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MRC-Holland Product Description version G1-03; Issued 23 February 2017 ® MLPA Product Description SALSA® MLPA® probemix P018-G1 SHOX To be used with the MLPA General Protocol. Version G1. One SHOX area probe has been replaced (CNE4) and four new probes have been included for the upstream SHOX enhancer sequences CNE-2, CNE-3 and CNE-5 and the downstream CNE7 enhancer sequence. For complete product history see page 10. Catalogue numbers: • P018-025R: SALSA® MLPA® probe mix P018 SHOX, 25 reactions. • P018-050R: SALSA® MLPA® probe mix P018 SHOX, 50 reactions. • P018-100R: SALSA® MLPA® probe mix P018 SHOX, 100 reactions. To be used in combination with a SALSA® MLPA® reagent kit, available for various number of reactions. MLPA reagent kits are either provided with FAM or Cy5.0 dye-labelled PCR primer, suitable for Applied Biosystems and Beckman capillary sequencers, respectively (see www.mlpa.com). Certificate of Analysis: Information regarding storage conditions, quality tests, and a sample electropherogram from the current sales lot is available at www.mlpa.com. Precautions and warnings: For professional use only. Always consult the most recent product description AND the MLPA General Protocol before use: www.mlpa.com. It is the responsibility of the user to be aware of the latest scientific knowledge of the application before drawing any conclusions from findings generated with this product. Intended use: The SALSA® MLPA® probemix P018 SHOX is an in vitro diagnostic (IVD)1 or research use only (RUO) assay for the detection of deletions or duplications in the human short stature homeobox (SHOX) gene and its regulatory regions located on Xp22.33/Yp11.32 as a cause for disorders associated with short stature, including Leri-Weill dyschondrosteosis (LWD), Langer mesomelic dysplasia (LMD), and Idiopathic short stature (ISS). This assay can be used with human DNA derived from peripheral blood and buccal swab. In the majority of patients, defects in the SHOX gene region are deletions, but point mutations can occur which will not be detected by MLPA. It is therefore recommended to use this SALSA® MLPA® probemix in combination with sequence analysis of the SHOX coding region. Copy number changes detected by only a single probe always require validation by another method. This probemix is not intended to be used as a standalone assay for clinical decisions. The results of this test should be interpreted by a clinical molecular geneticist or equivalent. 1 Please note that this probemix is for In Vitro Diagnostic use (IVD) in the countries specified at the end of this product description. In all other countries, the product is for Research Use Only (RUO). Clinical background: SHOX is located in the pseudoautosomal region 1 (PAR1) on the short arm of the X and Y chromosomes. Located upstream and downstream of SHOX are highly conserved non-coding elements (CNEs), important SHOX enhancer sequences. Mutations in SHOX or its regulatory regions cause a range of disorders associated with short stature, including LWD, LMD, and ISS, as SHOX is a known transcription factor highly expressed in tissues responsible for bone development (Benito-Sanz et al. 2012b). LWD is a dominant skeletal disorder characterised by short stature, mesomelic shortening of the limbs, and the characteristic Madelung deformity. LMD is a more severe form of LWD and is a result of mutations in both SHOX alleles (Bertorelli et al. 2007, Campos-Barros et al. 2007, Shears et al. 2002, Zinn et al. 2002). ISS classifies individuals with a height below the third centile when in the absence of an identified cause. Heterozygous mutations of SHOX and/or its regulatory elements are detected in approximately 60% of LWD patients and approximately 5-15% of ISS cases. Homozygous or compound heterozygous mutations of SHOX and/or its downstream enhancers are detected in 75% of LMD patients (Benito-Sanz et al. 2006, Benito-Sanz et al. 2012a, Chen et al. 2009, Huber et al. 2006). SALSA MLPA Probemix P018 SHOX Page 1 of 11 MRC-Holland Product Description version G1-03; Issued 23 February 2017 ® MLPA In individuals with a SHOX related disorder, 70-80% of mutations are due to a large deletion, 2-6% is from a partial deletion, and 20-25% are from point mutations (Binder 2011, Caliebe et al. 2012). Duplications have also been reported in LWD and ISS patients (Benito-Sanz et al. 2011b). The MLPA technique can detect most deletions and duplications and therefore complements sequence analysis of SHOX. More information is available on http://www.ncbi.nlm.nih.gov/books/NBK1215/. Gene structure and Transcript variants: SHOX spans 35 kb of the pseudoautosomal region 1 (PAR1) located on Xp22.33/Yp11.32. The Genbank chromosomal sequence is NG_009385.1. Two major transcript variants have been described: http://www.ncbi.nlm.nih.gov/gene/6473. SHOX transcript variant 1 (NM_000451.3, 3757 nt, coding sequence 692-1570) represents the longer transcript and encodes the longer active isoform (SHOXa). The ATG translation start site is located in exon 2 and the termination site is located in exon 6. The second transcript variant NM_006883.2 (1951 nt, coding sequence 692-1369) contains an alternate 3' terminal exon compared to transcript variant 1 and encodes a shorter isoform (SHOXb) with a different C-terminus compared to isoform SHOXa. Several regulatory sequences located outside of SHOX and affecting SHOX transcription have been described (e.g. Benito-Sanz et al. 2012b, Durand et al. 2010, Fukami et al. 2006, Sandbacka et al. 2011). Exon numbering: The exon numbering used in this P018-G1 SHOX product description and in the P018-G1 SHOX Coffalyser.Net analysis sheet is the exon numbering from the Genbank NG_009385.1 reference sequence. This exon numbering is different from the SHOX exon numbering in many articles where exon 7 is referred to as exon 6b. P018-G1 probemix content: This SALSA® MLPA® probemix P018 SHOX contains 48 MLPA probes with amplification products between 124 and 503 nt: 26 probes located in the SHOX + Xp22 areas (including SHOX and its regulatory regions); 13 probes elsewhere on the X-chromosome and 9 reference probes detecting autosomal chromosomes (table 1 and table 2). One probe is present for each exon of human SHOX. In addition, one probe is included detecting the area just before the SHOX promoter region. Furthermore, several probes are present for the regions that have been identified to be SHOX regulatory regions, located upstream and downstream of SHOX. Finally, several probes for 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. This probemix contains ten quality control fragments generating amplification products between 64 and 118 nt: four DNA Quantity Fragments (Q-Fragments), three DNA Denaturation Fragments (D-Fragments), one chromosome X and two chromosome Y-specific fragments (Table 1). The Q-fragments are only visible when less than 100 ng sample DNA is used. Low signal of the 88 or 96 nt fragment indicates incomplete DNA denaturation. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol. MLPA technique: The principles of the MLPA technique (Schouten et al. 2002) are described in the MLPA General Protocol (www.mlpa.com). MLPA technique validation: Internal validation of the MLPA technique using 16 DNA samples from healthy individuals of the same sex is required, in particular, when using MLPA for the first time, or when changing the sample handling procedure, DNA extraction method or instruments used. This validation experiment should result in a standard deviation <0.10 for all probes over the experiment. Required specimens: Purified DNA from peripheral blood or buccal swab, free from impurities known to affect MLPA reactions. For more information please refer to the section on DNA sample treatment found in the MLPA General Protocol. Reference samples: Reference DNA samples should be derived from the same tissue type, handled using the same procedure, and prepared using the same DNA extraction method as the patient samples. Reference samples should be derived from unrelated individuals who are from families without a history of short stature. All reference samples should be of the same sex. It is recommended to use reference and SALSA MLPA Probemix P018 SHOX Page 2 of 11 MRC-Holland ® MLPA Product Description version G1-03; Issued 23 February 2017 patient samples of the same sex to facilitate interpretation. More information regarding the selection and use of reference samples can be found in the MLPA General Protocol. Positive control DNA samples: MRC-Holland cannot provide positive DNA samples. Inclusion of a positive sample in each experiment is recommended. Performance characteristics: In individuals with a SHOX related disorder, 70-80% of all mutations are due to a large deletion and should be detected by this MLPA probemix (Binder 2011, Caliebe et al. 2012). The analytical sensitivity and specificity for the detection of deletions/duplications in SHOX and its surrounding enhancer regions (based on a 2006-2014 literature review) is very high and can be considered >99%. Analytical performance can be compromised by: SNPs or other polymorphisms (e.g. indels) in the DNA target sequence, impurities in the DNA sample, incomplete DNA denaturation, the use of insufficient or too much sample DNA, the use of insufficient or unsuitable reference samples, problems with capillary electrophoresis or a poor data normalisation procedure and other technical errors. The MLPA General Protocol contains technical guidelines and information on data evaluation/normalisation. Data analysis: Coffalyser.Net software must be used for data analysis in combination with the appropriate lot-specific MLPA Coffalyser sheet. For both, the latest version should be used which are freely downloadable at www.mlpa.com. Use of other non-proprietary software may lead to inconclusive or false results. For more details on MLPA quality control and data analysis, see the Coffalyser.Net Manual. Interpretation of results: The expected results for SHOX-specific MLPA probes are allele copy numbers of 2 (normal), 0 (homozygous deletion), 1 (heterozygous deletion), 3 (heterozygous duplication), and 4 (heterozygous triplication/homozygous duplication). Please see table 3 for examples of potential results. The standard deviation of all probes in the reference samples should be <0.10 and the dosage quotient (DQ) of the reference probes in the patient samples should be between 0.80 and 1.20. When these criteria are fulfilled, the following cut-off values for the DQ of the probes can be used to interpret MLPA results: Copy Number status Normal Homozygous deletion Heterozygous deletion Heterozygous duplication Heterozygous triplication/Homozygous duplication Ambiguous copy number - - - - - Dosage quotient 0.80 < DQ < 1.20 DQ = 0 0.40 < DQ < 0.65 1.30 < DQ < 1.65 1.75 < DQ < 2.15 All other values Arranging probes according to chromosomal location facilitates interpretation of the results and may reveal more subtle changes such as those observed in mosaic cases. Analysis of parental samples may be necessary for correct interpretation of complex results. False positive results: Please note that abnormalities detected by a single probe (or multiple consecutive probes) still have a considerable chance of being a false positive result. Incomplete DNA denaturation, due to salt contamination, can lead to a decreased probe signal, in particular for probes located within a CpG island in or near the SHOX gene. The use of an additional purification step or an alternative DNA extraction method may resolve such cases. False positive duplication results: Contamination of DNA samples with cDNA or PCR amplicons of individual exons can lead to false positive duplication results (Varga et al. 2012). Analysis of an independently collected secondary DNA sample can exclude these kinds of contamination artefacts. Copy number variation in healthy individuals has been found using this probemix. Normal copy number variation in healthy individuals is described in the database of genomic variants: http://dgv.tcag.ca/dgv/app/home. Users should always verify the latest updates of the database and scientific literature when interpreting their findings. Not all abnormalities detected by MLPA are pathogenic. In some genes, intragenic deletions are known that result in very mild, or no disease (Schwartz et al. 2007). For many genes, more than one transcript variant exists. Copy number changes of exons that are not present in all transcript variants may not SALSA MLPA Probemix P018 SHOX Page 3 of 11 MRC-Holland Product Description version G1-03; Issued 23 February 2017 - ® MLPA have clinical significance. Duplications that include the first or last exon of a gene (e.g. exons 1-3) might in some cases not result in inactivation of that gene copy. Copy number changes detected by reference probes are unlikely to have any relation to the condition tested for. Notes SHOX results: - Short stature and skeletal deformities due to SHOX defects are pseudoautosomal dominant disorders. Therefore, a heterozygous mutation of SHOX is expected to result in these phenotypes. - A recurrent 47.5 kb deletion downstream of SHOX has been described by Benito-Sanz et al. (2012b). This 47.5 kb deletion is covered by three probes in this P018-G1 probemix (Table 2). - Complete or partial duplications found within SHOX or its surrounding regulatory regions have been found in LWD and ISS patients (Benito-Sanz et al. 2011b). - Deletion of the SHOX intron 6 probes, which are located downstream of the stop codon, may not affect SHOX gene function. - Please note that single exon deletions have a considerable chance of being a false positive result, either due to non-pathogenic copy number variants (Benito-Sanz et al. 2011a), mutations within the probe binding site (Barca-Tierno et al. 2011) or due to impurities in the DNA. - Not all copy number changes detected by the “SHOX-area” probes will affect SHOX gene function. Analysis of family members may be required for correct interpretation of results. Limitations of the procedure: − MLPA cannot detect any changes that lie outside the target sequence of the probes and will not detect most inversions or translocations. Even when MLPA did not detect any aberrations, the possibility remains that biological changes in that gene or chromosomal region do exist but remain undetected. − Sequence changes (e.g. SNPs, point mutations, small indels) in the target sequence detected by a probe can cause false positive results. Mutations/SNPs (at least up to 20 nt from the probe ligation site) can reduce the probe signal by preventing ligation of the probe oligonucleotides or by destabilising the binding of a probe oligonucleotide to the sample DNA. Confirmation of results: Copy number changes detected by only a single probe always require confirmation by another method. An apparent deletion detected by a single probe can be due to e.g. a mutation/polymorphism that prevents ligation or destabilises the binding of probe oligonucleotides to the DNA sample. Sequence analysis can establish whether mutations or polymorphisms are present in the probe target sequence. The finding of a heterozygous mutation or polymorphism indicates that two different alleles of the sequence are present in the sample DNA and that a false positive MLPA result was obtained. Copy number changes detected by one or more than one consecutive probe should be confirmed by another independent technique such as long range PCR, qPCR, array CGH or Southern blotting, whenever possible. Deletions/duplications of more than 50 kb in length can often be confirmed by FISH. SHOX mutation database: https://grenada.lumc.nl/LOVD2/MR/home.php?select_db=SHOX. We strongly encourage users to deposit positive results in the X-chromosome gene database. Recommendations for the nomenclature to describe deletions/duplications of one or more exons can be found on www.hgvs.org/mutnomen/. Please report copy number changes detected by the reference probes, false positive results due to SNPs and unusual results (e.g. a duplication of SHOX exons 3 and 5, but not exon 4) to MRC-Holland: [email protected]. SALSA MLPA Probemix P018 SHOX Page 4 of 11 MRC-Holland ® MLPA Product Description version G1-03; Issued 23 February 2017 Table 1. SALSA® MLPA® P018-G1 SHOX probemix Length (nt) SALSA MLPA probe Chromosomal position(a) reference Outside PAR1 SHOX region / PAR1 64-70-76-82 Q-fragments (Only visible with <100 ng sample DNA) 88-92-96 D-fragments (Low signal of 88 or 96 fragment indicates incomplete denaturation) 100 X-fragment (X chromosome specific) 105 Y-fragment (Y chromosome specific) 118 Y-fragment (Y chromosome specific) 124 * Reference probe 15370-L13762 7q11 130 * SHOX AREA probe 18885-L24430 Xp22-PAR1 136 SHOX AREA probe 05642-L05096 Xp22-PAR1 142 IL3RA probe 13597-L15055 Xp22-PAR1 148 SHOX AREA probe 05648-L06218 Xp22-PAR1 154 SHOX AREA probe 13821-L14642 Xp22-PAR1 160 Reference probe 04966-L04696 1p22 166 SHOX probe 01145-L00702 Exon 1 Xp22-PAR1 172 * SHOX AREA probe 18886-L24431 Xp22-PAR1 178 SHOX AREA probe 05649-L20176 Xp22-PAR1 185 SHOX AREA probe 06293-L20177 Xp22-PAR1 191 Reference probe 06057-L05512 4p16 199 SHOX AREA probe 13296-L20175 Xp22-PAR1 204 SHOX probe 01146-L06220 Exon 2 Xp22-PAR1 211 PPP2R3B probe 09333-L10292 Xp22-PAR1 219 Reference probe 03247-L02684 13q14 226 SHOX probe 09336-L20178 Intron 6 Xp22-PAR1 231 SHOX probe 09337-L00911 Exon 6 Xp22-PAR1 238 ANOS1 (KAL1) probe 06402-L09795 Xp22.31 245 SHOX probe 01147-L00802 Exon 3 Xp22-PAR1 254 ARSF probe 16846-L20647 Xp22.33 261 Reference probe 00587-L20649 18q21 266 SHOX probe 01341-L20651 4 kb before SHOX-PAR1 274 FANCB probe 03906-L03066 Xp22.2 283 NLGN4X probe 05587-L04577 Xp22.31 290 SHOX AREA probe 06291-L06222 Xp22-PAR1 300 SHOX probe 01148-L15501 Exon 4 Xp22-PAR1 310 ASMT probe 01153-L00712 Xp22-PAR1 318 SHOX AREA probe 05645-L05099 Xp22-PAR1 328 PRKX probe 16898-L19768 Xp22.33 337 ∆ SHOX probe 01149-L19676 Exon 5 Xp22-PAR1 346 Reference probe 06560-L06118 1q32 355 VAMP7 probe 01156-L00659 Xq28-PAR2 364 * SHOX AREA probe 18889-L25087 Xp22-PAR1 379 ¥ SHOX AREA probe 14697-L24245 Xp22-PAR1 387 ¥ CSF2RA probe 10251-L24246 Xp22-PAR1 395 ¥ SHOX probe 09338-L24247 Intron 6 Xp22-PAR1 403 CRLF2 probe 13911-L19678 Xp22-PAR1 412 Reference probe 09793-L12593 15q21 420 ¥ AIFM1 probe 00820-L25090 Xq26.1 427 * SHOX AREA probe 18891-L25088 Xp22-PAR1 439 ¥ SHOX AREA probe 05646-L24249 Xp22-PAR1 445 ¥ SHOX AREA probe 09335-L24250 Xp22-PAR1 456 ¥ ZBED1 probe 16858-L25227 Xp22-PAR1 466 ¥ SHOX AREA probe 13297-L24253 Xp22-PAR1 474 Reference probe 09888-L10301 16p13 481 * SHOX AREA probe 18893-L25091 Xp22-PAR1 503 * Reference probe 09870-L19465 2p16 (a) The exon numbering used in this P018-G1 SHOX product description and in the P018-G1 SHOX Coffalyser.Net analysis sheet is the exon numbering from the Genbank NG_009385.1 reference sequence. This exon numbering is SALSA MLPA Probemix P018 SHOX Page 5 of 11 MRC-Holland ® MLPA Product Description version G1-03; Issued 23 February 2017 different from the SHOX exon numbering in many articles where exon 7 is referred to as exon 6b. * New in version G1 (from lot G1-0513 onwards). ¥ Changed in version G1 (from lot G1-0513 onwards). Small change in length, but no change in sequence detected. ∆ This probe has been reported to be more variable. Table 2. Xp/Yp probes arranged according to chromosomal location Length (nt) SALSA MLPA probe Gene Exon(a) Ligation site(b) Partial sequence(c) (24 nt adjacent to ligation site) p-telomere ------------------------------------------ Start of PAR1 -----------------------------------------211 ¬ 09333-L10292 PPP2R3B gene PAR1 CGTCCGAGTTCC-ACTCGCGCTACA 364 18889-L25087 SHOX AREA CNE-5 GAAATGTTAACA-GCTCCCCGAGCT 130 18885-L24430 SHOX AREA CNE-3 ATGGCAGAGCAT-TTGTACCCCTGG 427 18891-L25088 SHOX AREA CNE-2 TACACCGTTATG-CGGATGCTCGTT SHOX AREA 266 01341-L20651 Upstream of SHOX GCCTGGAACAGA-ACTTCCGCGGGG LOC159015 SHOX start codon 166 204 245 300 # 337 ∆ 231 01145-L00702 01146-L06220 01147-L00802 01148-L15501 01149-L19676 09337-L00911 Exon Exon Exon Exon Exon Exon 1 2 3 4 5 6 stop codon 226 09336-L20178 SHOX Intron 6 Distance to next probe 227.0 kb 91.6 61.7 56.3 63.5 4.7 kb NM_000451.3 692-694 (Ex 2) 99-100 920-921 1032-1033 1198-1199 1261-1262 1506-1507 TTTCTACTGCAA-ACAGAAATGGGA ACCACGTAGACA-ATGACAAGGAGA CGGGCAGACCAA-GCTGAAACAGAG CAGAACCGGAGA-GCCAAGTGCCGC ACAGCCAACCAC-CTAGACGCCTGC AAGCAACAGCAA-GAATTCCAGCAT 6.7 3.6 6.2 0.2 3.5 6.4 ¬ x ¬ 06402-L09795 ANOS1 (KAL1) gene FANCB gene AIFM1 gene kb kb kb kb kb kb TGGCTTCACGAG-TTCAGCCCATTG 6.4 kb 1568-1570 (Ex 6) 6.4 kb after Ex 6; 8 kb before Ex 7 1.4 kb before Ex 7 6.2 kb after CNE2 CNE3 CNE4 CNE5 CNE7 2 kb before CNE8 5.4 kb after CNE8 6 kb before CNE9 CNE9 CNE9 395 09338-L24247 SHOX Intron 6 TCCCACATTCTT-GGAATCACAATG 56.9 136 05642-L05096 SHOX AREA GCAGCAGTGAAA-GTGAGCATTCCC 19.8 154 13821-L14642 SHOX AREA GATGGCTGATAA-TTACTCCGTATG 19.4 172 18886-L24431 SHOX AREA GCCTCCGATACA-GTTTACGGCTTC 37.4 199 13296-L20175 SHOX AREA GGAAAACCACGT-TCCTATCGATCC 29.6 481 ^ 18893-L25091 SHOX AREA CAGACCAGGTCT-CCTGTTTCATGT 28.1 318 ^ 05645-L05099 SHOX AREA TGTTCCCACCGT-AAAACTCACTCC 8.4 439 ^ 05646-L24249 SHOX AREA TGCATGTCTGCT-TTTTGAATGGCC 10.7 466 13297-L24253 SHOX AREA TACAGCAAATGA-TACGTATAAATT 6.3 290 06291-L06222 SHOX AREA CTTGAAAGGGCA-GGAACTCTAATT 0.4 185 06293-L20177 SHOX AREA TAATTGATGAGA-TGCAGAAGCCAG 15.4 148 05648-L06218 Xp22-PAR1 Downstream TGGTGCTGAAAT-GAGGAAGCCCTG 48.7 178 05649-L20176 Xp22-PAR1 Downstream TGAGGAGGTACC-TCAAAGCTAAAC 64.4 445 09335-L24250 Xp22-PAR1 Downstream GAAATTCAGTTT-TAATAACACAGA 66.0 379 14697-L24245 Xp22-PAR1 Downstream CTCTGGTGAGAT-GCCATCTAGAGA 338.0 403 ¬ 13911-L19678 CRLF2 gene PAR1-Xp22PAR GAATGCCAGCAA-ATACTCCAGGAC 73.9 387 ¬ 10251-L24246 CSF2RA gene PAR1-Xp22PAR GACAAGCCTTCT-GCTCTGTGAGTT 69.8 142 ¬ 13597-L15055 IL3RA gene PAR1-Xp22PAR TGCACAGATAAG-TTTGTCGTCTTT 280.7 310 ¬ 01153-L00712 ASMT gene PAR1-Xp22PAR GACATCCCAGAA-GTGGTGTGGACG 706.4 456 ¬ 16858-L25227 ZBED1 gene PAR1-Xp22PAR TCGTCAAGAGCA-ACACGGAGCAGA 593.8 ------------------------------------------------- End of PAR1 ------------------------------------------------254 x ¬ 16846-L20647 ARSF gene Xp22.33 CATCCATATAAT-TATGGGTTTGAC 536.9 328 x ¬ 16898-L19768 PRKX gene Xp22.33 CGATTAGGAAAC-ATGAAGGTCAGT 2606.5 283 x ¬ 05587-L04577 NLGN4X gene Xp22.31 GACGGCTTGGGT-GATGCACGAAAT 2311.9 238 x kb kb kb kb Xp22.31 GTTTCCTGAAGC-GTGTGCCCACAA kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb 6304.2 kb 274 03906-L03066 Xp22.2 TCTCATCAGAAT-TCTCCCTATAAA 114321.4 kb 420 x ¬ 00820-L25090 Xq26.1 TATTGGTCTTGT-GGACAGTAGTTT 25732.3 kb -------------------------------------------------Start of PAR2 ------------------------------------------------355 + ¬ 01156-L00659 VAMP7 gene PAR2 TGTGGGAAAAGT-GTTTCCATTCTG 98 kb SALSA MLPA Probemix P018 SHOX Page 6 of 11 MRC-Holland Product Description version G1-03; Issued 23 February 2017 ® MLPA q-telomere (a) The exon numbering used in this P018-G1 SHOX product description and in the P018-G1 SHOX Coffalyser.Net analysis sheet is the exon numbering from the Genbank NG_009385.1 reference sequence. This exon numbering is different from the SHOX exon numbering in many articles where exon 7 is referred to as exon 6b. (b) Ligation sites of the P018 SHOX MLPA probes are indicated according to Refseq sequence NM_000451.3 containing 7 exons. (c) Only partial probe sequences are shown. Complete probe sequences are available at www.mlpa.com. Please notify us of any mistakes: [email protected]. ∆ This probe has been reported to be more variable. A founder SHOX point mutation in the Spanish Gypsy population, c.508G>C (p.A170P), results in a strongly reduced probe signal of the 300 nt exon 4 probe (Barca-Tierno et al. 2011). ^ The 481, 318 and 439 probes are within the recurrent ~47.5 kb deletion region described by Benito-Sanz et al. (2012b). x X-chromosome, outside PAR region. Gives 50% reduced signal in males as compared to females. + The VAMP7 probe at 355 nt is located very close to the X and Y q-telomere in PAR2. ¬ Flanking probe. Included to help determine the extent of a deletion/duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition tested. # CNE = Conserved Non-coding DNA Element. Locations of the upstream regulatory regions (CNE-2, CNE-3 and CNE-5) are based on Durand et al. (2010) and Benito-Sanz et al. (2012b). Locations of the downstream regulatory regions (CNE2 through CNE9) are based on Benito-Sanz et al. (2012b), Fukami et al. (2006), and Sabherwal et al. (2007). SALSA MLPA Probemix P018 SHOX Page 7 of 11 MRC-Holland ® MLPA Product Description version G1-03; Issued 23 February 2017 Table 3. Examples of probe signal ratios from potential results RATIO: when compared to Type of test sample Normal FEMALE DNA Normal MALE DNA FEMALE DNA with a deletion that includes one or more exons of the SHOX gene MALE DNA with a deletion that includes one or more exons of the SHOX gene FEMALE or MALE DNA with a deletion in the area upstream or downstream of SHOX, but not including SHOX gene probes FEMALE sample with Turner syndrome (45,X) MALE sample with Klinefelter syndrome (47,XXY) FEMALE sample with Triple X-syndrome (47,XXX). Probes All PAR1/PAR2 probes, incl. SHOX AREA, Xp22 X-chromosome probes OUTSIDE PAR1/2 region 105 & 118 nt Y-chromosome probes outside PAR1 + PAR2 All PAR1/PAR2 probes, incl. SHOX AREA, Xp22 X-chromosome probes OUTSIDE PAR1/2 region 105 & 118 nt Y-chromosome probes outside PAR1 + PAR2 One or more SHOX probes + possibly one or more SHOX AREA and/or Xp22 probes Deletions extending outside PAR1: one or more of the 6 chromosome X probes (marked x in Table 2) also show a lower copy number One or more SHOX probes + possibly one or more SHOX AREA and/or Xp22 probes Deletions extending outside PAR1: one or more of the 6 chromosome X probes (marked x in Table 2) also show a lower copy number One or more probes upstream or downstream of SHOX, indicated with SHOX AREA in table 2 All PAR1/PAR2 probes, incl. SHOX AREA, Xp22 X-chromosome probes OUTSIDE PAR1/2 region 105 & 118 nt Y-chromosome probes outside PAR1 + PAR2 All PAR1/PAR2 probes, incl. SHOX AREA, Xp22. X-chromosome probes OUTSIDE PAR1/2 region 105 & 118 nt Y-chromosome probes outside PAR1 + PAR2 All PAR1/PAR2 probes, incl. SHOX AREA, Xp22. X-chromosome probes OUTSIDE PAR1/2 region 105 & 118 nt Y-chromosome probes outside PAR1 + PAR2 SALSA MLPA Probemix P018 SHOX Explanation Male ref Female ref 1 1 2 1 0 - 1 1 1 0.5 1 ∞ 0.5 0.5 Heterozygous deletion in essential SHOX area in PAR1. PAR1 region is located on X and Y. This typically causes LWD or short stature. 1 0.5 Heterozygous deletion outside PAR1. Sequences present on only X. 0.5 0.5 Heterozygous deletion in essential SHOX area in PAR1. PAR1 region is located on X and Y. This typically causes LWD or short stature. 0 0 0.5 0.5 0.5 0.5 1 0.5 0 - 1.5 1.5 2 1 1 ∞ 1.5 1.5 3 1.5 0 - PAR1 & PAR2 regions are located on X and Y: normal females: 2 copies; normal males: 2 copies. Sequences present on only X: normal females: 2 copies; normal males: 1 copy. Sequences present on only Y: normal females: 0 copies; normal males: 1 copy. PAR1 & PAR2 regions are located on X and Y: normal females: 2 copies; normal males: 2 copies. Sequences present on only X: normal females: 2 copies; normal males: 1 copy. Sequences present on only Y: normal females: 0 copies; normal males: 1 copy. Heterozygous deletion outside PAR1. Sequences present on only X. Heterozygous deletion. PAR1 region is located on X and Y. Deletions in this region have been associated with LWD & ISS (Benito-Sanz et al. 2005, BenitoSanz et al. 2012a, Benito-Sanz et al. 2012b, Chen et al. 2009). Not all deletions detected by these probes will result in LWD or ISS! PAR1 & PAR2 regions are located on X and Y. Only one X is present in this case. Probe sequences located on only X. Only one X is present in this case. Probe sequences located on only Y. No Y present in this case. PAR1 & PAR2 regions are located on X and Y. Three instead of the normal two copies present. Probe sequences present on only X. Two Xs are present in this case. Probe sequences present on only Y. One Y present in this case. PAR1 & PAR2 regions are located on X and Y. Three instead of the normal two copies present. Probe sequences present on only X. Three Xs are present in this case. Probe sequences located on only Y. No Y present in this case. Page 8 of 11 MRC-Holland Product Description version G1-03; Issued 23 February 2017 ® MLPA Related SALSA® MLPA® probe mixes P329 CRLF2CSF2RA-IL3RA P216 GHD P217 IGF1R P262 GHI P026 Sotos P384 Human Height Probes for these PAR1 genes. Deletions of the IL3RA and CSF2RA genes, resulting in CRLF2 overexpression, are found in Acute Lymphoblastic Leukaemia samples. Probes for GH1, PROP1, POU1F1, GHRHR, HESX1, LHX3 and LHX4. Probes for IGF1R and IGFBP3. Probes for IGF1, GHR, JAK2 and STAT5B. Probes for NSD1. Probes for HHIP, HMGA2, ADAMTSL3, NPPC and IHH. References Barca-Tierno V et al. (2011). Identification of a Gypsy SHOX mutation (p.A170P) in Leri-Weill dyschondrosteosis and Langer mesomelic dysplasia. Eur J Hum Genet. 19:1218-1225. Benito-Sanz S et al. (2005). A novel class of Pseudoautosomal region 1 deletions downstream of SHOX is associated with Leri-Weill dyschondrosteosis. Am J Hum Genet. 77:533-544. 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. 27:1062. Benito-Sanz S et al. (2011a). A non-pathogenic pseudoautosomal region 1 copy number variant downstream of SHOX. Am J Med Genet A. 155A:935-937. Benito-Sanz S et al. (2011b). Clinical and molecular evaluation of SHOX/PAR1 duplications in Leri-Weill dyschondrosteosis (LWD) and idiopathic short stature (ISS). J Clin Endocrinol Metab. 96:E404-412. Benito-Sanz S et al. (2012a). Identification of the first PAR1 deletion encompassing upstream SHOX enhancers in a family with idiopathic short stature. Eur J Hum Genet. 20:125-127. Benito-Sanz S et al. (2012b). Identification of the first recurrent PAR1 deletion in Leri-Weill dyschondrosteosis and idiopathic short stature reveals the presence of a novel SHOX enhancer. J Med Genet. 49:442-450. Bertorelli R et al. (2007). The homozygous deletion of the 3' enhancer of the SHOX gene causes Langer mesomelic dysplasia. Clin Genet. 72:490-491. Binder G (2011). Short stature due to SHOX deficiency: genotype, phenotype, and therapy. Horm Res Paediatr. 75:81-89. Caliebe J et al. (2012). IGF1, IGF1R and SHOX mutation analysis in short children born small for gestational age and short children with normal birth size (idiopathic short stature). Horm Res Paediatr. 77:250-260. Campos-Barros A et al. (2007). Compound heterozygosity of SHOX-encompassing and downstream PAR1 deletions results in Langer mesomelic dysplasia (LMD). Am J Med Genet A. 143A:933-938. Chen J et al. (2009). Enhancer deletions of the SHOX gene as a frequent cause of short stature: the essential role of a 250 kb downstream regulatory domain. J Med Genet. 46:834-839. Durand C et al. (2010). Enhancer elements upstream of the SHOX gene are active in the developing limb. Eur J Hum Genet. 18:527-532. 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. 78:167-170. Huber C et al. (2006). High incidence of SHOX anomalies in individuals with short stature. J Med Genet. 43:735-739. Sabherwal N et al. (2007). Long-range conserved non-coding SHOX sequences regulate expression in developing chicken limb and are associated with short stature phenotypes in human patients. Hum Mol Genet. 16:210-222. Sandbacka M et al. (2011). Evaluation of SHOX copy number variations in patients with Mullerian aplasia. Orphanet J Rare Dis. 6:53. Schouten JP et al. (2002). Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res. 30:e57. Schwartz M et al. (2007). Deletion of exon 16 of the dystrophin gene is not associated with disease. Hum Mutat. 28:205. Shears DJ et al. (2002). Pseudodominant inheritance of Langer mesomelic dysplasia caused by a SHOX homeobox missense mutation. Am J Med Genet. 110:153-157. Varga RE et al. (2012). MLPA-based evidence for sequence gain: pitfalls in confirmation and necessity for exclusion of false positives. Anal Biochem. 421:799-801. Zinn AR et al. (2002). Complete SHOX deficiency causes Langer mesomelic dysplasia. Am J Med Genet. 110:158163. Selected publications using SALSA® MLPA® Probemix P018 SHOX Albuisson J et al. (2012). Clinical utility gene card for: Leri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LMD). Eur J Hum Genet. 20. Barroso E et al. (2010). Identification of the first de novo PAR1 deletion downstream of SHOX in an individual diagnosed with Leri-Weill dyschondrosteosis (LWD). Eur J Med Genet. 53:204-207. Fukami M et al. (2008). Identification and characterization of cryptic SHOX intragenic deletions in three Japanese patients with Leri-Weill dyschondrosteosis. J Hum Genet. 53:454-459. SALSA MLPA Probemix P018 SHOX Page 9 of 11 MRC-Holland Product Description version G1-03; Issued 23 February 2017 ® MLPA Funari MF et al. (2008). Cryptic intragenic deletion of the SHOX gene in a family with Leri-Weill dyschondrosteosis detected by Multiplex Ligation-Dependent Probe Amplification (MLPA). Arq Bras Endocrinol Metabol. 52:1382-1387. Funari MF et al. (2010). Usefulness of MLPA in the detection of SHOX deletions. Eur J Med Genet. 53:234-238. Gatta V et al. (2007). Identification and characterization of different SHOX gene deletions in patients with Leri-Weill dyschondrosteosys by MLPA assay. J Hum Genet. 52:21-27. Gatta V et al. (2014). Spectrum of phenotypic anomalies in four families with deletion of the SHOX enhancer region. BMC Med Genet. 15:87. Gervasini C et al. (2010). SHOX duplications found in some cases with type I Mayer-Rokitansky-Kuster-Hauser syndrome. Genet Med. 12:634-640. Hirschfeldova K et al. (2012). SHOX gene defects and selected dysmorphic signs in patients of idiopathic short stature and Leri-Weill dyschondrosteosis. Gene. 491:123-127. Iughetti L et al. (2010). Unexpected phenotype in a boy with trisomy of the SHOX gene. J Pediatr Endocrinol Metab. 23:159-169. Kant SG et al. (2013). Phenotypic characterization of patients with deletions in the 3'-flanking SHOX region. PeerJ. 1:e35. Rodriguez FA et al. (2013). Clinical and molecular characterization of Chilean patients with Leri-Weill dyschondrosteosis. J Pediatr Endocrinol Metab. 26:729-734. Sandoval GT et al. (2014). SHOX gene and conserved noncoding element deletions/duplications in Colombian patients with idiopathic short stature. Molecular genetics & genomic medicine. 2:95-102. Stuppia L et al. (2010). Different approaches in the molecular analysis of the SHOX gene dysfunctions. J Endocrinol Invest. 33:30-33. van Steensel MA et al. (2008). Contiguous gene syndrome due to a maternally inherited 8.41 Mb distal deletion of chromosome band Xp22.3 in a boy with short stature, ichthyosis, epilepsy, mental retardation, cerebral cortical heterotopias and Dandy-Walker malformation. Am J Med Genet A. 146A:2944-2949. Wolters B et al. (2013). Short stature before puberty: which children should be screened for SHOX deficiency? Horm Res Paediatr. 80:273-280. P018 Product history Version Modification G1 One SHOX area probe has been replaced (CNE4) and four new probes have been included for the upstream SHOX enhancer sequences CNE-2, CNE-3 and CNE-5 and the downstream CNE7 enhancer sequence. Three new probes near the PAR1 boundary have been included. One probe (GPR143) has been removed. The 88 and 96 nt control fragments have been replaced (QDX2). Six probes located on chromosome X and six reference probes have been replaced. One target and six reference probes have been replaced. Several reference probes replaced. Variable probes in the SHOX downstream region removed. Extra control fragments added. Many probes outside the SHOX gene have been added. First release. F1 E1 D1 C1 B1 A1 Implemented changes in the product description Version G1-03 – 23 February 2017 (03) - KAL1 gene name changed to ANOS1 in Table 1 & 2. - Added remark below Table 1 & 2 for the 337 nt SHOX exon 5 probe. Version G1-02 – 25 August 2016 (03) - All reference samples should be of the same sex added to the Reference samples section. - Ligation sites and remarks updated in Table 2. - Product description adapted to a new template. Version G1-01 – 07 July 2015 (02) - Product description restructured and adapted to a new template. Version 20 – 03 December 2013 (01) - Complete product description rewritten. Names of certain regulatory regions changed. P.3 rewritten; corrected error (Most defects in SHOX are point mutations into deletions); edited Publication list. Table 2: corrections in the names of regulatory regions detected by certain probes, denoted SHOX area extended. Table Interpretation of results and Table 3 – Change in relative probe signals as compared to normal male or female reference DNA samples added. Thanks to Karen Heath for the corrections. Version 19 - 03 December 2013 (01) - Product description completely rewritten. SALSA MLPA Probemix P018 SHOX Page 10 of 11 MRC-Holland Product Description version G1-03; Issued 23 February 2017 ® MLPA - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new picture included). More information: www.mlpa.com; www.mlpa.eu MRC-Holland bv; Willem Schoutenstraat 1 1057 DL, Amsterdam, The Netherlands E-mail Phone [email protected] (information & technical questions); [email protected] (orders) +31 888 657 200 SALSA MLPA Probemix P018 SHOX Page 11 of 11