The Eph receptor family is the largest known subgroup of receptor tyrosine kinases. This family is further subdivided into two distinct classes, EphA (A1 to A10) and EphB (B1 to B6), based on their binding affinities for two membrane-anchored ligand families with the corresponding names of type A (A1 to A5) and B (B1 to B3) ephrins 
. Following ligand binding, Eph receptors activate cell repulsion pathways to modulate cell compartmentalization and ordered cell migration in a variety of biological processes 
Mouse animal model studies have shown that, in the small intestine, EphB receptors mediate intestinal stem cell proliferation 
as well as epithelial cell migration and organization along the crypt-villous axis 
. Since loss of mitotic activity control, epithelial patterning and tissue architecture are hallmarks of tumorigenesis, disruption of normal EPHB receptor expression and function likely promotes colorectal carcinogenesis. Constitutive EPHB receptor expression may stimulate tumor initiation by disturbing proliferative stem cell homeostasis, and secondary silencing of EPHB receptor activity may permit expansion of cancer cells, beyond the spatial boundaries imposed by intact EPHB receptor function to populate adjacent tissue structures 
. In support of this hypothesis, we and others have recently shown a causal role for EphB inactivation in tumor progression 
. We found that EphB2
silencing in ApcMin/+
mice results in accelerated and more aggressive colorectal tumorigenesis.
In the current study, we screened 116 population-based familial cases of colorectal cancer for mutations in a candidate tumor suppressor gene, EPHB2, and identified three candidate variants (A438T, D679N, G787R), which were further characterized. Even though the A438T allele was not observed in control subjects and was found to segregate with disease in Family 1, biochemical characterization suggests that A438T is a rare neutral polymorphism; we cannot exclude the possibility that it affects more subtle aspects of EPHB2 signaling, such as the formation of higher order oligomers. The D679N variant has been previously reported to be associated with prostate cancer 
. Although, it remains possible that this variant modulates predisposition to prostate cancer, our data suggest that it occurs at a population frequency of approximately 1% and that it does not, on its own, increase susceptibility to colorectal cancer. We observed the D679N allele in a similar number of patients with colorectal cancer and population-matched controls. In contrast to these latter variants, our data suggest that the G787R variant is functionally compromised and may be a rare cause of hereditary colorectal cancer. The G787R variant was identified in a patient diagnosed with rectal cancer at 67 years of age, and biochemical characterization revealed that the G787R substitution markedly diminishes the receptor's intrinsic kinase activity.
There have been two other investigations examining the contribution of germline EPHB2
mutations to colorectal cancer susceptibility. Oba et al.
screened for EPHB2
mutations in colon tumors and respective normal colon tissues from 50 patients with colorectal cancer, and identified an intron 8 alteration in a single tumor sample, which results in a nonsense mutation. However, it is unclear if this is a somatic mutation, as there is no indication whether this genetic change was also observed in the paired normal colon sample 
. This investigation also identified 15 cases with LOH involving the EPHB2
gene and screened for mutations in the remaining EPHB2
allele. Since mutations in the remaining allele were not identified, Oba et al.
suggested that EPHB2
is not a classical tumor suppressor gene. However, since only 50 samples of likely sporadic cases of colorectal cancer were analyzed, a tumor suppressor role for EPHB2
cannot be excluded. In a more recent study, Kokko et al.
reported an association of three novel variants with colorectal cancer 
. Germline missense changes resulting in I361V, R568W, and D861N were observed in colorectal cancer patients, but not in healthy controls. However, it is possible that these three variants are rare neutral polymorphisms since the biological significance of the variants was not evaluated using direct functional assays. The patients screened in these latter two studies did not necessarily have significant family histories of colorectal cancer. In contrast to these two earlier reports, our study was designed to specifically evaluate the role of germline EPHB2
mutations in patients with familial colorectal cancer, and not in sporadic cases. Despite study design differences, together these three investigations suggest that EPHB2
germline mutations are not common occurrences in colorectal cancer. Further investigations of larger sample sizes are needed to confirm this observation.
In summary, we identified a germline EPHB2 variant (G787R) with diminished biological activity in a colorectal cancer patient, and suggest that EPHB2 mutations contribute to a small fraction of hereditary colorectal cancer. The rarity of germline EPHB2 mutations supports a more significant role for EPHB2 in colorectal tumor progression rather than in tumor initiation. Since the EPHB receptors (EPHB2, EPHB3 and EPHB4) follow a similar pattern of transcriptional silencing in colorectal cancers, all EPHB receptor family members probably play a similar role in this disease. Therefore, the EPHB family likely accounts for a minor proportion of genetic predisposition to colorectal cancer but has an important role in tumor progression. Although our findings suggest that the EPHB gene family should not be routinely screened for germline mutations in familial cases, the EPHB genes are candidate tumor suppressors, likely accounting for rare cases of familial colorectal cancer.