SALSA MLPA KIT P6-B2 SMA Lot 111, 511: As compared to the previous version B1 (lot 11), the 88 and 96 nt DNA Denaturation control fragments have been replaced (QDX2). Please note that, in contrast to the older version A2, the P6-B2 probemix contains probes for exon 7 and 8 of both SMN1 as well as SMN2. SPINAL MUSCULAR ATROPHY (SMA) is a neuromuscular disorder characterized by degeneration of the anterior horn cells of the spinal cord, leading to symmetrical muscle weakness and atrophy. SMA is the second most common lethal autosomal recessive disorder in Caucasians, after cystic fibrosis. There are two (highly-similar) genes playing a pivotal role in SMA: SMN1 and SMN2. This new SALSA MLPA kit P6-B2 SMA kit will detect copy number changes of exon 7 and 8 of the SMN1 and SMN2 genes. Most individuals have two copies each of SMN1 and SMN2. The SMN1 and SMN2 genes can only be distinguished by two single nucleotide differences: one in exon 7 and one in exon 8. Both genes are located close to each other in a complicated inverted repeat area on chromosome 5q13. The two genes encode the same protein. However, the single nucleotide difference in exon 7 affects the mrna splicing. As a result, the SMN2 gene is only 1-15% as efficient in making functional SMN protein as compared to SMN1. More than 95% of SMA patients show homozygous deletion of at least exon 7 of the telomeric SMN1 gene. The great majority of SMA carriers can be identified by the presence of only a single SMN1 exon 7 copy. The one copy frequency in the US is estimated to be 1:37 for Caucasians, 1:46 for Ashkenazi Jews, 1:56 for Asians, 1:91 for African-Americans and 1:125 for Hispanics (Hendrickson, B.G. et al, 29: J.Med.Genet. 46:641-644). The SMN2 copy number is very variable with only 6-7% of individuals having two copies. Provided that at least one functional SMN1 copy is present, complete absence of the centromeric SMN2 gene seems to have no clinical consequences. Establishing the number of the SMN2 copy number is however important for SMA patients: the more SMN2 copies, the less severe the disease is expected to be. Absence of SMN1 combined with a SMN2 copy number of only 1 or 2 usually results in early onset severe disease. Healthy individuals lacking SMN1 but having 5 or more SMN2 copies have been described. Four probes in this P6-B2 SMA probemix detect SMN1 or SMN2 sequences. All other probes are reference probes on other chromosomes. Most important is the probe at 183 nt which is specific for SMN1 exon 7. Heterozygous deletion of this probe indicates that the individual is a SMA carrier. The SMN1 exon 8 probe at 218 nt will confirm a copy number change of the exon 7 probe in approximately 95% of all cases. Please note that only the exon 7 nucleotide difference between SMN1 and SMN2 has an effect on the production of a functional SMN protein. Therefore, in case only the SMN1 exon 8 probe appears to be deleted, it is most likely that the individual is not a SMA carrier. This new P6-B2 version also contains probes for SMN2 exon 7 (282 nt) and exon 8 (31 nt). Results from these SMN2 probes have no effect on the SMA carrier status but are important for the prognosis of SMA patients. This SALSA kit is designed to detect deletions/duplications of one or more sequences in the SMN1 and SMN2 gene in a DNA sample. Heterozygous deletions of probe recognition sequences should give a 35-5% 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. Finally, note that other defects such as small point mutations 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 kits are supplied with all necessary buffers and enzymes. Purchase of the SALSA test kits includes a limited license to use these products for research purposes. More information Website : www.mlpa.com E-mail : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 6, 157 DN Amsterdam, the Netherlands SALSA kit P6 SMA page 1 of 7
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 3, e57 (22). Related SALSA kits P21 SMA: Spinal Muscular Atrophy (SMA), to determine SMN1 and SMN2 copy number changes (patients). P58 IGHMBP2: Autosomal recessive distal spinal muscular atrophy 1 (DSMA1), gene included IGHMBP2. References for MLPA used in SMA research: Yoon, S. et al. (21) determination of SMN1 and SMN2 copy numbers in a Korean population using Multiplex Ligation-dependent Probe Amplification. Korean J.Lab.Med. 3:93-96. Arkblad, E. et al (29) A population-based study of genotypic and phenotypic variability in children with spinal muscular atrophy. Acta Paedatr. 98:865-872. Eggermann, T. et al. (28) A new splice site mutation in the SMN1 gene causes discrepant results in SMN1 deletion screening approaches. Neuromuscul. Disord. 18:146-149. Zapletalova E. (27). Analysis of point mutations in the SMN1 gene in SMA patients bearing a single SMN1 copy. Neuromuscul Disord. 27 Apr 3. Arkblad EL. et al. (26). Multiplex ligation-dependent probe amplification improves diagnostics in spinal muscular atrophy. Neuromuscul Disord. 26 Oct 16. Scarciolla O. et al. (26). Spinal muscular atrophy genotyping by gene dosage using multiple ligationdependent probe amplification. Neurogenetics. 26 Nov;7(4):269-276. Tomaszewicz K et al. (25). Detection of homozygous and heterozygous SMN deletions in a single assay with multiplex ligation-dependent probe amplification. Beijing Da Xue Xue Bao. 18;37(1):55-7. SMN region The SMA disorder is usually divided into three clinical groups. Patients with type I SMA disease (MIM# 2533) show onset at birth or before six months, and usually die of respiratory insufficiency within two years. Type I SMA patients are never able to sit or walk. Patients with type II SMA (MIM# 25355) show onset after 6 months. They can sit but will never able to walk unaided, and life expectancy is significantly reduced. Type III SMA (MIM# 2534) patients show the first symptoms after 18 months and are able to stand and walk, but often become wheelchair-bound during youth or adulthood. The SMA region on 5q13.2, containing the telomeric SMN1 and the centromeric SMN2 genes, displays high instability, leading to frequent deletions and gene conversions. The full-length cdnas of SMN1 and SMN2 are completely identical, except for two nucleotide differences one in exon 7 and one in exon 8. The exon 8 difference has no effect on the transcript, however the exon 7 difference disrupts a putative exonic splicing enhancer in SMN2. As a result, most SMN2 transcripts lack exon 7 and are not functional. Absence of any functional SMN1 copy results in insufficient amounts of full length transcripts, and is the cause of 95% of all SMA cases. The presence of SMN2 results in a small amount of full length transcripts. The more SMN2 copies a patient has, the more functional SMN protein is produced and the milder the SMA phenotype is. The majority of type I SMA patients carry a homozygous deletion of SMN1 and a normal or reduced number of SMN2 copies. The majority of the type II and III SMA patients show homozygous absence of SMN1 and an increased number of SMN2 copies (3 4 copies). A homozygous deletion of SMN2 is found in about 5% of healthy individuals. This has no clinical phenotype when at least one functional SMN1 copy is present. Description of the SMN probes in P6-B2 SMA - The SMN1 Exon 7 probe 14919-L1781 (183 nt) is the most important probe in this probemix. This probe is specific for SMN1 and will give no significant signal on SMN2. The probe has its ligation site at the C-to-T transition in exon 7, which is a site that affects RNA splicing. The presence of SMN1 sequences (cytosine at the ligation site) in the sample will result in a peak at 183 nt. This probe can be used to determine the SMN1 copy number which is important for the determination of a SMA carrier status. Of the 5q13-linked SMA patients, 96.4% show a homozygous deletion of SMN1 exon 7 and 8, or exon 7 only. The remaining 3.6% present a compound heterozygosity with a subtle mutation on one chromosome and a deletion/gene conversion on the other. Such a subtle mutation is likely not to be detected by this P6 SMA SALSA kit P6 SMA page 2 of 7
MLPA test and should be identified by sequencing. In a very small number of patients the SMN1 defect is a copy number change of SMN1 exons 1-6 which may be detectable with the P21 SMA MLPA kit. - The SMN1 Exon 8 probe 14881-L1782 (218 nt) is able to distinguish between SMN1 and SMN2 at the exon 8 (G-to-A transition). The signal of this probe indicates the copy number of SMN1 exon 8. In approximately 95% of the samples, the copy number detected by the SMN1 exon 7 probe and the SMN1 exon 8 probe is identical. This SMN1 exon 8 probe cannot be used to quantify to number of SMN1 copies, as an exon 8 mutation will still result in a functional protein. Only the SMN1 exon 7 probe should be used to determine the SMN1 copy number. In the majority of the remaining 5% of the samples, gene conversion between SMN1 and SMN2 has resulted in a chimeric gene containing the SMN1 exon 7 sequence and the SMN2 exon 8 sequence. Such a hybrid gene results in a functionally identical protein as the SMN1 protein. - The SMN2 Exon 7 probe 14921-L1783 (282 nt) shows the SMN2 copy number which is important for SMA patients, but which has no influence on SMA carrier status. The SMN2 copy number is much more variable than the SMN1 copy number. Approximately 5% of individuals lack the SMN2 gene. - The SMN2 Exon 8 probe 14878-L1784 (31 nt) confirms the results obtained with the SMN2 exon 7 probe in most individuals. In case the copy number detected by this exon 8 probe does not correspond to that found by the exon 7 probe, only the exon 7 probe should be used to determine the SMN2 copy number. The summary of these findings and what they mean for carrier/patient status can be found in table 1. Table 1: Overview of expected results and the corresponding conclusion Finding Conclusion Explanation SMA symptoms - SMN1 exon 7: copies. - SMN1 exon 8: copies. confirms this. SMA symptoms - SMN1 exon 7: copies. - SMN1 exon 8: 1 or more copies. SMA symptoms - SMN1 exon 7: 1 copy. No SMA symptoms - SMN1 exon 7: 1 copy. - SMN1 exon 8: 1 copy. No SMA symptoms - SMN1 exon 7: 1 copy. - A: SMN1 exon 8 copies > 1. - B: SMN1 exon 8 copies =. No SMA symptoms - 2 copies of SMN1 exon 7. SMA patient SMN1 is absent, as no copies of the distinct SMN1 exon 7 are present. The absence of both SMN1 exon 8 copies SMA patient SMA patient SMA carrier SMA carrier Most likely not a SMA carrier False negative results SMA carrier screening SMN1 is absent, as no copies of the determining SMN1 exon 7 sequence are found. Due to gene conversion, 1 or more copies of the characteristic SMN1 exon 8 sequence appear to have become incorporated in the SMN2 gene. If the patient has SMA symptoms, but one copy of SMN1 exon 7 is present, the patient may belong to the 3.6% group presenting compound heterozygosity. Sequencing might reveal a defect in the remaining SMN1 copy. One copy of SMN1 is absent, making the person a carrier. The absence of one copy of the SMN1 exon 8 sequence confirms this. One copy of SMN1 is absent, making the person a carrier. A: due to gene conversion, 1 (or more) copies of the characteristic SMN1 exon 8 have become incorporated in the SMN2 gene. B: an SMN2 exon 8 copy has replaced the characteristic SMN1 exon 8 copy. Most likely this person is not a carrier. However, there is a possibility that both SMN1 copies lie on one chromosome. If there is reason to believe that the person is a carrier (i.e. child is SMA-patient), he/she may belong to the 4% of carriers where this is indeed the case. Be aware that for carrier screening, false negative results can be obtained. The presence of two SMN1 exon 7 copies per cell suggests that the person tested is not a carrier. However, this test result can also be due to the presence of two SMN1 copies on one chromosome and on the other, in which case the person tested is in fact a SMA carrier. Detection of some carriers is therefore compromised, as MLPA and other techniques are not able to identify carriers who have one chromosome lacking SMN1 with the other chromosome carrying two copies of SMN1. In most populations, approximately 3.3-8.1 % of individuals have a chromosome 5 strand with two SMN1 copies. In some populations however, this percentage is much higher. In a recent survey (Hendrickson, B.G. SALSA kit P6 SMA page 3 of 7
et al, 29: J.Med.Genet. 46:641-644), 27% of African-Americans was shown to have three or more copies of SMN1. As a consequence, carrier testing of African-Americans is compromised as a larger number of individuals with two SMN1 copies may in fact be a carrier with two copies on the same chromosome. Therefore, one should keep in mind that a person with two SMN1 copies can STILL be a carrier: if both copies lie on one chromosome arm, there is a 5% chance that not a single SMN1 copy is passed on to the offspring. If a parent of a confirmed patient is found to have two copies, this option is a likely explanation. False positive results SMA carrier screening Please note that individual MLPA probes can be affected differently by changes in experimental procedures or impurities in samples. Highly unlikely results such as an unusual high frequency of SMN1 exon 7 loss (carrier) or SMN1 exon 7 gain, without loss or gain of the exon 8 probe in most of these samples, should be treated with caution. Data analysis The P6-B2 SMA probemix contains 21 MLPA probes with amplification products between 154 and 342 nt. In addition, it contains 9 control fragments generating an amplification product smaller than 12 nt: four DNA Quantity fragments (Q-fragments) at 64-7-76-82 nt, three DNA denaturation control fragments (Dfragments) at 88-92-96 nt, one X-fragment at 1 nt and one Y-fragment at 15 nt (QDX2). 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 probe s amplification product by the total area of only the reference probes in this probemix (block normalization). 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 normalization assumes that no changes occurred in the genomic regions targeted 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 blots or long range PCR. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website www.mlpa.com. Reference samples The choice of reference samples is important for the correct determination of the SMN1 and SMN2 copy numbers. is not able to provide reference DNA samples. One reason is that MLPA reactions on all samples, including reference samples, should be done on DNA extracted by the same method. It is strongly advised to first make a selection of suitable reference samples with known copy numbers before SMA testing is started. One method of doing this is to test a number (e.g. 16) of healthy individuals. Identification of samples having two copies of both SMN1 and SMN2 should usually be simple as in most populations these will constitute the great majority of the samples. Please note that in some populations, such as African-Americans, the number of individuals with a total of three SMN1 copies may be almost identical to that with two copies (Hendrickson, B.G. et al, 29: J.Med.Genet. 46:641-644). This product was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA kit P6 SMA page 4 of 7
Table 2. SALSA MLPA P6-B2 SMA probemix Length Chromosomal position SALSA MLPA probe (nt) reference SMN1 / SMN2 64-7-76-82 Q-fragments: DNA quantity; only visible with less than 1 ng sample DNA 88-92-96 D-fragments: Low signal of 88 or 96 nt fragment indicates incomplete denaturation 1 X-fragment: Specific for the X chromosome 15 Y-fragment: Specific for the Y chromosome 154 Reference probe 2595-L1785 5q35 163 Reference probe 2291-L1786 3p14 172 Reference probe 2978-L1787 4q25 183 SMN1 probe 14919-L1781 SMN1 exon 7 191 Reference probe 559-L1788 11q22 2 Reference probe 976-L17298 11p13 28 Reference probe 1249-L1796 1q32 218 SMN1 probe 14881-L1782 SMN1 exon 8 228 Reference probe 14498-L1711 2p12 237 Reference probe 2334-L1731 12q23 245 Reference probe 14293-L171 15q13 255 Reference probe 13128-L1799 9q34 264 Reference probe 763-L1791 1q26 272 Reference probe 14361-L1798 4q35 282 SMN2 probe 14921-L1783 SMN2 exon 7 292~ Reference probe 824-L1797 3q25 31 SMN2 probe 14878-L1784 SMN2 exon 8 311 Reference probe 6425-L1792 6p22 321 Reference probe 142-L1793 8q24 331 Reference probe 143-L1794 8q13 342 Reference probe 13399-L17297 6q12 ~ This probe has been reported to be more variable. Table 3. SMN1 and SMN2 probes in P6-B2 Length SALSA Partial sequence (24 nt Distance to Gene / Exon (nt) MLPA probe adjacent to ligation site) next probe 183 ٨ 14919-L1781 SMN1 exon 7 TTACAGGGTTTC-AGACAAAATCAA.7 kb 218 + 14881-L1782 SMN1 exon 8 GTAAAAGACTGG-GGTGGGGGTGGG > 1 kb 282 14921-L1783 SMN2 exon 7 TTACAGGGTTTT-AGACAAAATCAA.7 kb 31 14878-L1784 SMN2 exon 8 GTAAAAGACTGA-GGTGGGGGTGGG ٨ One copy in most carriers; none in most patients. + Confirms SMN1 exon 7 results in 95 % of carriers. Note: The identity of the genes detected by the reference probes and the complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA kit P6 SMA page 5 of 7
8 5. 7 5 8 5. 7 4 8 5. 8 4 9 6. 2 7 9 1. 3 2 8 5. 8 4 9 1. 3 9 9 1. 3 2 9 6. 2 5 9 1. 3 9 9 6. 3 8 1 5. 7 8 1. 9 2 9 6. 3 7 1. 9 1. 9 6 1 5. 8 1 1. 9 6 1 5. 8 1 1 5 3. 1 1 1 6 1. 6 6 1 5 3. 1 2 1 6 1. 6 5 1 5 3. 1 1 6 1. 6 6 1 5 3. 1 1 7. 5 1 1 6 1. 6 7 1 7. 5 5 1 7. 5 1 7. 5 3 1 9. 9 3 1 8 1. 6 9 1 8 1. 7 1 1 8 1. 6 7 1 8 1. 7 1 9. 9 2 1 9. 9 5 1 9 9. 3 1 9. 9 4 2 6. 8 7 1 9 9. 1 1 9 9. 5 2 6. 8 8 2 6. 9 5 1 9 9. 4 2 6. 9 9 2 1 6. 7 9 2 1 6. 7 7 2 1 6. 7 9 2 1 6. 8 2 3 6. 5 6 2 2 7. 3 7 2 2 7. 3 8 2 3 6. 5 7 2 2 7. 3 8 2 2 7. 4 3 2 3 6. 5 6 2 3 6. 6 1 2 4 5. 3 2 4 5. 3 2 4 5. 4 2 4 5. 8 2 5 3. 9 7 2 5 4. 2 5 3. 9 7 2 5 4. 5 2 6 2. 5 5 2 6 2. 5 4 2 6 2. 5 6 2 6 2. 5 9 2 7 1. 3 4 2 7 1. 3 3 2 7 1. 3 3 2 7 1. 3 9 2 8. 7 3 2 8. 7 2 2 8. 7 2 2 9. 8 5 2 9. 8 2 2 9. 8 4 2 9. 8 7 3. 3. 7 3. 3 1 1. 2 9 3 1 1. 2 7 3 1 1. 2 7 3 1 1. 2 7 3 2 1. 3 2 1. 2 3 2. 9 7 3 2 1. 3 4 1. 3 3 3. 7 3 3 3. 7 2 3 3. 7 1 3 4 1. 3 3. 6 9 3 4 1. 1 3 4. 9 6 Description version 15; 24-9-211 SALSA MLPA P6 SMA sample pictures 7 6 5 4 3 2 1 5 1 15 2 25 3 35 4 Figure 1. Capillary electrophoresis pattern of a sample of approximately 5 ng human male control DNA (2 copies each of SMN1 and SMN2) analyzed with SALSA MLPA kit P6-B1 SMA (lot 21). 12 11 1 9 8 7 6 5 4 3 2 1 5 1 15 2 25 3 35 4 Figure 2. Capillary electrophoresis pattern of a sample of approximately 5 ng human male SMA-carrier (1x SMN1, 2x SMN2) DNA analyzed with SALSA MLPA kit P6-B1 SMA (lot 21). 7 6 5 4 3 2 1 5 1 15 2 25 3 35 4 Figure 3. Capillary electrophoresis pattern of a sample of approximately 5 ng human female DNA (3x SMN1, 1x SMN2) analyzed with SALSA MLPA kit P6-B1 SMA (lot 21). 175 15 125 1 75 5 25 5 1 15 2 25 3 35 4 Figure 4. Capillary electrophoresis pattern of a sample of approximately 5 ng human male DNA (4x SMN1; x SMN2) analyzed with SALSA MLPA kit P6-B1 SMA (lot 21). SALSA kit P6 SMA page 6 of 7
SALSA MLPA kit P6-B2 SMA sample picture 12 96.17 11 216.98 1 15.36 181.6 9 8 85.57 9.92 17.27 19.95 26.54 245.16 262.76 7 152.88 161.52 199.7 236.68 254.2 29.93 28.8 321.58 33.47 311.27 6 227.45 271.47 299.36 34.71 5 4 1.52 3 D ye Sign al 2 1 1 15 2 25 3 35 Figure 1. Capillary electrophoresis pattern of a sample of approximately 5 ng human male control DNA analyzed with SALSA MLPA kit P6-B2 SMA (lot 111). Implemented changes - compared to the previous product description version. Version 15 (46) - Product description adapted to a new lot (lot number added, new picture included). Version 14 (46) - Product description adapted to a new product version (version number changed, lot number added, explanation of changes as compared to the previous version on page 1). New sample picture added. - Warning added in Table 1, 292 nt probe 824-L1797. Version 13 (45) - Product description adapted to a new product lot (version number changed, lot number added). Version 12 (45) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new pictures included). - Major changes in the text of this complete product description. SALSA kit P6 SMA page 7 of 7