The aims of this study were to localize and identify the gene predisposing to JP, and to evaluate its role in hereditary and sporadic colorectal tumorigenesis.
In study I, we analysed whether PTEN, gene frequently mutated in CS and BRR, could also be the gene predisposing to JP. In total, 25 unrelated JP patients were screened for the presence of PTEN mutations and no mutations were found. Furthermore, linkage analysis in eight informative families excluded linkage to PTEN, and hence, excluded the possibility of PTEN promoter or 3'-UTR mutations and whole gene deletions at least these eight JP families. Thus, although germline mutation has been detected both in CS and BRR (also characterized by hamartomatous gastrointestinal polyps), our results suggest that JP is not allelic to these syndromes.
To localize the gene predisposing to JP, a whole genome wide search was done in three Finnish JP families (families 2, 4 and 5, data not shown). However, simultaneously and independently, Howe and colleagues (1998b) succeeded in localizing the gene predisposing to JP in chromosome band 18q21.1 (Howe et al., 1998b). This chromosomal region contains two putative tumor suppressor genes: DCC and SMAD4, and in collaboration with Howe and colleagues (study II) we managed to identify germline mutations in SMAD4 gene in JP patients. Conclusively, SMAD4 mutations were found in 8 of 12 JP cases studied (5 familial and 3 sporadic). SMAD4 mutations were not detected in two families and in two sporadic cases, that were negative also in the previous PTEN mutation analysis. JP is obviously a genetically heterogeneous condition, as evidenced by the fact that not all families are linked to 18q markers and not all patients studied had germline SMAD4 mutations. It has been hypothesized that the germline mutations in genes (other than SMAD4) encoding different components of the TGF-β signalling pathway may be present in JP. To study this hypothesis further, we focused our screening on SMAD genes, which are involved in TGF-β signaling via TGF-β type II and I receptors. In study III cDNA based mutation screening was performed for SMAD2, SMAD3 or SMAD7 genes and no mutations were detected in these genes in any of our patients.
One syndrome, which is occasionally detected among JP patients, is HHT. Two genes have been identified for HHT. Endoglin, which maps to chromosome 9q3 is identified as the gene for HHT1 and ALK1, which maps to chromosomal locus 12q11-14 is the gene for HHT2, respectively. Both endoglin and ALK1 are members of TGF-β receptor superfamily and are therefore considered as good candidates for JP gene. We screened endoglin and ALK1 mutations among those JP patients, where SMAD2, SMAD3, SMAD4, SMAD7 or PTEN mutations have not been detected. No mutations were detected in either one of these genes in any of patients analyzed (unpublished data).
Loss of growth inhibition by transforming growth factor β (TGF-β) is an important step in colorectal tumorigenesis. As SMAD proteins play a key role in intracellular TGF-β signaling, mutations in some of the SMAD genes may disrupt this pathway. To investigate whether germline mutations in SMAD2, SMAD3 or SMAD4 could predispose to HNPCC, we analyzed mutations in these genes from 14 HNPCC kindreds in which germline mutations of MSH2 and MLH1 had been ruled out. SMAD gene mutations were not found in any of patient analyzed, suggesting that these genes do not have a major role in HNPCC (study IV).
It is generally considered that the genes involved in hereditary cancer syndromes are also mutated in the same type of sporadic tumors. However, SMAD4 mutations seem to be relatively rare in the sporadic colon cancers. In study V, we examined whether hypermethylation of the promoter could be an alternative mechanism for SMAD4 inactivation in colorectal cancers. In total, 26 MSI and 16 MSS tumors and corresponding normal DNAs were analyzed and no evidence of hypermethylation was found. SMAD4 mutation screening was also performed among 22 primary CRCs and the only change detected was a polymorphism in exon 2. Our results together with previously published data suggests that SMAD4 is not frequently mutated in primary colorectal carcinoma and that the hypermethylation of SMAD4 promoter region is not a crucial mechanism in colorectal tumorigenesis.