Since the discoveries of the genes responsible for Familial Adenomatous Polyposis, Lynch syndrome, and the hamartomatous polyposes, little progress has been made in the identification of genetic causes for the remaining unexplained majority of familial CRC. One of the reasons for this lack of success is likely to be extensive genetic heterogeneity, which has proved to be a difficult obstacle to overcome for investigators undertaking linkage studies on collections of CRC families. Other than exclusion of known syndromes, approaches aimed at limiting the heterogeneity within a sample set have included enrichment for families with young-onset cases [8
], exclusion of multiple polyposis phenotypes [8
], exclusion or inclusion of adenomas and hyperplastic polyps [41
], and stratification by occurrence of extracolonic cancers [41
]. While these approaches certainly limit phenotypic heterogeneity, the extent to which they reduce genetic heterogeneity is unknown.
The recent identification of a familial predisposition to colonic malignancies arising through the serrated pathway [13
] has presented an opportunity to study a subset of familial CRC in which genetic heterogeneity is more limited. Serrated neoplasia families show distinct features, such as variable levels of MSI and frequent somatic BRAF
mutation in tumours and a mixture of serrated and adenomatous polyps, that allow them to be distinguished from the remainder of familial CRC. Such features characterised families with evidence of linkage to 2q. Incomplete penetrance, small family size, under-reporting due to a proportion of individuals being asymptomatic, and practical difficulties in obtaining tumour and polyp samples for all patients, however, mean that all features will not be observed in all families. In an effort to account for this, a set of criteria was developed for this study that allowed for some flexibility while still requiring families to show a clustering of CRC and polyps in conjunction with characteristics that define Jass syndrome. In this attempt to include phenotypically homogeneous families, the possibility remains that a minority of these families may have a genetic predisposition different to that responsible for the majority of Jass syndrome. While this leaves open the possibility of some families being incorrectly included, these are likely to be a minority.
A linkage strategy was selected that would maximise the possibility of identifying linkage, while minimising the effects of any heterogeneity that may remain within the selected families. To this end, the family considered the most likely to allow the identification of a strong linkage signal was subjected to a genome-wide linkage screen, followed by finemapping of regions of interest in the remaining 10 families. Loci at 2p25.2-p25.1, 2q24.3-q37.1 and 8p21.2-q12.1 were considered worthy of further investigation, with 2q24.3-q37.1 showing the strongest evidence of linkage. This region has also been identified in three previous genome-wide linkage studies [4
] and one association study [40
The consensus region at 2q32.2-q33.3 identified through finemapping linkage and haplotype analyses contains five genes that stand out as potential candidates and as a result were prioritised for sequencing. CFLAR
are located in close proximity to each other at 2q33.1 and are all involved in the death receptor-induced apoptotic pathway [44
somatic mutation has been reported in CRC [45
], and a CASP8
promoter polymorphism has been reported to be associated with susceptibility to multiple cancers including CRC [40
], although the association has not been replicated in other populations [46
has attracted attention as a therapeutic target as well as a potential oncogene due to its role in the TRAIL pathway [48
is a member of the Frizzled family which plays an important role in Wnt signalling [49
], central to the development and progression of CRC [50
], and has recently been shown to activate the Wnt pathway in CRC cell lines [51
inactivation leads to epithelial cell proliferation and mixed polyp formation in the mouse colon [52
] and reduced protein expression associated with somatic mutation is observed in human CRC and cell lines [53
]. Although sequencing of these genes did not reveal any variants segregating with disease, only coding regions and exon–intron boundaries were sequenced, leaving noncoding regions, which may harbour regulatory elements and potential splicing variants, largely unexplored. With current re-sequencing technology and exome capture, the investigation of the remaining genes and regulatory regions becomes more feasible.
Several studies have reported evidence of linkage among a proportion of non-syndromic CRC families, with 3q22, 7q31 and 9q22 prominent [4
]. As well as suggesting that they are unlikely to contribute to Jass syndrome, the failure to find evidence of linkage to these loci in the present study suggests that further characterisation of the 3q22, 7q31 and 9q22 regions should aim to exclude families demonstrating molecular and/or histological evidence of serrated neoplasia.
Approximately half of the families studied showed evidence of linkage to 2q32.2-q33.3. The balance of the families did not demonstrate linkage, suggesting that other as yet unidentified loci may contribute to families with serrated neoplasia. Continuing efforts to identify and characterise serrated neoplasia families will serve the dual purpose of allowing a greater understanding of the phenotypic presentation of the syndrome, and facilitating further genetic studies which will allow confirmation and refinement of the linked region, paving the way for more comprehensive analysis of the locus, and potentially the identification of the underlying genetic defect.