Most severely affected will be the probe for exon 15. Please keep an eye on the D-fragments (especially the 96 nt fragment).

Transcription

1 SALSA MLPA probemix P343-C3 Autism-1 Lot C As compared to version C2 (lot C2-0312) five reference probes have been replaced, one reference probe added and several lengths have been adjusted. Warning: All SHANK3 probes are located in an extremely CG-rich chromosomal area. Such CpG islands are more difficult to denature than the rest of the human genome. The presence of salt in DNA samples can result in incomplete denaturation of these CpG islands, which may result in false positive results: apparent deletions of several consecutive SHANK3 probes, while reference probes are normal. Such false positive results are even more likely when DNA has been extracted by the Qiagen EZ1, M48 or M96 system, as these leave a higher salt concentration in the sample. High salt concentrations can also be due to evaporation (dried out samples; SpeedVac concentration). Most severely affected will be the probe for exon 15. Please keep an eye on the D-fragments (especially the 96 nt fragment). Multiple studies postulate that at least some autism cases have a genetic basis and many different loci have been implicated in autism. This P343-C3 probemix contains MLPA probes for three of these chromosomal regions: the 15q11-q13 (including UBE3A, GABRB3 and the 15q13 microdeletion region with CHRNA7), the 16p11 microdeletion region and the SHANK3 gene at 22q13. Within the 15q11 region, two probes have been included for the SNRPN-HB2-85 cluster, five for the UBE3A gene, two for ATP10A, seven for GABRB3 and two for OCA2. The great majority of these probes differ from the probes present in the ME028 Prader-Willi-Angelman probemix. This P343 probemix may therefore also be useful for further characterisation of large deletions in Prader-Willi-Angelman patients. In addition, nine probes are present detecting 15q13 sequences, including three probes that are located within the common 15q13 microdeletion region described by Sharp A.J. et al (Nature Genetics 2008, 40: ). The 16p11.2 region is covered by 11 probes detecting sequences in the Mb region. Genomic imbalances of an approximately 600 kb region in 16p11.2 ( Mb) have been associated with autism, intellectual disability, congenital anomalies, and schizophrenia. Please note that 15q11, 15q13 and 16p11.2 deletions and duplications have also been described in healthy individuals. Phenotype prediction for abnormalities detected in these regions is very difficult. The SHANK3 gene (22 exons) spans ~58 kb of genomic DNA and is located on 22q13.33, 49 Mb from the p-telomere. Three probes are included for SHANK3 gene (exons 3, 14, and 21). Finally, ten reference probes are included in this probemix, detecting different autosomal chromosomal locations. This SALSA probemix is designed to detect/duplications of one or more sequences in the above mentioned chromosomal regions 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. Finally, note that most defects in these regions 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 Acid Research 30, e57 (2002). SALSA probemix P343 Autism-1 Page 1 of 7

2 Related SALSA probemixes ME028 PWS/AS: Contains probes to detect Prader-Willi and Angelman syndrome (15q region). P188 22q13: Broad screening of the 22q13 region. P325 OCA2: Contains probes for nearly all OCA2 exons. P336 UBE3A: Contains probes for all UBE3A exons. P339 SHANK3: Contains probes for all SHANK3 exons. P396 SHANK2: Contains probes for all SHANK2 exons. References Moreira, DP et al., Investigation of 15q11-q13, 16p11.2 and 22q13 CNVs in autism spectrum disorder Brazilian individuals with and without epilepsy. PloS one, 9(9), e Rodriguez-Lopez, J et al., An efficient screening method for simultaneous detection of recurrent copy number variants associated with psychiatric disorders. Clinica Chimica Acta, 445, Moreira, ES et al., Detection of small copy number variations (CNVs) in autism spectrum disorder (ASD) by custom array comparative genomic hybridization (acgh). Res Autism Spectr Disord, 23, SzczaĂ, K et al., Paternally Inherited GABRB3 Intragenic Deletion in a Boy with Autistic Features and Angelman Syndrome Phenotype Case Report and Literature Review. Autism-Open Access, 1-4. More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands Data analysis The P343-C3 Autism probemix contains 50 MLPA probes with amplification products between 121 and 500 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. 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 this 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 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 exon 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 probemix P343 Autism-1 Page 2 of 7

3 Table 1. SALSA MLPA P343-C3 Autism-1 probemix Length Chromosomal position SALSA MLPA probe (nt) reference 15q11-q13 / Exon 16p11 SHANK 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 105 Y-fragment: Specific for the Y chromosome 121 * Reference probe L p * Reference probe L q ATP10A probe L q12 Exon UBE3A probe L q11 Exon GABRB3 probe L q12 Exon Reference probe L q UBE3A probe L q11 Exon KLF13 probe L q13 Exon HIRIP3 probe L14670 Exon NDNL2 probe L q13 Exon GABRB3 probe L q12 Exon * Reference probe L q UBE3A probe L q11 Exon APBA2 probe L q13 Exon SEZ6L2 probe L12439 Exon SNRPN-HB2-85 probe L q GABRB3 probe L q12 Exon DOC2A probe L12447 Exon «SHANK3 probe L07383 Exon «MAZ probe L12440 Exon UBE3A probe L q11 Exon Reference probe L q UBE3A probe L q11 Exon * Reference probe L p ATP10A probe L q12 Exon CHRNA7 probe L q13 Exon GABRB3 probe L q12 Exon «TJP1 probe L q13 Intron «SHANK3 probe L14007 Exon GABRB3 probe L q12 Exon * Reference probe L q CD2BP2 probe L12442 Exon MVP probe L22423 Exon GABRB3 probe L q12 Exon SPN probe L12443 Exon TRPM1 probe L q13 Exon GABRB3 probe L q12 Exon «SHANK3 probe L15800 Exon * Reference probe L q Reference probe L p «MAZ probe L29557 Exon SCG5 probe L q13 Exon OCA2 probe L q12-13 Exon OCA2 probe L q12-13 Exon HIRIP3 probe L12445 Exon MAPK3 probe L12446 Exon SNRPN-HB2-85 probe L q LAT probe L12448 Exon SCG5 probe L q13 Exon Reference probe L q22 * New in version C3 (from lot C onwards). Changed in version C3 (from lot C onwards). Small change in length, no change in sequence detected. «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. SALSA probemix P343 Autism-1 Page 3 of 7

4 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: Table 2. P343-C3 probes arranged according to chromosomal location Table 2a. 15q11-15q13 area Length SALSA MLPA Ligation site Partial sequence (24 nt Distance to Gene Exon (nt) probe NM_sequence adjacent to ligation site) next probe L29483 SNRPN-HB2-85 AAGAAATCCCTT-CCAGGAGGGCTC 10.2 kb L13795 SNRPN-HB2-85 AAGTTCTTTAAC-GTCATCGGCTTG kb UBE3A gene (NM_ ) stop codon (ex10) L12925 UBE3A Exon 9(13) TCATTCATTTAC-AGATGAACAGAA 14.2 kb L14677 UBE3A Exon 8(12) TCTGTTCTGATT-AGGTGAGGTACT 2.4 kb L11553 UBE3A Exon 5(9) TCTACAGGAAGC-TAATGGGGAAAA 14.8 kb L14668 UBE3A Exon 3(7) TCTTCCTCAAGG-ATAGGTGATAGC 4.2 kb L11550 UBE3A Exon 2(6) CTACCACCAGTT-AACTGAGGGCTG kb start codon (ex1) ATP10A gene (NM_ ) stop codon (ex21) L12883 ATP10A Exon TGCAGTGCCGAA-ATTCCGATACCT kb L14669 ATP10A Exon GGCCAACGTGTA-CTTTGTCTTCAT kb start codon (ex1) GABRB3 gene (NM_ ) stop codon (ex9) L09339 GABRB3 Exon 9(12) CGATACCAGGAA-TTCAGCAATATC 13.1 kb L11545 GABRB3 Exon 8(11) 10 nt before exon 8(11) CACCACTTTGTT-TCTTTTCTAGGG 6.5 kb L11544 GABRB3 Exon 7(10) AGGAACATTGGA-TACTTCATTCTT 12.6 kb L11542 GABRB3 Exon 6(9) 18 nt after exon 6(9) CCTGCATCCACT-TATAGTCCCTTC 3.1 kb L11540 GABRB3 Exon 5(8) 29 nt before exon 5(8) CAGCCCTTCTTT-AATATCTTCCCT 38.0 kb L11538 GABRB3 Exon 4(7) GGGATCCCTCTC-AACCTCACGCTT kb L11537 GABRB3 Exon 3(4) GTCCCCCGGTCT-GCGTGGGGATGA kb start codon (ex1) OCA2 gene (NM_ ) stop codon (ex24) L01553 OCA2 Exon CCGCTCATGTAT-GCCCTGGCCTTC kb L03725 OCA2 Exon reverse TGCACTTTACCT-GCGCACTTGCAG kb start codon (ex2) L00867 APBA2 CACCACCCACTT-GATTTTTTTCAT kb L08231 NDNL2 CTCTTGGGTTCA-AGTTCCACCAGC kb 300 «08389-L14671 TJP1 CACAGGCTGAGT-GGAGTGTTTTGC kb L14672 TRPM1 ATGGACATCCTA-GGAATGTGAAAT kb L08230 KLF13 TTGAACCCCCTT-TCTCAGGGATGG kb L08237 CHRNA7 AGACTGTTCGTT-TCCCAGATGGCC kb L14464 SCG5 Exon 3 NM_ ; TGACTGGAGACA-ACATTCCTAAGG 16.8 kb L29660 SCG5 Exon 6 NM_ ; TCAGCATGGCTT-ATGTGCACGTGT The TRPM1, KLF13 and CHRNA7 probes are located within the common 15q13 microdeletion region that has been described by Sharp A.J. et al (Nature Genetics : ). Please note that Helbig identified 15q13 duplications not only in 12 out of 1223 epilepsy patients but also in 23 out of 3699 control samples. 15q13 deletions were identified in 12 out of 1223 individuals with idiopathic generalized epilepsy by Helbig et al (Nature Genetics : ) and in 9 out of 3391 schizophrenia patients (Nature : ). No 15q13 deletions were detected in 3181 control samples. SALSA probemix P343 Autism-1 Page 4 of 7

5 The NM_ sequence represents transcript variant 1. The NM_ sequence is a reference standard in the NCBI RefSeqGene project. The NM_ sequence represents transcript variant 2 and is a reference standard in the NCBI RefSeqGene project. The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2b. 16p11 area Length (nt) SALSA MLPA probe Gene detected Partial sequence (24 nt adjacent to ligation site) Mapview 36 location (hg18) Distance to next probe p-telomere L12448 LAT ACCAGTTTGTAT-CCAAGGGGCATC kb L12443 SPN CCATCAAGATGT-CATCAGTGCCCC kb 238 «11669-L12440 MAZ CCACGGCAGCAT-ACCTGCGCATCC kb 420 «11673-L29557 MAZ GAAGAAATGTTT-TCTTAGGGGAAT kb L22423 MVP GTCGTGGAGATC-ATTCAGGCCACC kb L12439 SEZ6L2 GCAGCCAGATTA-CTTAGAGAGGCA kb L12445 HIRIP3 GGCGAGCCTCAA-AGGCAGTTGAGG kb L14670 HIRIP3 CCAGGGAAGACA-AACTGGACCTTA kb L12447 DOC2A CACTTGCTGCCT-GGAGCCTGTAAG kb L12446 MAPK3 CTGGATCAGCTC-AACCACATTCTG kb L12442 CD2BP2 GGAAGGCCACTT-TGATGCCGATGG centromere A recurrent microdeletion syndrome on 16p11.2-p12.2 has been described by Ballif BC et al (2007, Nature Genetics 39: ). Phenotype included developmental delay. Size of the deletion is different in the five subjects described, however all included the PALB2 and IL21R genes. Table 2c. SHANK3 gene Length (nt) SALSA MLPA probe SHANK3 Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon 1-3 (ex 1) 310 «20567-L14007 Exon AAGCGGCGAGTT-TATGCCCAGAAC 27.6 kb 391 «14190-L15800 Exon 14(15) GAGGGCTTTGGT-TTTGTGCTCCGG 18.1 kb 232 «06787-L07383 Exon 21(22) ACCAACTGTGAT-CAGTGAGCTCAG stop codon (ex 22) The SHANK3 gene is complicated and many SHANK3 probes have a higher than average standard deviation in many of our tests. Apparent deletions and duplications observed by only one or two of these probes should be treated with caution. All SHANK3 probes are located in an extremely GC rich chromosomal area. Such CpG islands are more difficult to denature than the rest of the human genome. The presence of salt in DNA samples can result in incomplete denaturation of these CpG islands, and may result in false positive results (apparent deletions of several consecutive SHANK3 probes, while the reference probes are normal). Such false positive results can in particular be obtained when DNA is purified by the Qiagen EZ1, M48 and M96 systems. High salt concentrations can also be due to evaporation (dried out samples; SpeedVac concentration). The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Changed in version C3 (from lot C onwards). Small change in length, no change in sequence detected. «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 UBE3A, GABRB3 and SHANK3 exon numbering has changed. From description version 9 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for these genes. 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 2. SALSA probemix P343 Autism-1 Page 5 of 7

6 The identity of the genes detected by the reference probes is available on request: SALSA MLPA probemix P343-C3 Autism-1 sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P343-C3 AUTISM-1 (lot C3-1016). Implemented Changes compared to the previous product description versions. Version 09 4 November 2016 (55) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - New references added on page 1. - Exon numbering of the UBE3A GABRB3 and SHANK3 genes have been changed on page 3 and 4. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. Version 08 (48) - Figure(s) based on the use of old MLPA buffer (replaced in December 2012) removed. Version 07 (48) - Textual change below Table 2. Version 06 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 05 (48) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). Version 04 (48) - Exon numbering of the GABRB3 gene has been changed on page 3 and 4. - Remark on RefSeqGene standard added below Table 2. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Small correction of chromosomal locations in Table 1 and 2. - Various minor textual and layout changes. Version 03 (46) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Warning added on page 1. and below Table 2c that the SHANK3 probes are sensitive to high salt concentrations. - New related probemixes added on page 1. SALSA probemix P343 Autism-1 Page 6 of 7

7 - Warning added about salt-sensitive reference probe (124 nt). SALSA probemix P343 Autism-1 Page 7 of 7