Four microsatellite markers flanking the PTEN locus (markers D10S219, D10S551, D10S579, and D10S541) were used to generate haplotypes for 47 individuals of eight informative JP families originating from the United States and the United Kingdom.
For the microsatellite markers D10S219 and D10S541, the maximum two-point lod score was 0 at a recombination fraction of θ = 0.5 for both models (model 1: allele frequency 0.002 and penetrance 0.5; model 2: allele frequency 0.0002 and penetrance 0.85). For markers D10S551 and D10S579, the maximum two-point lod scores were 0.50 and 0.72, respectively, at θ = 0 for the first model and 0.63 and 0.20 at θ = 0 and θ = 0.4, respectively for the second model. Multipoint analysis revealed lod scores < -2.0 over the entire region, so the linkage of JP to PTEN locus was excluded in the eight families studied.
PTEN mutations were analyzed among probands from 14 JP families and 11 sporadic cases by either direct sequencing or DGGE analysis and mutations were not detected. A frequent polymorphism in intron 8 was observed in 36 % (4 of 11) of sporadic JPs cases. The presence of this sequence variant in its heterozygous state in four cases excludes whole gene deletion as a cause of JP in these patients.
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). 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. Since both of these genes were good candidates for JP, the mutation screening was performed for both of them. After sequencing 14 DCC exons and 11 SMAD4 exons, a 4 base pair (bp) deletion was detected in SMAD4 exon 9 (between nucleotides 1372 and 1375, GenBank accession number U44378). This mutation was first detected in one affected individual from the Iowa kindred. Next, the SMAD4 exon 9 was screened from all 46 members of the Iowa JP kindred, the same 4 bp deletion being present in all 13 affected and 4 of 26 at risk individuals, but not in any of 7 spouses (study II).
Then, eight additional unrelated JP patients were analyzed for mutations of all exons of SMAD4 by SSCP or genomic sequencing, and the mutation was found in four of them. Two JP kindreds (originating from United States and Finland) were segregating the same 4 bp deletion in exon 9 that was detected in the Iowa kindred. This deletion causes a frameshift that creates a new stop codon at codon 434. In total, 242 controls were analyzed for the presence of this alteration and the altered allele was not observed in any of them (study II). Mutations were also detected in two sporadic JP cases. The first one was a 2 bp deletion in exon 8 at nucleotides 1170 to 1171 (codon 348). This deletion causes a frameshift that creates a stop codon at codon 350. The change was not detected in any of 101 controls analyzed. Another patient was found to have a 1 bp insertion between nucleotides 815 and 820 of exon 5, this change added a guanine to a stretch of six sequential guanines in the wild-type sequence and created a frameshift and a new stop codon at codon 235. For this change, 107 controls were analyzed and again, this change was not detected in any of them (study II).
In study III, seven unrelated JP families or sporadic patients were analyzed for mutations of all exons of SMAD4 by genomic sequencing. Four out of these seven cases were previously analyzed for SMAD4 mutations in study II (by SSCP), and were then reported mutation negative (JP1/1, JP2/13, JP4/1, and JP6/1, study II). In this study, three different germline defects were detected. In family 3, we detected the same 4-base-pair deletion in exon 9, which has been previously described in three JP kindreds. In the sporadic case number 2 (JP6/1 in study II) a C to a G transversion at nucleotide 661 was detected. This mutation changes serine to a stop codon at codon 177. Forty-nine controls were analyzed (AlwI digestion) and the change was not detected in any of them. The change detected in JP family 1 (JP 4/1 in study II) was an A to C transition at nucleotide 1186, which is predicted to convert tyrosine to serine at amino acid 353. This variant was present in both cases with JP (see Material and Methods, Patients) but also in one unaffected at risk individual. For this variant, 55 Finnish controls were analyzed by restriction enzyme digestion (EcoRI), and none of them displayed the change. These two base pair changes were missed in study II, because they did not show in SSCP analysis. No SMAD4 mutations were detected in families 2 and 4, or in sporadic cases number 1 and 3.
In total, a set of 12 independent JP cases were analyzed for SMAD4 mutations (studies II and III). Among these, mutations were detected in 5 families and 3 sporadic cases.
Linkage analysis using markers D18S970, D18S474, D18S1099, D18S851, D18S484, D18S858 and D18S977 (SMAD4 is located close to markers D18S474 and D18S1099) resulted in a clear exclusion (Z £ -2) of the SMAD4 region in family 2 (see Materials and Methods, Patients). However, formal exclusion could be obtained only at, and in the near vicinity of, marker D18S858 in family 4 while the rest of the marker map produced inconclusive lod scores.
SMAD2, SMAD3 and SMAD7 mutations were analyzed among those JP families or sporadic cases where SMAD4 or PTEN mutations had not been detected (families 2 and 4 and patients 1 and 3). Mutation analysis was performed by automated sequencing covering the translated region of these genes. No SMAD2, SMAD3 or SMAD7 mutations were detected in any of these patients. The only variant identified was an A to G change at the third position of codon 103 in the SMAD3 gene. The change was homozygous in all of our four samples. This polymorphism has been reported earlier and the variant does not cause any amino acid change (Arai et al., 1998).
SMAD2, SMAD3 and SMAD4 mutations were analyzed among 14 familial colon cancer kindreds, eleven of these displaying at least one MSS tumor. Previous studies had evaluated MLH1 and MSH2 mutations in these families, all of them being mutation negative (Nyström-Lahti et al., 1996; Holmberg et al., 1997). SMAD gene mutation analysis was performed by automated sequencing covering the translated region of the genes. Genetic alterations were not detected in SMAD2 or SMAD4 genes in any of these patients.
In the SMAD3 gene, three discrepancies were detected between GenBank sequence (U76622) and sequences from our patients. The first was an A to G change at the third position of codon 103 (exon 2), this silent change has been reported earlier (study II). A second, silent, change detected was C to T transition at nucleotide 907 (exon 6). The frequency of these two variants in the normal population was not analyzed, 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. This change was detected in two patients. For this variant, 110 Finnish controls were analyzed by restriction enzyme digestion (HgaI). Seven out of 110 control individuals displayed the change (6.4 %). To further compare the frequency of this polymorphism in colon cancer patients versus control individuals, 132 patients were included in analysis. Taken together 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 analyzed in two of these families where DNA from multiple family members was available, and the polymorphism was not segregating with cancer in these families.
In this study, the possible hypermethylation of the SMAD4 promoter was analyzed by using a HpaII and MspI digestion. Using this assay, we examined the methylation status for SMAD4 promoter region in a group of 26 MSI and 16 MSS colorectal tumors. The fragment selected for this analysis was a CG-rich region published by Hagiwara and colleagues (submitted), and the amplified sequence contained altogether 55 CpG dinucleotides. It was possible to determine the methylation status for six CCGG sites by restriction. No PCR product was detected from any of HpaII digested DNA, suggesting that the SMAD4 promoter is unmethylated in all the cases studied.
Twenty-two primary colon cancers were analyzed for mutations of all exons of SMAD4 by genomic sequencing. The only change detected was a G to A transition at the third position of codon 118 (exon 2). This silent change was present in one tumor sample and also in corresponding normal DNA.
Mutations in SMAD4 5'-untranslated region (331 bp fragment downstream from the transcription start site) were analyzed among those JP families/cases where SMAD4 or PTEN mutations had not been detected (families 2 and 4 and sporadic cases 1 and 3). Mutation analysis was performed by automated sequencing, and no mutations were detected in any of patients analysed.
Genomic ALK1 and endoglin mutation screening was performed among those four JP patient, where SMAD2, SMAD3, SMAD4, SMAD7 or PTEN mutations were not detected. Mutations were not detected in either one of these genes in any of those four patients.