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Editor—Transforming growth factor-β (TGF-β) family members are known to be involved in the regulation of cell proliferation, differentiation, and apoptosis.1Members of the TGF-β family include TGF-βs, activins, and bone morphogenetic proteins (BMPs). Their signals are mediated to the cell nucleus by a network of transmembrane serine/threonine kinase receptors and their downstream effectors, the SMAD proteins.2 SMAD proteins play a key role in intracellular TGF-β signalling and inactivating mutations of SMADs, such asSMAD2, SMAD3, andSMAD4, provide resistance of cells to TGF-β induced growth inhibition.
To date, eight human SMADs have been identified. Two of them, SMAD2 andSMAD4, have been reported to be mutated in a subset of colorectal carcinomas.3-6 Germline mutations ofSMAD4 have been found in patients with juvenile polyposis, a condition predisposing to colorectal cancer.7-10
SMAD3 mutations have not been reported in human cancers. In a recent study by Arai et al,11 SMAD3 mutations were analysed in 35 sporadic colorectal and 15 HNPCC cancers and no mutations were found. Targeted disruption of the SMAD3 gene in mice has been reported to lead to development of colorectal cancer,12 though other studies have not detected a clear association.13 14 No genetic alterations in otherSMADs have been reported in malignancy.
Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominantly inherited cancer susceptibility syndrome, associated with germline mutations in five DNA mismatch repair genes:MLH1, PMS1, PMS2, MSH2, and MSH6.15-19Inactivation of both alleles of a mismatch repair gene results in microsatellite instability (MSI) that is a hallmark of HNPCC tumours.20-23 The genes responsible for microsatellite stable (MSS) HNPCC are still unknown.
Loss of growth inhibition by TGF-β is an important step in colon tumorigenesis and in HNPCC tumours with MSI this is mainly the result of frameshift mutations within a polyadenine sequence repeat in the TGF-β type II receptor (TGFβRII) gene.24 It has been proposed that mutations inTGFβRII could underlie the cancer predisposition in MSS HNPCC,25 and also that other genes involved in the TGF-β pathway are candidates for MSS HNPCC.26
Chromosomal deletions are common genetic alterations in cancer and they are targeted at tumour suppressor loci.27 28 Previous studies have shown that one copy of chromosome 18q is lost in over 70% of sporadic colorectal cancers.29-32 TheDCC (deleted in colorectal cancer) gene has been suggested as a candidate target gene in this region and loss of expression of DCC has also been reported in colorectal cancers.33 However, mutations in the coding region of DCC seem to be rare34and the position of DCC as a candidate tumour suppressor is not clear. Two other candidate genes,SMAD4 and SMAD2, have recently been identified at the same 18q region3 35emphasising the possible role of the SMADgenes in colorectal tumorigenesis. The aim of the present study was to investigate whether germline mutations in SMAD2, SMAD3, and SMAD4 underlie microsatellite stable HNPCC.
Mutation screening was performed in 14 Finnish HNPCC kindreds from which lymphoblastoid cell lines were available. Based on genealogical evidence the families are unrelated, though the existence of early common ancestors cannot be excluded. One affected subject per family was included in the study. Of the kindreds, six fulfilled the Amsterdam criteria for HNPCC.36 Other patients represent familial HNPCC-like colorectal cancer (CRC); the number of patients with CRC or endometrial cancer ranged from two to six per family (average three) (table 1). All kindreds selected for this study have previously been shown to be MLH1 andMSH2 mutation negative.37 In three kindreds, DNA from tumour tissue had not been available. From 10 families one and in one family two colorectal cancer samples were available and no evidence of MSI had been detected (table 1). The study was approved by the ethical committee of the Department of Medical Genetics, University of Helsinki.
Total cellular RNA was extracted from lymphoblasts by RNA extraction kit (QIAGEN). The SMAD2, SMAD3, and SMAD4 genes were amplified from RNA using an RT-PCR procedure. First, 20 μl cDNA was created from 0.8 μg of RNA using standard random priming methods with Mu-MLV reverse transcriptase (Promega) and RNAse inhibitor (Promega). The cDNA sequences for SMAD2,SMAD3, and SMAD4were derived from GenBank database (accession numbers U65019, U76622, and U44378, respectively). PCR primers for cDNA amplification were designed using the Primer3 server (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi). Each gene was divided into five fragments, covering the whole coding region of the gene. The forward (F) and reverse (R) primers and size of each PCR product are listed in table 2.
The PCR reactions were carried out in a 50 μl reaction volume including 2 μl of cDNA, 1 × PCR reaction buffer (Perkin Elmer Applied Biosystems Division), 200 μmol/l of each dNTP (Finnzymes), 0.8 μmol/l of each primer, and 2 units of AmpliTaq GOLD polymerase (PE/ABI). The MgCl2 concentration was 1.5 mmol/l forSMAD2 fragments 1 and 2,SMAD3 fragment 3, and allSMAD4 fragments. For all other fragments the MgCl2 concentration was 2.5 mmol/l.SMAD3 fragment 2 reaction also included 10% of DMSO. The PCR conditions are available upon request.
After PCR, 5 μl of the PCR product was run on a 3% agarose (NuSieve) gel to verify the specificity of the PCR reaction. The rest of the PCR product was purified using QIAquick PCR purification Kit (QIAGEN). Direct sequencing of the PCR products was performed using the ABI PRISM Dye Terminator or ABI PRISM dRhodamine cycle sequencing kits (PE/ABI). Cycle sequencing products were electrophoresed on 6% Long Ranger gels (FMC Bioproducts) and analysed on an Applied Biosystems model 373A or 377 DNA sequencer (PE/ABI).
To screen for the presence of a base substitution inSMAD3 in controls, restriction enzyme digestion was performed. HgaI (New England BioLabs) digestion was used to detect A to G change inSMAD3 exon 3 at codon 170. New PCR primers for genomic exon 3 amplification were designed using the Primer3 server. The primers were: 5′-ATCGACACTGAGCCACCTCT (forward) and 5′-CCCACGTGCCTACCTCTG (reverse). The PCR reactions were carried out in a 50 μl reaction volume including 100 ng genomic DNA, 1 × PCR reaction buffer (PE/ABI), 200 μmol/l of each dNTP (Finnzymes), 0.8 μmol/l of each primer, 2 units of AmpliTaqGOLD polymerase (PE/ABI), and 1.5 mmol/l of MgCl2.The following PCR cycles were used for amplification: 10 minutes at 95°C, 40 cycles of 45 seconds at 95°C, 45 seconds at 56°C, one minute at 72°C, and final extension for 10 minutes at 72°C.HgaI cuts the PCR fragment (187 bp) that contains the substitution into two fragments (134 bp and 53 bp in size) whereas the wild type fragment lacks the restriction site and is not digested. The digestion was performed in 1 × NEBuffer (New England BioLabs) at 37°C overnight. After digestion, the PCR products were electrophoresed through a 3% agarose gel.
In this work we analysed SMAD2,SMAD3, and SMAD4mutations in 14 familial colon cancer kindreds, 11 of these displaying at least one MSS tumour. The microsatellite analysis data derived typically from one single tumour per family suggest that these kindreds do not segregate DNA mismatch repair gene mutations, but does not exclude this possibility. Previous studies had evaluatedMLH1 and MSH2mutations in the series with negative results.37 38 SMAD gene mutation analysis was performed by automated sequencing covering the translated region of the genes. Genetic alterations were not detected in SMAD2or SMAD4 in any of these patients. InSMAD3, three discrepancies were detected between GenBank sequence (U76622) and sequences from our patients, firstly the A to G change at the third position of codon 103 (exon 2). Homozygous A to G change was seen in 11 of our 14 patients and in three of them the substitution was heterozygous. This discrepancy has been reported earlier and the variant does not cause any amino acid change.9 11 A second, silent change detected was a C to T transition at nucleotide 907 (exon 6). This change was homozygous and it was present in one of our 14 HNPCC patients (in family 31). The frequency of these variants in the normal population was not analysed, as the changes were silent. The third change was an adenine to guanine transition at nucleotide 545, which is predicted to convert isoleucine to valine at amino acid 170 (fig 1). This change was detected in two patients (in families 65 and 75). For this variant, 110 Finnish controls were analysed by restriction enzyme digestion (HgaI). Seven out of 110 controls displayed the change (6.4%). To compare further the frequency of this polymorphism in colon cancer patients and controls, 132 patients were included in the analysis. Taken together, in the 14 HNPCC patients and 132 colon cancer patients the frequency of this polymorphism was 8.9% (13/146). From those 13 cancer patients who had valine instead of isoleucine at codon 170, four turned out to be familial. Segregation of the polymorphism was analysed in two of these families where DNA from multiple family members was available and the polymorphism did not segregate with cancer in these families.
SMAD2, SMAD3, orSMAD4 mutations were not found in any of our patients using a cDNA based mutation analysis. It should be noted that like all other mutation detection methods, this method may miss a subset of mutations. Also, the potential existence of founder mutations in the Finnish population may have hampered our efforts to detectSMAD gene defects in HNPCC. However, it is likely that defects of SMAD2, SMAD3, orSMAD4 are not a common cause of familial colon cancer. Further work is necessary to unravel the molecular background of MSS HNPCC.
We thank Siv Lindroos, Liisa Suksi, and Päivi Laiho for technical assistance. This study was supported by grants from the Finnish Cancer Society, the Academy of Finland, Emil Aaltonen Foundation, Finnish Cultural Foundation, Sigrid Juselius Foundation, and Biocentrum Helsinki.