ME024 9p21 CDKN2A/2B region

SALSA^® MLPA^® Probemix ME024 9p21 CDKN2A/2B region detects copy number variations and the methylation status of the 9p21 region, including CDKN2A and CDKN2B.

Specifications

Contents: 48 MLPA probes, including 23 for the CDKN2A and CDKN2B region, 2 for PAX5, 2 for MTAP and 2 for MIR31. Of these probes, 10 provide information on the methylation status of the CDKN2A and CDKN2B promoter regions and 1 on the methylation status of the MIR31 gene.

Tissue: human genomic DNA, including DNA from FFPE tissue.

Application: research on the 9p21.3 region.

For research use only (RUO). Not for use in diagnostics.

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General information

The SALSA MLPA Probemix ME024 9p21 CDKN2A/2B region is a research use only (RUO) assay for the detection of aberrant methylation of one or more sequences of the CDKN2A and CDKN2B genes on chromosome band 9p21. This probemix can also be used to detect deletions/duplications in the aforementioned chromosomal region including MIR31, MTAP, CDKN2A and CDKN2B genes, and PAX5 gene on 9p13.

Genomic losses of the 9p21.3 region, encompassing the CDKN2A/2B genes, are frequent events in many human cancers. This locus encodes three cyclin-dependent kinase inhibitors p14^ARF, p15^INK4B and p16^INK4A. Genomic deletion of one or both copies of these important cell cycle regulatory genes is the main inactivation mechanism in various cancers. CDKN2A deletion can extend to the MTAP gene, located 110 kb downstream. The MTAP gene encodes methylthioadenosine phosphorylase, an important enzyme for the salvage of both adenine and methionine. It is known that many tumour cells require addition of methionine to their growth medium, because their MTAP gene is co-deleted with CDKN2A. Cells lacking MTAP are expected to be sensitive to purine synthesis inhibitors and/or methionine starvation, and therefore homozygous co-deletion of the CDKN2A and MTAP genes might open possibilities for alternative treatment for cancer patients. Other genes that are frequently co-deleted with CDKN2A/2B are CDKN2B-AS1, PAX5, and microRNA 31 (MIR31). Loss of MIR31 has been shown to have pro-tumorigenic effects on e.g. breast and ovarian cancer (Creighton et al. 2010). CDKN2B-AS1 (non-protein coding CDKN2B antisense RNA 1) is suggested to act as an epigenetic silencer of the CDKN2B gene (Yu et al. 2008). The PAX5 gene, at 9p13, which is essential for normal B-cell lymphopoiesis, is frequently co-deleted with CDKN2A in B-ALL (Kim et al. 2011).

An alternative mechanism of inactivation of the CDKN2A/2B genes is hypermethylation of the promoter regions leading to lack of expression of p14^ARF, p15^INK4B and p16^INK4A proteins, which further results in uncontrolled cell proliferation and tumour development and progression (Wolter et al. 2001).

Alterations of the CDKN2A/2B genes have been also described at the germline level. Germline mutations in the CDKN2A gene are frequently associated with predisposition to malignant cutaneous melanoma and pancreatic cancer (Chan et al. 2021). Up to 40% of familial melanomas are associated with CDKN2A mutations (Hewitt et al. 2002), including point mutations and various intragenic deletions.

The image below shows a schematic representation of the CDKN2A and CDKN2B gene structure and encoded proteins.

Regulatory status

SALSA MLPA Probemix ME024 9p21 CDKN2A/2B region is for research use only (RUO) in all territories.

Product documentation

Positive samples

Inclusion of a positive sample is usually not required, but can be useful for the analysis of your experiments. MRC Holland has very limited access to positive samples and cannot supply such samples. We recommend using positive samples from your own collection. Alternatively, you can use positive samples from an online biorepository, such as the Coriell Institute.

The commercially available positive samples below have been tested with the current (B3) version of this product and have been shown to produce useful results.

Copy number alterations (CNAs) affecting reference or control probes are not reported. Indicated chromosomal bands accommodate genes targeted by MLPA probes, however, the whole extent of CNAs present in the samples below cannot be determined by this ME024 probemix. ”Methylated” refers to cases where methylation-specific probes have ratios higher than baseline methylation.

Germline samples

Coriell NA03226, Coriell NA05067: Heterozygous duplication affecting the probes for 9p. CDKN2A/2B are unmethylated.
Coriell NA02819, Coriell NA01750: Heterozygous duplication affecting the probes for DOCK8, GLDC, MLLT3, KLHL9, MIR31, MTAP, CDKN2A, CDKN2B-AS1 and CDKN2B. CDKN2A/2B are unmethylated.

Cancer cell line samples

DSMZ ACC-200 (COLO-824): Gain affecting all probes for 9p. CDKN2A/2B are unmethylated.
DSMZ ACC-29 (MOLT-16): Homozygous deletion affecting the probes for CDKN2A, CDKN2B-AS1 and CDKN2B.
DSMZ ACC-255 (CADO-ES1): Homozygous deletion affecting the probes for MTAP, CDKN2A, CDKN2B-AS1 and CDKN2B. The 465 nt probe targeting MIR31 shows 100% signal loss upon HhaI digestion.
DSMZ ACC-437 (MOLT-14): Homozygous deletion affecting the probes for MTAP, CDKN2A, CDKN2B-AS1 and CDKN2B.
DSMZ ACC-203 (SK-N-MC): CDKN2A is unmethylated; CDKN2B is methylated.
DSMZ ACC-573 (SU-DHL-8), DSMZ ACC-42 (697): CDKN2A: NM_000077.5 is methylated, NM_058195.4 is unmethylated; CDKN2B is methylated.
DSMZ ACC-581 (HCT-116): CDKN2A: NM_000077.5 is methylated, NM_058195.4 is unmethylated. CDKN2B is unmethylated.
DSMZ ACC-511 (ALL-SIL): Heterozygous deletion affecting the probes for MIR31 and MTAP.Heterozygous deletion affecting the probes for CDKN2A, CDKN2B-AS1 and CDKN2B.
DSMZ ACC-40 (CTV-1): Homozygous deletion affecting the probes for MTAP, CDKN2A and CDKN2B-AS1. Homozygous deletion affecting the 229 and 458 nt probes for CDKN2B.CDKN2B is methylated.
DSMZ ACC-713 (GRANTA-452): Heterozygous deletion affecting the probes for MLLT3, KLHL9, MIR31 and PAX5. Homozygous deletion affecting the probes for MTAP, CDKN2A, CDKN2B-AS1 and CDKN2B.
DSMZ ACC-282 (JURKAT): Homozygous deletion affecting the probes for KLHL9, MIR31, MTAP, CDKN2A, CDKN2B-AS1 and CDKN2B.
DSMZ ACC-742 (BALL-1): Heterozygous deletion affecting the probes for MIR31, MTAP, CDKN2A (except for the 265, 280 and 259 nt probes), CDKN2B-AS1 and CDKN2B. Homozygous deletion affecting the 265, 280 and 259 nt probes for CDKN2A. CDKN2A/2B are unmethylated.
DSMZ ACC-339 (MHH-CALL-3): Heterozygous deletion affecting the probes for MIR31, MTAP, CDKN2A (only the 136, 157 and 273 nt probes), CDKN2B and PAX5. Homozygous deletion affecting the probes for CDKN2A (except for the 136, 157 and 273 nt probes) and CDKN2B-AS1.CDKN2B is methylated.
DSMZ ACC-22 (REH): Homozygous deletion affecting the probes for KLHL9, MIR31, MTAP, CDKN2A, CDKN2B-AS1 and CDKN2B.
DSMZ ACC-508 (RS4;11): Homozygous deletion affecting the probes for MLLT3, KLHL9, MIR31, MTAP, CDKN2A, CDKN2B-AS1 and CDKN2B.
DSMZ ACC-578 (TOM-1): Heterozygous deletion affecting the probes for MTAP, CDKN2A (only the 357 and 472 nt probes), PAX5 and PCSK5. Homozygous deletion of CDKN2A (except for the 357 and 472 nt probes).CDKN2A is unmethylated; CDKN2B is methylated.

Related products

Various related products

P419 CDKN2A/2B-CDK4: Targets CDKN2A, CDKN2B, CDK4, and the MITF p.E318K (c.952G>A) variant.
P088 Oligodendroglioma 1p-19q: Targets CDKN2A, CDKN2B, chr. 1p and 19q, and the most common somatic IDH1 and IHD2 point mutations.
P105 Glioma: Targets several genes, regions and variants involved in gliomas, including CDKN2A.
ME042 CIMP: Methylation status and copy number of the promoter regions of CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1.
ME001 Tumour suppressor mix: Methylation status and copy number of 25 tumour suppressor genes, including CDKN2A and CDKN2B.

Resources

General MLPA resources

MLPA education

Publications

References

Chan SH et al. (2021). CDKN2A germline alterations and the relevance of genotype-phenotype associations in cancer predisposition. Hered Cancer Clin Pract. 19:21.
Creighton CJ et al. (2010). Molecular profiling uncovers a p53-associated role for microRNA-31 in inhibiting the proliferation of serous ovarian carcinomas and other cancers. Cancer Res. 70:1906-15.
Hewitt C et al. (2002). Germline mutation of ARF in a melanoma kindred. Hum Mol Genet. 11:1273-9.
Kim M et al. (2011). PAX5 deletion is common and concurrently occurs with CDKN2A deletion in B-lineage acute lymphoblastic leukemia. Blood Cells Mol Dis. 47:62-6.
Wolter M et al. (2001). Oligodendroglial tumors frequently demonstrate hypermethylation of the CDKN2A (MTS1, p16INK4a), p14ARF, and CDKN2B (MTS2, p15INK4b) tumor suppressor genes. J Neuropathol Exp Neurol. 60:1170-80.
Yu W et al. (2008). Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA. Nature. 451:202-6.