At baseline, a structured interview schedule was used to obtain information about potential risk factors including country of birth, education, smoking history, alcohol consumption, and for women, reproductive history and use of hormone replacement therapy. Current usual diet was assessed by a 121-item food frequency questionnaire.19 A blood sample www.selleckchem.com/products/acalabrutinib.html was collected and weight, height, waist, and hip circumferences were measured.20 Addresses and vital status of the subjects were determined by record linkage to electoral rolls, the National

Death Index, Victorian death records, and from electronic phone books and responses to mailed questionnaires and newsletters. Cancer cases were identified by linkage to population-based cancer registries in all Australian states. Blood samples were stored either in liquid nitrogen or dried on Guthrie cards. DNA was extracted from Guthrie cards by the Chelex method and from buffy coats using a guanidinium isothiocyanate–based method (Corbett Buffy Coat CorProtocol 14102). All samples were genotyped for the single-nucleotide Aloxistatin research buy polymorphism in the HFE gene that is responsible for the C282Y substitution in the HFE protein (rs1800562) using real-time polymerase chain reaction. Those samples with one copy of

the variant leading to C282Y were also genotyped for the variant leading to the H63D substitution (rs1799945).21 Therefore, there were four HFE genotype groups: (1) C282Y homozygotes, (2) simple heterozygotes with one copy of the C282Y variant and no copies of the H63D variant, (3) compound heterozygotes with one copy each of the C282Y and H63D variants, and (4) other HFE genotype with no copies selleck inhibitor of the C282Y variant and unknown number of copies of the H63D variant. The H63D and C282Y variants have only very rarely been reported to occur together on a single chromosome.22 For participants classified as C282Y homozygotes by this genotyping, additional genotyping was performed for confirmation. All participants homozygous for the C282Y variant (as part of an HFE-genotype stratified

random sample) were invited to participate in a study of iron and health (the “HealthIron” study) from 2004–2007, where we collected a cheek swab, with subsequent genotyping for C282Y and H63D in an independent laboratory. For those who did not participate in HealthIron, additional genotyping was done on a baseline plasma sample. Only those participants classified as C282Y homozygotes by the initial and confirmatory genotyping were considered to be homozygotes for this analysis, otherwise they were classified according to the results of the confirmatory genotyping. Hazard ratios were estimated using Cox regression with age as the time axis. Follow-up began at baseline and ended at death, date of diagnosis, date left Australia, or end of follow-up, whichever came first.