Initially, we developed a TaqMan assay (Applied Biosystems, Foster City, CA) for the T36M allele and assessed its prevalence in a cohort of European derived ARPKD patients. This served to both validate the assay and to supply a baseline T36M prevalence rate in an ARPKD patient cohort, a sample of individuals with classical ARPKD and their parents(Rossetti et al. 2003
). In our cohort, we derived an allele frequency of 13.1%; 95% CI: 8.9–18.8%. The total number of alleles is an odd number, because in one case we only had one parent and no DNA from an affected individual. The 95% confidence level overlaps with the 17.6% incidence rate observed by others(Bergmann et al. 2005
). All mutations were confirmed by sequencing.
We next screened 1842 subjects with a history of sporadic colorectal adenocarcinoma (US residents of European origin) and 1601 clinic-based control subjects (similar in age, gender and state of residence to the cancer cohort), see . Six T36M heterozygotes were found in the control sample (0.37%), and none (0.0%) in the colorectal cancer sample (p=0.01 Fisher's exact test).
Incidence of T36M change in the ARPKD cohort, a cohort with proven colorectal carcinoma and a clinic-based control group without a history of colonic carcinoma.
To confirm the initial study, which showed an unexpected protective effect of T36M on colorectal cancer, we assembled a new cohort of control and colorectal cancer patients (of European origin) with no overlap with the initial sample. In this second study, we found nine T36M heterozygotes in the control sample (0.45%, n=2002 subjects) and one in the colorectal cancer sample (0.05%, n=1925 subjects) (p=0.022). This individual was confirmed to have stage 3 colorectal carcinoma. Together, the data show that the prevalence of T36M is 15:3603 (0.42%) in the control and 1:3767 (0.027%) in the colorectal sample (p=0.0002) (). This indicates that the T36M allele is not associated with an increased risk of colon cancer; indeed, the opposite hypothesis that the T36M mutation is protective becomes extremely significant, with an odds ratio =0.072, 95% CI: 0.003–0.36.
To calculate the carriage rate of all PKHD1 mutations in the European sample, we used the allelic incidence of T36M of 1:480 alleles (0.42% of genomes) in controls and the incidence of T36M in our ARPKD sample, (1 in 7.6 PKHD1 mutations are T36M (13.1%)), to project a 3.2% (7.6 × 0.42), 1:31 carrier frequency for PKHD1 in the European population (3.2%, 95% CI:1.7–5.9%). The previous estimate of ARPKD mutation carrier rate is 1:70 or 1.43%, which is just outside the 95% confidence limits of our study. These sixteen T36M heterozygotes were confirmed by Dye-terminator DNA sequencing.