General information
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,
CDKN2B and
CDK4 genes
, which are associated with familial cutaneous melanoma, neural system tumour- 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 p16
INK4A and p14
ARF. The majority of
CDKN2A mutations detected in familial melanoma patients affect exons 1 and 2, which code for p16
INK4A. However, exon 1 mutations specific for p14
ARF 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 and
CDKN2B 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) in the
MITF gene has been suggested to associate with predisposition to familial melanoma (Yokoyama et al. 2011; Bertolotto et al. 2011).
This SALSA MLPA probemix is not CE/FDA registered for use in diagnostic procedures. Purchase of this product includes a limited license for research purposes.
Probemix content
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 the
CDK4 gene and 10 flanking probes in total for the
CDKN2A and
CDKN2B 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 and the identity of the genes detected by the reference probes are available in Table 3 and online (
www.mrcholland.com).
This 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, 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.
SALSA Binning DNA SD008
The SD008 Binning DNA provided with this probemix can be used for binning of all probes including the
MITF mutation-specific probe (probe 17808-L23191 at 162 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 for quantification of mutation signals. 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, available online:
www.mrcholland.com.
This product is for research use only (RUO).
Sample DNA
Sample DNA developed for this product: