The SALSA MLPA
Probemix P419 CDKN2A/2B-CDK4 is a research use only (RUO)
assay for the detection of deletions or duplications in the CDKN2A
which are associated with familial cutaneous melanoma and pancreatic cancer syndromes. This probemix can also be used to detect the presence of a MITF
p.E318K (c.952G>A) point mutation and the status of codon 24 of the CDK4
gene with a WT probe.
Malignant melanoma is estimated to be hereditary in 5-10% of the cases. Familial cutaneous melanoma arises in an autosomal-dominant pattern within the affected families and germline alterations in the CDKN2A
gene (9p21.3) are detected in up to 40% of cases (Goldstein et al. 2007). The CDKN2A
gene encodes for two proteins read in alternative reading frames, namely p16INK4A
. The majority of CDKN2A
mutations detected in familial melanoma patients affect exons 2 and 3, which code for p16INK4A
. However, exon 1 (previously known as 1β) mutations specific for p14ARF
have also been reported (Hewitt et al. 2002), as well as larger genomic deletions of CDKN2A
(Randerson-Moor et al. 2001; Knappskog et al. 2006; Lesueur et al. 2008). Moreover, both heterozygous and homozygous deletions harbouring both CDKN2A
are frequently detected in somatic melanoma samples (Flores et al. 1996).
Another high-penetrance, but low-frequency, melanoma susceptibility gene is CDK4
(12q14.1), which is mutated in 2% of the melanoma families (Goldstein et al. 2007). Two point mutations (p.R24H and p.R24K) of CDK4
have been detected in familial melanoma cases (Zuo et al. 1996). In addition, a germline mutation p.E318K (c.952G>A) of the MITF
gene has been suggested to be associated with predisposition to familial melanoma (Yokoyama et al. 2011; Bertolotto et al. 2011).
The SALSA MLPA Probemix P419-B1 CDKN2A/2B-CDK4 contains 57 MLPA probes with amplification products between 121 and 504 nucleotides (nt). This includes 14 probes for the CDKN2A
gene, nine probes for the CDKN2B
gene, nine probes for CDK4
gene and in total 10 flanking probes for the CDKN2A
genes. Furthermore, this probemix contains one probe specific for the MITF
p.E318K (c.952G>A) point mutation, which will only generate a signal when the mutation is present, and one wildtype probe for CDK4
codon 24, which will have a drop in signal in case of a CDK4
codon 24 mutation. In addition, 13 reference probes are included that detect relatively copy number stable regions in various cancer types, including cutaneous melanoma. Complete probe sequences are available online (www.mlpa.com
) and the identity of the genes detected by the reference probes is available in Table 2d.
This probemix contains nine quality control fragments generating amplification products between 64 and 120 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.mlpa.com
SALSA Binning DNA SD008:
The SD008 Binning DNA provided with this probemix can be used for binning of the MITF
mutation-specific probe (probe 17808-L23191 at 161 nt). SD008 Binning DNA is a mixture of genomic DNA from healthy individuals and plasmid DNA that contains the target sequence detected by the above mentioned probe. Inclusion of one reaction with 5 μl SD008 Binning DNA in initial MLPA experiments is essential as it can be used to aid in data binning of the peak pattern using Coffalyser.Net software. Furthermore, Binning DNA should be included in the experiment whenever changes have been applied to the set-up of the capillary electrophoresis device (e.g. when capillaries have been renewed). Binning DNA should never be used as a reference sample in the MLPA data analysis, neither should it be used in quantification of mutation signal, as for this purpose true mutation positive patient samples or cell lines should be used. It is strongly advised that all samples tested are extracted with the same method and derived from the same source of tissue. For further details, please consult the SD008 Binning DNA product description provided. This product is for research use only (RUO).
Sample DNA developed for this product: