer. Am. J. Hum. Genet. 78: Huber et al (2006). High incidence of SHOX anomalies in individuals with short stature. J Med Genet. 43(9): Schiller et al (2000) Phenotypic variation and genetic heterogeneity in Léri-Weill syndrome. Eur. J. Hum. Genet. 8: Exon numbering The exon numbering from the Genbank mrna reference sequences is used in this product description. This exon numbering is different from the SHOX exon numbering in many articles. At least 2 alternatively spliced SHOX mrna forms are known, encoding proteins with different patterns of expression. The 2 mrnas, SHOXa and SHOXb, encode for proteins of 292 and 225 amino acids, respectively. Both transcripts have a common 5' end. Transcript variant 1, as exemplified by the Genbank NM_ reference sequence, contains exons 1-6. Transcript variant 2, as exemplified by reference sequence NM_006883, contains exons Exon 6 is called 6a and exon 7 is called exon 6b in most SHOX articles. Exon 7 contains the last 42 nt of the variant 2 coding sequence. As this exon 7 sequence is entirely composed of two Alu repeats, we designed a probe in a unique intron sequence located in front of exon 7. Data analysis The P018-E1 SHOX probemix contains 43 MLPA probes with amplification products between 130 and 490 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-fragment 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 intra-normalized by dividing the peak area of each amplification product by the total area of only the reference probes in the probemix (block normalization). Ratios can be obtained by dividing the intra-normalized probe ratio in a sample by the average intra-normalized probe ratio of all reference runs. This type of normalization assumes that no changes occur in the genomic regions targeted by the reference probes. It is strongly recommended using reference and patient samples of the same sex to minimize variation, as intersex comparison makes analysis more difficult. 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 blots or long range PCR. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website This probemix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA kit P018 SHOX Page 2 of 6

3 Table 1. SALSA MLPA P018-E1 SHOX probemix Length (nt) SALSA MLPA probe Chromosomal position reference Outside PAR1 SHOX region / PAR 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 (AMOT gene) 105 Y-fragment: Specific for the Y chromosome (UTY gene) 118 Y-fragment: Specific for the Y chromosome (DBY gene) 130 Reference probe L q SHOX-area probe L05096 Xp22.33-PAR1 142 * IL3RA probe L15055 Xp22.33-PAR1 148 SHOX-area probe L06218 Xp22.33-PAR1 154 * SHOX-area probe L14642 Xp22.33-PAR1 160 * Reference probe L p SHOX probe L00702 Exon 1 PAR1 172 ± SHOX-area probe L15705 Xp22.33-PAR1 178 SHOX-area probe L15335 Xp22.33-PAR1 184 SHOX-area probe L06219 Xp22.33-PAR1 191 * Reference probe L p * SHOX-area probe L15336 Xp22.33-PAR1 204 SHOX probe L06220 Exon 2 PAR1 211 ~ PPP2R3B probe L10292 Xp22.33-PAR1 219 * Reference probe L q SHOX probe L00708 Intron 6 PAR1 231 SHOX probe L00911 Exon 6 (6a) PAR KAL1 probe L09795 Xp SHOX probe L00802 Exon 3 PAR1 257 Reference probe L q LOC probe L kb before SHOX-PAR * FANCB probe L03066 Xp NLGN4X probe L04577 Xp SHOX-area probe L06222 Xp22.33-PAR1 300 SHOX probe L15501 Exon 4 PAR1 310 ~ ASMT probe L00712 Xp22.33-PAR1 318 SHOX-area probe L05099 Xp22.33-PAR GPR143 (OA1) probe L10170 Xp SHOX probe L00910 Exon 5 PAR1 346 * Reference probe L q ~~ VAMP7 (SYBL1) probe L00659 Xq28-PAR2 364 Reference probe L p * SHOX-area probe L16348 Xp22.33-PAR1 386 CSF2RA probe L15502 Xp22.33-PAR1 392 SHOX probe L kb before exon 7 (6b) 400 * CRLF2 probe L16505 Xp22.33-PAR1 412 * Reference probe L q AIFM1 (PDCD8) probe L15506 Xq SHOX-area probe L15507 Xp22.33-PAR1 439 SHOX-area probe L15508 Xp22.33-PAR1 463 * SHOX-area probe L15510 Xp22.33-PAR1 474 * Reference probe L p * Reference probe L q31 * New in version E1 (from lot 1009 onwards). Changed in version E1 (from lot 1009 onwards). Change in length but no change in sequence detected. + X-chromosome, outside PAR region. Gives 50 % reduced signal in males as compared to females. ~ This probe has been found to be duplicated in an apparently healthy individual in one of our quality tests. ~~ This probe has been found to be deleted in an apparently healthy individual in one of our quality tests. ± The 172 nt probe detects the same sequence as the 160 nt probe in P018-D1 but has a longer hybridizing sequence. We expect that the probe signal is no longer influenced by SNP rs In case of apparent deletions, it is recommended to sequence the region targeted by this probe. SALSA kit P018 SHOX Page 3 of 6

4 Table 2. SHOX probes arranged according to chromosomal location Length SALSA MLPA Ligation site Partial sequence (24 nt Distance to Gene / exon (nt) probe NM_ adjacent to ligation site) next probe P-telomere / start PAR1 region 227 kb 211 ~ L10292 PPP2R3B gene CGTCCGAGTTCC-ACTCGCGCTACA kb L06221 SHOX region 4.7 kb before start LOC SHOX gene GCCTGGAACAGA-ACTTCCGCGGGG 4.7 kb SHOX startcodon L00702 SHOX exon TTTCTACTGCAA-ACAGAAATGGGA 6.7 kb L06220 SHOX exon ACCACGTAGACA-ATGACAAGGAGA 3.6 kb L00802 SHOX exon CGGGCAGACCAA-GCTGAAACAGAG 6.2 kb L15501 SHOX exon CAGAACCGGAGA-GCCAAGTGCCGC 0.2 kb L00910 SHOX exon ACAGCCAACCAC-CTAGACGCCTGC 3.5 kb L00911 SHOX exon 6 (6a) AAGCAACAGCAA-GAATTCCAGCAT 6.4 kb SHOX stopcodon L00708 SHOX intron kb after exon 6 TGGCTTCACGAG-TTCAGCCCATTG 6.4 kb L15503 SHOX exon 7 (7b) 1.4 kb before exon 7 (only present in TCCCACATTCTT-GGAATCACAATG 56.8 kb NM_ ) L05096 Xp22.32-PAR1 GCAGCAGTGAAA-GTGAGCATTCCC 19.8 kb L14642 Xp22.32-PAR1 GATGGCTGATAA-TTACTCCGTATG 11.4 kb 172 ± L15705 Xp22.32-PAR1 ACACCACCAGAGT-TACTTGAATCAA 45.4 kb L15336 Xp22.32-PAR1 GGAAAACCACGT-TCCTATCGATCC 57.7 kb L05099 Xp22.32-PAR1 TGTTCCCACCGT-AAAACTCACTCC 8.5 kb L15507 Xp22.32-PAR1 TGCATGTCTGCT-TTTTGAATGGCC 10.7 kb L15510 Xp22.32-PAR1 TACAGCAAATGA-TACGTATAAATT 6.3 kb L06222 Xp22.32-PAR1 CTTGAAAGGGCA-GGAACTCTAATT 0.4 kb L06219 Xp22.32-PAR1 TAATTGATGAGA-TGCAGAAGCCAG 15.4 kb L06218 Xp22.32-PAR1 TGGTGCTGAAAT-GAGGAAGCCCTG 48.7 kb L15335 Xp22.32-PAR1 TGAGGAGGTACC-TCAAAGCTAAAC 64.4 kb L15508 Xp22.32-PAR1 GAAATTCAGTTT-TAATAACACAGA 66.0 kb L16348 Xp22.32-PAR1 CTCTGGTGAGAT-GCCATCTAGAGA kb L16505 CRLF2 gene GAATGCCAGCAA-ATACTCCAGGAC 73.9 kb L15502 CSF2RA gene GACAAGCCTTCT-GCTCTGTGAGTT 69.8 kb L15055 IL3RA gene TGCACAGATAAG-TTTGTCGTCTTT kb 310 ~ L00712 ASMT gene GACATCCCAGAA-GTGGTGTGGACG kb End of PAR1 region L04577 NLGN4X gene Xp22.32 GACGGCTTGGGT-GATGCACGAAAT kb L09795 KAL1 gene Xp22.31 GTTTCCTGAAGC-GTGTGCCCACAA kb L10170 GPR143 gene Xp22.2 GGGCGCTGGAGT-CCCAACTTACCT kb L03066 FANCB gene Xp22.2 TCTCATCAGAAT-TCTCCCTATAAA kb L15506 AIFM1 gene Xq25 TATTGGTCTTGT-GGACAGTAGTTT kb Start of PAR2 region Very close to 355~~ L00659 VAMP7 gene (PAR2) Xq28 TGTGGGAAAAGT-GTTTCCATTCTG q-telomere + X-chromosome, outside PAR region. Gives halve the signal in males as compared to females. ~ This probe has been found to be duplicated in an apparently healthy individual in one of our quality tests. ~~ This probe has been found to be deleted in an apparently healthy individual in one of our quality tests. ๑ There are two major SHOX transcripts. The Genbank NM_ transcript includes exon 6 (Va) but lacks exon 7. An alternative transcript, exemplified by sequence NM_ , includes exon 7 but lacks exon 6. ± The 172 nt probe detects the same sequence as the 160 nt probe in P018-D1 but has a longer hybridizing sequence. We expect that the probe signal is not longer influenced by SNP rs In case of apparent deletions, we recommend to sequence the region targeted by this probe. The VAMP7 probe at 355 nt is located very close to the q-telomere of X and Y in the PAR2 pseudoautosomal region. These five probes are in the putative SHOX regulatory region (CNE7-9) described by Chen J. (2009) J.Med.Genet. online. The 290 and 184 nt probes are within the putative regulatory region identified by Fukami, M. et al (2006) Am. J. Hum. Genet. 78: SALSA kit P018 SHOX Page 4 of 6

5 Note: Exon numbering is according to the NCBI NM_ reference sequences and might be different from the literature! Several probes are only included to facilitate determination of the extend of a deletion / duplication. Copy number alterations of certain probes may not be related to the condition being tested. Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. The following changes in the relative probe signals (as compared to the autosomal reference probes indicated in table 1) are expected when MALE reference DNA is used: 1. An approx. 100% increase in signal of the chromosome X probes outside the PAR regions (marked + in table 2) and a complete loss of the Y probe signals in female DNA. The signals of the SHOX probes, the other probes in the PAR1 region and the VAMP7 (SYBL) probe in the PAR2 region at the q telomere should remain unchanged as the Y chromosome also contains a copy of the PAR1 and PAR2 regions. 2. A 40-60% reduction in signal of one or more SHOX probes, in case of a (partial) deletion of the SHOX gene. These deletions will in general cause Leri-Weill dyschondrosteosis. 3. A 40-60% reduction in signal of one or more probes downstream of SHOX, indicated with Xp22.32-PAR1 in table 2. Deletions in this region have been associated with Leri-Weill dyschondrosteosis (Benito-Sanz et al (2005) Am. J. Hum. Genet. 77, ). We included numerous probes in this region, as the exact location of the SHOX regulatory region is not 100% certain. The most likely region is marked with in table 2. Please note that not all deletions detected by the Xp22.32-PAR1 probes will result in Leri Weill dyschondrosteosis. 4. A 40-60% reduction in signal of the SHOX probes and all other PAR1 probes and a complete loss of one or more other Xp probes in case of deletions of Xp that extend outside PAR1. 5. A complete loss of the Y probe signals and a 40-60% reduction of the SHOX, the PAR1 and the PAR2 probes in Turner syndrome (X0). 6. An approx. 100 % increase in signal of the X probes outside the PAR regions and a 40-60% increase in signal of the SHOX and the PAR1 and PAR2 probes in Klinefelter syndrome (XXY). 7. An approx. 200 % increase in signal of the X probes outside the PAR regions, the disappearance of the Y probe signals and a 40-60% increase in signal of the SHOX and the PAR1 and PAR2 probes in Triple X syndrome (XXX). The following changes in the relative probe signals (as compared to the autosomal reference probes indicated in table 1) are expected when FEMALE reference DNA is used: 1. A 40-60% decrease in signal of the chromosome X probes outside the PAR regions (marked + in table 2) and the appearance of Y probe signals in male DNA. The signals of the SHOX probes, the other probes in the PAR1 region and the VAMP7 (SYBL) probe in the PAR2 region at the q telomere should remain unchanged as the Y chromosome also contains a copy of the PAR1 and PAR2 regions. 2. A 40-60% reduction in signal of one or more SHOX probes, in case of a (partial) deletion of the SHOX gene. These deletions will in general cause Leri-Weill dyschondrosteosis. 3. A 40-60% reduction in signal of one or more probes downstream of SHOX, indicated with Xp22.32-PAR1 in table 2. Deletions in this region have been associated with Leri-Weill dyschondrosteosis (Benito-Sanz et al (2005) Am. J. Hum. Genet. 77, ). We included numerous probes in this region, as the exact location of the SHOX regulatory region is not 100% certain. The most likely region is indicated in table 2. Please note that not all deletions detected by the Xp22.32-PAR1 probes will result in Leri Weill dyschondrosteosis. 4. A 40-60% reduction in signal of the SHOX probes and all other PAR1 probes and a 40-60% decrease in signal of one or more other Xp probes in case of deletions of Xp that extend outside the PAR1 region. 5. A 40-60% reduction of all chromosome X probes in Turner syndrome (X0). 6. The appearance of Y probe signals and a 40-60% increase in signal of the SHOX and the PAR1 and PAR2 probes in Klinefelter syndrome (XXY). 7. A % increase in signal of all chromosome X probes (all probes in table 2) in Triple X syndrome (XXX). SALSA kit P018 SHOX Page 5 of 6

6 SALSA MLPA kit P018-E1 SHOX sample pictures ,83 134,05 146,38 158,45 141,48 177, ,01 105,37 128,37 171,88 211,43 230,30 434, ,72 100,49 115,31 197,76 225,00 190,56 152,09 264,67 183,49 218,03 204,16 257,72 164,85 244,65 289,56 298,94 309,58 334,35 393,34 369,81 412,37 345,66 386,60 363,53 400,76 439,79 464,42 475,64 491,80 238,05 317,61 354, ,55 327,67 421,18 274, Dye Signal Size (nt) Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analyzed with SALSA MLPA kit P018-E1 SHOX (lot 1009) ,96 158,47 146, ,46 95,14 134,03 141,45 128,29 171,90 177,52 211,51 230,47 225, ,76 204,22 238,09 334,38 434,40 190,62 152,10 218,14 264,65 439,89 298,93 197,75 257,69 393,38 183,55 345,58 289,55 244,79 327,66 369,84 421,12 164,89 281,54 309,54 412,42 386,56 274,18 363,58 464,37 475,54 491,71 354,51 400,75 317, Dye Signal Size (nt) Figure 2. Capillary electrophoresis pattern from a sample of approximately 50 ng human female control DNA analyzed with SALSA MLPA kit P018-E1 SHOX (lot 1009). Implemented Changes the following has been altered compared to the previous product description version(s). Version 14 (45) - Minor textual change of page 5 (The following changes.). Version 13 (45) - This product description has been changed to incorporate a new version (lot number added, changes in Table 1 and Table 2, new pictures included). - Various minor textual changes on page 1; various minor layout changes. Tables have been numbered. - Small changes of probe lengths in Table 1 & 2. Version 12 (43) - This product description version belonged to P018-X1, a non-commercial test version SALSA kit P018 SHOX Page 6 of 6