General information
The SALSA MLPA Probemixes P251-P252-P253 NB mix 1, 2 & 3 are research use only (RUO) assays for the detection of copy number changes of several chromosomal regions that frequently show copy number changes in neuroblastoma tumours.
Neuroblastoma (NB) is a relatively common paediatric cancer that usually occurs sporadically and frequently originates from one of the adrenal glands. Neuroblastoma is characterized by striking clinical heterogeneity, including cases that show spontaneous tumour regression. Neuroblastoma accounts up to 10% of all paediatric cancers. Several acquired genetic alterations such as amplification of the
MYCN oncogene, deletions of chromosome bands 1p36 and 11q23 and unbalanced gains of 17q regions have been well-characterized and show correlation with tumour behaviour, including response to treatment. For review please see e.g. Ambros et al. (2009) and Ahmed et al. (2017).
These SALSA MLPA probemixes are not CE/FDA registered for use in diagnostic procedures. Purchase of these products includes a limited license for research purposes.
Probemix content
The SALSA MLPA Probemix P251-C2 NB mix 1 contains 49 probes, and this includes 36 probes in total for chromosomes 1, 3 and 11. The SALSA MLPA Probemix P252-D1 NB mix 2 contains 49 probes, and this includes 34 probes in total for chromosomes 2 (
MYCN region) and 17. The SALSA MLPA Probemix P253-D1 NB mix 3 contains 47 probes and this includes 33 probes in total for chromosomes 4, 7, 9, 12 and 14. In addition, 13 reference probes are included in P251, 15 reference probes are included in P252 and 14 reference probes are included in P253 that target relatively copy number stable regions in various cancer types including neuroblastoma. However, it should be noted that neuroblastoma karyotypes can harbour multiple numerical and structural aberrations, which can complicate interpretation of these reference probes. Complete probe sequences and the identity of the genes detected by the reference probes is available online (
www.mrcholland.com).
The P251 probemix contains probes for chromosomes 1, 3 and 11:
- 1p36: A deletion of the 1p36 region is present in 20-40% of NB patients with near-diploid/tetraploid tumours and is often associated with
MYCN amplification (in 60% of cases). A probe for 1p36 tumour suppressor gene
CHD5 is included.
- 3p21-p22: Deletions on the 3p arm have been described in neuroblastomas, and the
RASSF1A gene is a candidate tumour suppressor gene in this region. The presence of 3p deletions appears to correlate with higher age at diagnosis.
- 11q: Deletions of the 11q arm, and in particular 11q23, are common in NB patient samples and associated with higher a disease stage and poor prognosis.
The P252 probemix contains probes for chromosomes 2 and 17:
- 2p24: Amplification of the proto-oncogene
MYCN is found in 20-30% of all neuroblastomas.
MYCN amplified tumours follow a very aggressive course and are associated with additional structural abnormalities - in particular with loss of 1p, gain of 17q and near-triploidy or -tetraploidy.
MYCN amplification is often used for identification of high-risk patients. Additional probes for the nearby
NBAS,
DDX1 and
ALK genes are included.
- 2q33: Loss of 2q33 has been reported in neuroblastomas and has been associated with loss of expression of
CASP8.
- 17p: Gains of the 17p probes together with gains of the 17q probes would indicate complete chromosome 17 gains, in contrast to the frequent unbalanced 17q gains that are often associated with translocations. Three probes for
TP53 have been included but
TP53 mutations and deletions might be rare in neuroblastomas.
- 17q: Unbalanced gain of 17q is present in approximately 50% of patients and is associated with a poor outcome in neuroblastomas. It often results from an unbalanced translocation with 1p or 11q. Gain of 17q, in unbalanced translocations or as part of whole chromosome gain, is seen in 80% of neuroblastomas. Whole chromosome 17 gain is typically seen in near-triploid tumours with favourable prognosis.
Please note that triploidy of all chromosomes cannot be detected by MLPA as only
relative gains or losses are detected.
The P253 probemix contains probes for chromosomes 4, 7, 9, 12 and 14:
- Partial copy number changes of chromosomes 4, 7, 9, 12 and 14 have been described in neuroblastomas. Probes for
CDKN2A and
PTPRD on chromosome 9 have been included as
CDKN2A is deleted (often homozygously) in many cancer types and the
PTPRD gene has also been identified as a candidate tumour suppressor gene in neuroblastoma.
Each probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at
www.mrcholland.com.
P252 competitor mix information
Samples with very high levels of MYCN amplification exhibit very high signals for the MYCN probes and low signals for other probes, making it difficult to analyse the latter. Therefore, the P252 probemix is shipped together with a vial of CM002 (P252 competitor mix). When a sample shows a very high level of MYCN amplification it can be retested with the competitor mix. This competitor mix contains oligonucleotides that can be included at the start of the MLPA reaction. Adding the competitors specifically reduces the signal of the eight MYCN region probes, making it possible to examine changes in other genes/chromosomal areas.
Instructions for use:
1. Denature 4 µl sample DNA by heating 5 minutes at 98°C.
2. Add 1.5 µl MLPA Buffer + 1.5 µl P252 probemix + 1 µl of P252 competitor mix.
3. Proceed with the MLPA protocol starting with one minute incubation at 95°C and 16 hour incubation at 60°C followed by the ligation and PCR steps.