Epidemiology/Health Services Research ORIGINAL ARTICLE Prevalence, Characteristics, and Prognostic Significance of HFE Gene Mutations in Type 2 Diabetes The Fremantle Diabetes Study 1 3 TIMOTHY M.E. DAVIS, FRACP GARY P. JEFFREY, FRACP variants of the hemochromatosis (HFE) 2 2 JOHN BEILBY, PHD ENRICO ROSSI, PHD gene product are important determinants 1 2 WENDY A. DAVIS, PHD CONCHITA BOYDER, MSC 1 1 of iron storage. HFE-related hemochro- JOHN K. OLYNYK, FRACP DAVID G. BRUCE, FRACP matosis is considered to include C282Y homozygous and compound C282Y/ H63D heterozygous genotypes (9). H63D OBJECTIVE — To examine the relationship between iron status, hereditary hemochromato- and C282Y have been reported to be in- sis (HFE) gene mutations, and clinical features and outcomes of type 2 diabetes in a well- dependent risk factors for diabetic ne- characterized representative sample of community-based patients. phropathy (10) and proliferative retinopathy (11), respectively. RESEARCH DESIGN AND METHODS — HFE genotype data were available for 1,245 type 2 diabetic patients from the longitudinal observational Fremantle Diabetes Study (FDS), Because most published studies have representing 96.2% of the total FDS type 2 diabetes cohort. Data were collected at recruitment been small-scale and cross-sectional, with between 1993 and 1996 and annually until the end of June 2001. Hospitalization and mortality a restricted number of potentially explan- data were available until the end of June 2006. The presence of the C282Y HFE mutation was atory variables, there is a need for detailed determined in all subjects and H63D in C282Y heterozygotes. Fasting serum iron, transferrin, large-scale longitudinal studies examin- and ferritin were measured in all C282Y homozygotes and C282Y/H63D heterozygotes and in ing the relationship between iron metab- 286 randomly selected wild-type subjects. Multiple logistic regression analysis was performed to olism, HFE mutations, and the clinical determine independent baseline associates of prevalent complications (myocardial infarction, features and complications of diabetes cerebrovascular disease, retinopathy, neuropathy, and nephropathy), as was Cox proportional hazards modeling to determine predictors of incident complications and mortality. (4). Such studies are essential before di- rected screening for iron overload can be RESULTS — Although there were expected positive associations between HFE gene muta- recommended in diabetic patients (12). tions and serum iron and transferrin saturation, there were no independent positive associations We have, therefore, analyzed data from between HFE gene status and either microvascular or macrovascular complications in cross- the observational Fremantle Diabetes sectional and longitudinal analyses. HFE gene status did not independently predict cardiac or Study (FDS) to assess the effects of iron all-cause mortality. Measures of iron metabolism including serum ferritin were not associated status and HFE mutations on the charac- with combined microvascular or macrovascular end points. teristics and outcome of type 2 diabetes. CONCLUSIONS — Directed screening for iron overload and/or HFE mutations appears unwarranted in patients with type 2 diabetes. RESEARCH DESIGN AND Diabetes Care 31:1795–1801, 2008 METHODS —The FDS took place in a postal code–defined urban community of lthough early reports suggested that hemoglobin (2) and has been suggested to 120,097 people in the state of Western hemochromatosis protects against be an additional component of the meta- Australia. Descriptions of recruitment A the chronic complications of diabe- bolic syndrome (3). Hyperglycemia, and and details of nonrecruited patients have tes (1), recent studies have identified ad- other effects of excess tissue iron includ- been published (13). Of 2,258 diabetic verse metabolic and vascular effects that ing oxidant stress, angiogenesis, and fi- patients identified between 1993 and could be associated with iron overload brosis (4–6), could promote the 1996, 1,426 (63%) entered the FDS and (2–11). Serum ferritin correlates posi- development of complications such as ne- 1,294 had type 2 diabetes based on age at tively with insulin resistance and glycated phropathy (7,8). The C282Y and H63D diagnosis, history of insulin treatment, ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● adiposity, and other features including is- From the 1School of Medicine and Pharmacology, Fremantle Hospital, University of Western Australia, let autoantibody status if required (13). Fremantle, Australia; the 2Department of Clinical Biochemistry, PathWest, and the School of Surgery and Eligible patients who were not recruited Pathology, University of Western Australia, Perth, Australia; and the 3School of Medicine and Pharma- were a mean of 1.4 years older than par- cology, Sir Charles Gairdner Hospital, University of Western Australia, Perth, Australia. ticipants, but their sex distribution and Corresponding author: Professor T.M.E. Davis, [email protected]. Received 1 February 2008 and accepted 7 June 2008. the distributions of diabetes types and Published ahead of print at http://care.diabetesjournals.org on 19 June 2008. DOI: 10.2337/dc08-0248. treatment modalities were similar (13,14). © 2008 by the American Diabetes Association. Readers may use this article as long as the work is properly The FDS protocol was approved by the cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons. Fremantle Hospital Human Research Ethics org/licenses/by-nc-nd/3.0/ for details. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby Committee, and all subjects gave informed marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. consent. DIABETES CARE, VOLUME 31, NUMBER 9, SEPTEMBER 2008 1795 HFE gene mutations in type 2 diabetes Clinical assessment Laboratory measurements structed using Kaplan-Meier estimates At baseline and annual reviews up to No- HFE gene mutations were determined in and compared by a log-rank test. Cox pro- vember 2001, a questionnaire was com- buffy-coat DNA by PCR amplification portional hazards modeling (forward con- pleted, a physical examination was using published primers (18), followed ditional entry at P Ͻ 0.05, removal at P Ͼ performed, and fasting biochemical tests by restriction enzyme cleavage and 0.05) was used to determine independent were carried out in a single laboratory us- analysis on a 3% agarose gel. All sub- predictors of these events, with the validity ing standard methods (13). Assessments jects were assessed for C282Y using the of the proportional hazards assumption as- in a small minority of patients (Յ3%) unique RsaI digestion site. Because it is sessed from ln[Ϫln(survival)] curves and with self-reported acute intercurrent ill- not clinically significant in the absence time-dependent covariates. All clinically ness or with clinical signs of an acute in- of C282Y (9,19), H63D was determined plausible univariate variables with P Ͻ 0.20 fection were deferred until after recovery. only in C282Y heterozygotes using the were considered for entry. Ethnic background was assessed as north- unique MboI digestion site. We divided ern European (principally Anglo-Celts), subjects into 1) wild types, 2) C282Y/ RESULTS southern European, Asian, African, Ab- wild-type heterozygotes, 3) compound original, or other (13). Identification of C282Y/H63D heterozygotes, and 4) Baseline patient characteristics the metabolic syndrome was by the defi- C282Y homozygotes. The characteristics of the 1,245 type 2 di- nitions of the World Health Organization, Fasting serum iron, transferrin, and abetic patients with complete HFE geno- the National Cholesterol Education Pro- ferritin were measured in available sam- type data (96.2% of the total type 2 gram’s Adult Treatment Panel III, and the ples from C282Y homozygotes and het- diabetes cohort) are summarized in Table International Diabetes Federation Con- erozygotes and in 286 randomly selected 1. Major mutations were most frequent sensus Group (14). wild-type subjects, using established as- Ϫ among the 786 patients of Anglo-Celt eth- Complications were identified using says (18). All sera were stored at 80°C nicity and least in the 229 Southern Eu- standard criteria (15). Peripheral neurop- until analysis. Serum iron was measured, ropeans. The significant relationship Ͼ athy was defined as a score 2of8onthe after separation from transferrin in an between education and HFE genotype re- Michigan Neuropathy Screening Instru- acidic guanidinium chloride solution and flects the fact that Anglo-Celts were more ment clinical portion. Retinopathy was reduction with ascorbic acid, from the ab- likely to have progressed beyond primary taken as any grade detected by direct/ sorbance of the colored complex formed school. Apart from weak associations be- indirect ophthalmoscopy and/or ophthal- with ferroZine (Hitachi 917 analyzer; tween serum lipids and genotype, there mologist assessment. Self-reported Boehringer Mannheim, Sydney, Austra- were no significant between-group differ- stroke/transient ischemic attack was lia). Serum transferrin was determined by ences in a range of demographic, biophys- amalgamated with prior hospitalizations rate immunoturbidity (Hitachi 917 ana- ical, and diabetes-specific variables. to define baseline cerebrovascular disease lyzer). Transferrin saturation was calcu- Of the eight C282Y