SALSA MLPA KIT P029-A1

Transcription

1 SALSA MLPA KIT P029-A1 Williams-Beuren Syndrome Lot 1207: As compared to the previous lots 0403 and 0606, two DNA Denaturation control fragments (Dfragments) at 88 and 96 nt have been added. Small changes have been made to three probes as indicated on page 3. Williams-Beuren Syndrome (WBS) is an autosomal dominant disorder which in full-blown form includes supravalvular aortic stenosis (SVAS), multiple peripheral pulmonary arterial stenoses, elfin face, mental and statural deficiency, characteristic dental malformation, and infantile hypercalcemia. These traits are often not all present in patients. Although WBS patients usually perform poorly on standard IQ tests, they have often remarkable musical and verbal abilities as well as an engaging personality. The most frequent behavioural problems are poor concentration, attention-seeking behaviour and restlessness. The Williams-Beuren syndrome is estimated to occur at a frequency of approximately 1 in 7500 live births. A deletion of the 7q11.23 chromosomal region, including the ELN gene is found in approximately 90-95% of the clinically typical WBS patients but in a lower percentage of atypical cases. The commonly deleted chromosomal region has a size of approximately 1.5 Mb and is flanked by two highly homologous DNA sequences. However, smaller deletions involving only the ELN gene or the ELN and LIMK1 genes have also been described in SVAS and atypical WBS patients. The P029-A1 probemix contains probes for the detection of copy number changes of genes located in WBS critical region at 7q More information about genes in this probemix can be found on page 2. This SALSA kit is designed to detect deletions/duplications of one or more sequences in the aforementioned genes in a DNA sample. Heterozygous deletions of probe recognition sequences should give a 35-50% reduced relative peak area 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 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 most defects in this gene are expected to be small (point) mutations which will not be detected by this SALSA 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). References for SALSA kit P029 WBS Depienne C, Heron D, Betancur C, Benyahia B, Trouillard O, Bouteiller D, Verloes A, Leguern E, Leboyer M, Brice A. (2007) Autism, language delay and mental retardation in a patient with 7q11 duplication. J Med Genet. 44: 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 P029 Williams Page 1 of 7

2 Genes detected by P029-A1 WBS This probemix includes probes for the following genes located in the WBS critical region: ELN: Elastin gene. Hemizygous deletion of the ELN gene is found in % of the WBS patients. Disruption of the ELN gene is also the cause of supravalvular aortic stenosis (SVAS). However, only a minority of WBS patients have severe clinical SVAS. CLIP2 (CYLN2): The gene for the cytoplasmic linker 2, or the cytoplasmic linker protein 115 (CLIP115). The cytoplasmic linker proteins are supposed to mediate the interaction between specific membranous organelles and microtubules. The neurological features of WBS may be related to haploinsuffiency of CLIP2 (Hoogenraad et al., Nature Genet. 32: , 2002). FZD9: A homologue of the Drosophila frizzled wnt receptor. The potential function of FZD9 in transmitting a Wnt protein signal in the human brain and other tissues suggested to Wang et al. (Hum Mol Genet., 6:465-72, 1997) that heterozygous deletion of the FZD9 gene could contribute to the Williams syndrome phenotype. TBL2: A transducin-beta like gene. STX1A: The Syntaxin 1A gene. The protein plays a central role in neurotransmitter release. LIMK1: The LIM Kinase gene. This gene is strongly expressed in brain and is also a candidate for some neurologic features of WBS. RFC2 is the gene for subunit 2 of the replication factor C, which is involved in DNA replication. It has been suggested (Am J Hum Genet. 58: , 1996), that deletion of one copy of the RFC2 gene might lead to reduced efficiency of DNA replication, which might account for growth deficiency as well as developmental disturbances. FKBP6: The FKBP6 protein shows structural homology to FKBP immunophilins, which are cellular receptors for the immunosuppressive drugs FK506 and rapamycin. Deleted in all or most of Williams syndrome cases (Meng, X. et al., Genomics 52: , 1998). Data analysis The P029-A1 WBS probemix contains 32 MLPA probes with amplification products between 157 and 445 nt. In addition, it contains 7 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at nt and three DNA denaturation control fragments (Dfragments) at nt. More information on how to interpret observations on these control fragments can be found in the MLPA protocol. Data generated by this probemix can first be normalised intra-sample by dividing the peak area of each probe s amplification product by the total area 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 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. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website This probemix was developed by C.J. McElgunn & J.P. Schouten at. In case the results obtained with this probemix lead to a scientific publication, it would be very much appreciated if the first probemix designer could be made a coauthor. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA kit P029 Williams Page 2 of 7

3 Table 1. SALSA MLPA P029-A1 WBS probemix Length Chromosomal position SALSA MLPA probe (nt) reference gene 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 157 Reference probe L q FKBP6 probe L q Reference probe L p Reference probe L q Reference probe L q Reference probe L q Reference probe L q CLIP2 (CYLN2) probe L q Reference probe L p ± Reference probe L q FZD9 probe L q Reference probe L p TBL2 probe L q Reference probe L p STX1A probe L q Reference probe L q21 310± ELN probe L q11.2, exon Reference probe L q ELN probe L q11.2, exon Reference probe L q ELN probe L q11.2, exon Reference probe L q ELN probe L q11.2, exon Reference probe L q LIMK1 probe L q Reference probe L p RFC2 probe L q Reference probe L p CLIP2 (CYLN2) probe L q Reference probe L q Reference probe L p Reference probe L p16 Changed from lot 1207 onwards. Small change in length, no change in sequence detected. ± These probes are located within, or close to, a very strong CpG island. A low signal of these probes can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. + This probe may give a signal on a related sequence on chromsome 1. T/G mismatch at ligation site and at -9, +4, +5, +30. The gene targeted by this probe has changed its name. The previous name can be found within brackets. SNPs at -2 and -4 (freq.0.1) could influence the probe signal. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. Note: Exon numbering used here may differ from literature! Please notify us of any mistakes. The identity of the genes detected by the reference probes is available on request: info@mlpa.com. SALSA kit P029 Williams Page 3 of 7

4 Table 2. P029 region probes arranged according to chromosomal location Length (nt) SALSA MLPA probe Gene / Exon L01215 FKBP L00881 FZD L00882 TBL L00883 STX1A Ligation site ELN NM_ NM_ ; NM_ ; NM_ ; NM_ ; Partial sequence (24 nt adjacent to ligation site) CTGTTTTCCGTG-TTCGTAACAGAA GCTCATGGTCAA-GATCGGGGTCTT TACCTGTGCAGA-TGATCGCACCAT AACAGCATCCGT-GAGCTACACGAC Distance to next probe/exon 77 kb 139 kb 129 kb 325 kb L00876 ELN, exon GGGGATAAAACG-AGGTGCGGAGA 14 kb L00877 ELN, exon GGTGGAGTGGCT-GACGCTGCTGCA 14 kb L00878 ELN, exon TTTCCCGGCTTT-GGTGTCGGAGTC 12 kb L00879 ELN, exon ACCTCATCAACG-TTGGTGCTACTG 28 kb L00880 LIMK1 NM_ ; TGTGGGACCTTT-ATCGGTGACGGG 135 kb L01240 RFC2 NM_ ; CTTTTGCAGATG-GCAGGCCTCCTG 106 kb L01133 CLIP2 (CYLN2) NM_ ; CTGGGGGACTTT-GTGGTGGGCGA 59 kb L00885 CLIP2 (CYLN2) NM_ ; GGAGGCCAATCG-TCACTCCCCAGG 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 kit P029 Williams Page 4 of 7

5 SALSA MLPA kit P029-A1 WBS sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analyzed with SALSA MLPA kit P029-A1 WBS (lot 1207). SALSA kit P029 Williams Page 5 of 7

6 Figure 2. Capillary electrophoresis patterns from samples with approximately 50 ng human male control DNA analyzed with SALSA MLPA kit P029-A1 WBS (lot 1207). A) Deletion of complete WBS region and B) Deletion of the region from FZD9 to LIMK1 (including ELN). CYLN2 gene is normal. SALSA kit P029 Williams Page 6 of 7

7 Implemented Changes compared to the previous product description version(s). Version 08 (46) - Various minor textual changes. - Reference added on page 1. - Ligation sites of the probes targeting the FKBP6, FZD9, TBL2, STX1A, LIMK1, RFC2 and CLIP2 genes added to table 2. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Warning added in Table 1, 247 nt probe (01164-L00720), 310 nt probe (01333-L00876) and 391 nt probe (01160-L00716). - The CYLN2 gene name changed to CLIP2. - The text Many copy number alterations in healthy individuals are described in... added to page 2. - Additional information added on gene FZD2 on page 2. - Change in disorder name from Williams to WBS. Version 07 (44) - Various minor textual changes on page 1. - Minor changes in the data analysis section on page 2. - Tables have been numbered. - Data analysis paragraph When only a small has been changed. - Added between brackets which standard product description has been used for the update of the product description, e.g. 03 (43) SALSA kit P029 Williams Page 7 of 7