Tumor second hit analysis
As far as the LOH is concerned, two FFPE tumor blocks could be studied:
the CRC of member II:6 and the breast tumor of member II:4. No
significant loss of any of the alleles was observed in the CRC, although
a slight reduction of the mutant allele was detected in the breast tumor
(Supplementary Figure S5). On the other hand, a promoter methylation
assay was used to study the methylation status of PTPRT promoter
in the available tumors at two different sites previously reported
(Laczmanska et al., 2013; Peyser et al., 2016). This assay showed that
the CRC tumor DNA of one of the mutation carriers (II:6) was
hypermethylated at the two PTPRT promoter sites tested, as
compared to healthy colon DNA from the same carrier, as well as to a
pool of healthy colon samples used as a control (Figure 3A). Although to
a lesser extent, the same result was observed when DNA from the breast
tumor and healthy breast from member II:4 was studied at the promoter
site described by Peyser et al. (Peyser et al., 2016) (Figure 3B).
However, no methylation was observed in any of the breast samples at the
methylation site reported by Laczmanska et al. (Laczmanska et al.,
2013). A pyrosequencing assay targeting PTPRT promoter confirmed
the hypermethylation observed in the CRC of member II:6 compared to its
healthy colon tissue (Figure 3C). Although once again more subtle, the
breast cancer also showed an increased methylation compared to the
healthy breast tissue of member II:4.