Common chromosomal fragile sites (CFSs) are unstable genomic loci that show increased sensitivity to replication stress and are prone to breaks and gaps on metaphase chromosomes upon exposure with carcinogens. Several studies have shown that CFSs are sites of frequent chromosome breakage and rearrangement in cancer cells. The two most active CFSs - FRA3B and FRA16D - comprise the two large tumor suppressor genes FHIT and WWOX, respectively. It is suggested that loss of WWOX or FHIT initiates genomic instability and can contribute to tumourigenesis and cancer progression (Glover T et al. 2017, Nat Rev Cancer. 17:489-501).
Moreover, intragenic or complete deletions of the WWOX gene generating a premature stop codon are found to be associated with severe early-onset epileptic encephalopathy, characterised by epilepsy with lasting deleterious impact in the developing brain (Valduga M et al. 2015, J Hum Genet. 60:267-71; Mignot C at al. 2015, J Med Genet. 52:61-70).
The FHIT gene (10 exons), encompassing the FRA3B region, spans ~1500 kb of genomic DNA and is located on 3p14.2, ~60 Mb from the p-telomere. FHIT gene is a member of histidine triad gene family, whose expression is lost or reduced in many human cancers, and the loss of FHIT is suggested to initiate genome instability in preneoplastic lesions.
The WWOX gene (9 exons), encompassing the FRA16D region, spans ~1100 kb of genomic DNA and is located on 16q23.1, ~80 Mb from the p-telomere. WWOX (WW domain-containing oxidoreductase) is a member of the short-chain dehydrogenase/reductase (SDR) protein family, and is commonly affected by deletions in wide range of solid tumours, and additionally by translocations in multiple myeloma (Aldaz CM et al. 2014, Biochim Biophys Acta. 1846:188-200).
The P063-B1 probemix contains two probes for each exon of the FHIT gene, and one probe for each exon of the WWOX gene with two probes for exon 1. Furthermore, nine probes have been included for the flanking regions of the FHIT and WWOX genes to aid in determination of the extent of the copy number change. In addition, 14 reference probes are included, detecting different autosomal chromosomal locations which are relatively quiet in most tumour types. However, it should be noticed that tumour genomes can harbour multiple numerical and structural aberrations, which can complicate interpretation of these reference probes and data normalisation.
This SALSA® MLPA® probemix is designed to detect copy number changes of one or more sequences in the aforementioned genes in a DNA sample. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak height of the amplification product of that probe. Note that a mutation or a polymorphism in the sequence detected by a probe can also cause a reduction in relative peak height, 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. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. Finally, note that most defects in these genes are expected to be small (point) mutations which will not be detected by this SALSA® MLPA® test.