SALSA MLPA KIT P018-D1 SHOX Lot 0409, As compared to version C, several changes have been made as indicated on page 3.

Transcription

1 SALSA MLPA KIT P018-D1 SHOX Lot 0409, As compared to version C, several changes have been made as indicated on page 3. Mutations or deletions of the SHOX gene, located in the PAR1 region, are involved in idiopathic growth retardation. The SHOX gene escapes X inactivation in females. The growth failure of Turner Syndrome (X0) females is most likely the result of the presence of only one SHOX gene copy in each diploid cell (haploinsufficiency). The tall stature observed in Klinefelter (XXY) syndrome and in Triple X females (XXX) is probably the result of the presence of a third copy of the SHOX gene. Deletions of the p-end of the X chromosome, encompassing the SHOX gene, are detected in the majority of Leri-Weill dyschondrosteosis (LWD) patients. LWD is characterised by a disproportionate short stature with predominantly mesomelic limb shortening and Madelung deformity of the arm (Schiller et al Europ. J. Hum. Genet. 8: 54-62). Deletion sizes found by these investigators varied from 100 Kb to 9 Mb and often did not correlate with the severity of the phenotype. Mutations in and deletions of the SHOX gene were also detected in a significant percentage of patients with short stature whom at the time of diagnosis showed no obvious skeletal features reminiscent of the Leri-Weill syndrome. In persons with a height for age below the third percentile, Rappold et al (J.Clin.Endocrinol. Metab. (2002) 87: ), found a silent missense mutation, a nonsense mutation or a small deletion in the SHOX coding sequence in approximately one out of 80 patients. Using Fluorescent In Situ Hybridisation (FISH), a large deletion of the SHOX gene was apparent in one out of 50 patients. This would imply a prevalence of at least 1 in 2000 children. In contrast, classic growth hormone deficiency is found to be present in 1 in 3500 children, and Turner syndrome in approximately 1 in 2500 females. This P018 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 (Benito-Sanz et al (2005) Am. J. Hum. Genet. 77, ; Fukami, M. et al (2006) Am. J. Hum. Genet. 78, ). 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 MLPA kit is designed to detect deletions/duplications of one or more exons of the SHOX gene. 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 area. However, mutations and/or polymorphisms very close to the probe ligation site may also result in a reduced relative peak area. Therefore, apparent deletions detected by a single probe always require confirmation by other methods. Please note that mutations/polymorphisms very close to the probe ligation site may also result in a reduced relative peak area. Apparent deletions detected by a single probe therefore always require confirmation by other methods. Please note that other defects in the SHOX gene, such as small (point) mutations, will not be detected by this 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 MLPA 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). More information Website : info@mlpa.com Fax : Mail : bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands SALSA kit P018 SHOX Page 1 of 7

2 References of SALSA MLPA kit P018 SHOX 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. 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. Please note 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. According to the OMIM database and the NCBI Genbank description in entry NM_000451, the SHOX gene is composed of 6 exons ranging in size from 58 nt to 1,146 nt, and the two transcripts diverge after exon IV. Apparently one non-coding exon before the start codon is not counted. We use a different exon designation. Transcript variant A, as exemplified by the NM_ sequence, contains exons 1-6a. Transcript variant B, as exemplified by sequence NM_006883, contains exons b. (Exon 6b = exon Vb in some articles.) Exon 6b contains the last 42 nt of the variant B coding sequence. This exon 6b sequence is entirely composed of two Alu repeats. The exon 6b probe was therefore designed in a unique intron sequence next to exon 6b. Data analysis The P018-D1 SHOX probemix contains 44 MLPA probes with amplification products between 130 and 463 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 normalised intra-sample by dividing the peak area of each amplification product by the total area 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 targeted by the reference probes. It is strongly recommended to use 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. Comparison of results should preferably 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, long range PCR or FISH. 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 7

3 Table 1. SALSA MLPA P018-D1 SHOX probemix Length (nt) SALSA MLPA probe Chromosomal position reference Outside PAR SHOX region 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 0797-L q SHOX-area probe 5642-L05096 Xp Reference probe 3088-L p SHOX-area probe 5648-L06218 Xp * Reference probe 3404-L q SHOX-area probe 5643-L12715 Xp SHOX probe 1145-L00702 Exon ± IL3RA probe 9334-L00711 Xp SHOX-area probe 5649-L05103 Xp SHOX-area probe 6293-L06219 Xp Reference probe 3246-L q SHOX-area probe 5644-L05098 Xp SHOX probe 1146-L06220 Exon PPP2R3B probe 9333-L10292 Xp * Reference probe 2698-L q SHOX probe 9336-L00708 Exon SHOX probe 9337-L00911 Exon KAL1 probe 6402-L09795 Xp SHOX probe 1147-L00802 Exon Reference probe 0587-L q LOC probe 1341-L kb before SHOX 274 STS probe 5186-L04567 Xp NLGN4X probe 5587-L04577 Xp SHOX-area probe 6291-L06222 Xp SHOX probe 1148-L01331 Exon ASMT probe 1153-L00712 Xp SHOX-area probe 5645-L05099 Xp GPR148-OA1 probe 2566-L10170 Xp SHOX probe 1149-L00910 Exon Reference probe 3580-L p VAMP7 (SYBL1) probe 1156-L00659 Xq Reference probe 1838-L p SHOX-area probe 5651-L05105 Xp CSF2RA probe L12977 Xp SHOX probe 9338-L12978 SHOX region 394 * HDHD1A probe 5195-L12979 Xp CDKL5 probe 6465-L05991 Xp AIFM1 (PDCD8) probe 0820-L00338 Xq SHOX-area probe 5646-L05100 Xp SHOX-area probe 9335-L05104 Xp ARSF probe 9339-L05349 Xp SHOX-area probe 5647-L05101 Xp * Reference probe 4141-L p * Reference probe 4258-L q31 * New in version D1 (from lot 0408 onwards) The 160 nt probe has a C>G SNP at 16 nt from the ligation site. It detects the same sequence as the 154 nt probe in P018-C but because of the longer hybridizing sequence of the current probe, the SNP is no longer expected to influence the signal. Results should be treated with caution though. ± The 172 nt IL3RA probe has reported to be variable, possibly due to sensitivity to salt in the sample. Changed in version D1. Change in length but no change in sequence detected. The 196 nt probe 5644-L5098 carries a G/A SNP 2 nt upstream from ligation site, not very common (3 in 200 patients). X-chromosome, outside PAR region. Gives halve the signal in males as compared to females. The 445 has been reported to be variable in healthy controls (0, 1, 2 copies). Reports from Germany, Spain, Holland. SALSA kit P018 SHOX Page 3 of 7

4 Table 2. P018-D1 SHOX probes arranged according to chromosomal location Length (nt) SALSA MLPA probe Gene / exon Ligation site NM_ * Partial sequence (24 nt adjacent to ligation site) Distance to next probe/exon 227 Kb P-telomere L10292 PPP2R3B gene CGTCCGAGTTCC-ACTCGCGCTACA 273 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 L01331 SHOX exon CAGAACCGGAGA-GCCAAGTGCCGC 0.2 Kb L00910 SHOX exon ACAGCCAACCAC-CTAGACGCCTGC 3.5 Kb L00911 SHOX exon 6 (=exon 6a) AAGCAACAGCAA-GAATTCCAGCAT 6.4 Kb SHOX stopcodon L00708 SHOX region 6.4 Kb downstream (SHOX exon 7) exon 6 TGGCTTCACGAG-TTCAGCCCATTG 6.4 Kb L12978 SHOX region 1.4 Kb before alt. (SHOX exon 6b) exon 6b (=Vb) TCCCACATTCTT-GGAATCACAATG 56.8 Kb L05096 Xp22.32-PAR GCAGCAGTGAAA-GTGAGCATTCCC 31.2 Kb L12715 Xp22.32-PAR ACACCACCAGAGT-TACTTGAATCAA 60.0 Kb L05098 Xp22.32-PAR CATCCGCTTCGT-TTTGGAGGGTTT 43.1 Kb L05099 Xp22.32-PAR TGTTCCCACCGT-AAAACTCACTCC 8.5 Kb L05100 Xp22.32-PAR TGCATGTCTGCT-TTTTGAATGGCC 5.5 Kb L05101 Xp22.32-PAR Less reliable AGAAACCTCATT-TTAACAAACCGC 11.6 Kb L06222 Xp22.32-PAR CTTGAAAGGGCA-GGAACTCTAATT 0.4 Kb L06219 Xp22.32-PAR TAATTGATGAGA-TGCAGAAGCCAG 15.4 Kb L06218 Xp22.32-PAR TGGTGCTGAAAT-GAGGAAGCCCTG 48.7 Kb L05103 Xp22.32-PAR TGAGGAGGTACC-TCAAAGCTAAAC 64.4 Kb L05104 Xp22.32-PAR GAAATTCAGTTT-TAATAACACAGA 66.0 Kb L05105 Xp22.32-PAR TGACTTCTCTAG-ATGGCATCTCAC Kb L12977 CSF2RA gene GACAAGCCTTCT-GCTCTGTGAGTT 82.4 Kb L00711 IL3RA gene AATCCTGGAACG-TACACAGTACAA Kb L00712 ASMT gene GACATCCCAGAA-GTGGTGTGGACG Kb End of PAR region L05349 ARSF gene CATCCATATAAT-TATGGGTTTGAC Kb L04577 NLGN4X gene GACGGCTTGGGT-GATGCACGAAAT Kb L12979 HDHD1A gene CTTGCAGCTCCC-GATGTCCAAAGA Kb L04567 STS gene CCTATCTGCATC-GCAGGGGCTGTG Kb L09795 KAL1 gene GTTTCCTGAAGC-GTGTGCCCACAA Kb L10170 GPR143 gene GGGCGCTGGAGT-CCCAACTTACCT Kb L05991 CDKL5 gene ATTGACCAACTT-TTTACTATTCAG Kb L00338 AIFM1 gene TATTGGTCTTGT-GGACAGTAGTTT Kb L00659 VAMP7 gene TGTGGGAAAAGT-GTTTCCATTCTG * There are two major SHOX transcripts. We used the Genbank NM_ transcript which includes exon 6a (Va) but lacks exon 6b. An alternative transcript, exemplified by sequence NM_ , includes exon 6b but lacks exon 6a. The 388 nt probe is located close to this alt. exon 6b. These two probes are in the putative SHOX regulatory region that was described by Fukami, M. et al (2006) Am. J. Hum. Genet. (January 2006, online). The SYBL1 probe at 355 nt is located very close to the q-telomere of X and Y in the second pseudoautosomal region (PAR2). The 160 probe has a C>G SNP at16 nt from the ligation site. This probe detects the same sequence as the 154 nt probe in P018-C. We expect no influence of the SNP on the probe signal as the probe now has a longer hybridizing sequence. See also the notes on the previous probe table. Note: Exon numbering might be different as compared to literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA kit P018 SHOX Page 4 of 7

5 Compared to MALE control DNA, you may notice the following changes in relative signals of these probes: 1. An approx. 100% increase in signal of the X probes and a complete loss of the Y probe signals in female DNA. Please note that the signals of the (autosomal) 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. 2. A 40-60% reduction in signal of one or more SHOX probes, in case of a deletion of part or the complete SHOX gene. These deletions will in general cause Leri-Weill dyschondrosteosis. Deletions of the SHOX area probe 1341-L06221 might affect the SHOX promotor. 3. Recently, a novel class of pseudoautosomal region 1 deletions downstream of SHOX were also found to be associated with Leri Weill dyschondrosteosis (Benito-Sanz et al (2005) Am. J. Hum. Genet. 77, ). The exact location of the regulatory region is not 100% certain. We therefore included numerous probes in this region. 4. A 40-60% reduction in signal of the SHOX probes as well as of the other XpPAR probes in case the complete PAR1 region is lost. 5. A complete loss of the Y probe signals and a 40-60% reduction of the SHOX, the PAR1 and the PAR2 probes in Turners syndrome (X0). 6. An approx. 100 % increase in signal of the X probes outside the pseudo-autosomal (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, 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). Compared to FEMALE control DNA, you may notice the following changes in relative signals of these probes: 1. A 40-60% reduction in signal of one or more SHOX probes, in case of a deletion of part or the complete SHOX gene. These deletions will in general cause Leri-Weill dyschondrosteosis. Deletions of the LOC probe sequences might affect the SHOX promotor. 2. Recently a novel class of pseudoautosomal region 1 deletions downstream of SHOX were also found to be associated with Leri Weill dyschondrosteosis (Benito-Ananz, S. et al (2005) Am. J. Hum. Genet. 77, ). 3. A 40-60% reduction in signal of the SHOX probes as well as of the other XpPAR probes in case the complete PAR1 region is lost. 4. A 40-60% decrease in signal of the X probes and the appearance of Y probe signals in male DNA. Please note that the signals of the SHOX probes, the five other probes in the PAR1 region and the SYBL probe in the PAR2 region at the q telomere should remain unchanged. 5. A 40-60% reduction of the SHOX, the X, the PAR1 and the PAR2 probes in Turners 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 the X, the SHOX and the PAR1 and PAR2 probes in Triple X syndrome (XXX). SALSA kit P018 SHOX Page 5 of 7

6 SALSA MLPA kit P018-D1 SHOX sample pictures 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-D1 SHOX (lot 0409) 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-D1 SHOX (lot 0409). SALSA kit P018 SHOX Page 6 of 7

7 Implemented Changes the following has been altered compared to the previous product description version(s). Version 12 (44) - Minor changes in the product description on page 1. - Minor changes in the data analysis section on page 2. - Minor changes on table titles. SALSA kit P018 SHOX Page 7 of 7