HFE mutations in Caucasian participants of the Hemochromatosis and Iron Overload Screening study with serum ferritin level < 1000 μ g / L

1London Health Sciences Centre, London, Ontario; 2Department of Epidemiology, University of California, Irvine, California, USA; 3Department of Epidemiology & Biostatistics, Western University, London, Ontario; 4Department of Veterans Affairs Long Beach Healthcare System, Long Beach, and Division of Hematology/Oncology, Department of Medicine, University of California, Irvine, California; 5Southern Iron Disorders Center, Birmingham, Alabama; 6Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota, USA Correspondence: Dr Paul C Adams, Department of Medicine, University Hospital, 339 Windermere Road, London, Ontario N6A 5A5. Telephone 519-685-8500 ext 35375, fax 519-663-3549, e-mail padams@uwo.ca Received for publication February 5, 2013. Accepted February 22, 2013 Many patients are now referred to specialists because of mild elevations in serum ferritin level. The test may have been ordered to screen for iron deficiency in a fatigued patient or as a cause of liver disease, or may have been part of a multitest panel of blood tests in an annual examination (1). Elevations in serum ferritin levels often cause anxiety in patients and the magnitude of serum ferritin may be hundreds of μg/L above the upper end of the reference range, adding to the patients’ concern. Patients are frequently advised that they may have hemochromatosis and iron overload and, by the time of further assessment, they may have donated blood, switched to an iron-reduced diet and joined an Internet advocate society for patients with iron overload. In the present subanalysis of data from the Hemochromatosis and Iron Overload Screening (HEIRS) study (2), we illustrate the proportions of participants in serum ferritin concentration intervals who potentially have iron loading HFE mutations of the hemochromatosis gene.

Participants underwent postscreening clinical examinations if they had elevated transferrin saturation and ferritin levels, or were HFE C282Y homozygotes.The analyses were also limited to Caucasian participants because C282Y homozygotes were rare in other races in the HEIRS study (2).Patients who had a serum ferritin level <1000 µg/L were considered because this is a common clinical problem and there were a small number of participants with a ferritin level ≥1000 µg/L (76 men, 30 women).Data were grouped according to serum ferritin levels into seven intervals for men and eight intervals for women (200 µg/L to 1000 µg/L).The prevalences of C282Y homozygotes, compound heterozygotes (C282Y/H63D) and H63D homozygotes within each interval for men and women were determined.A subgroup analysis was also performed for participants with both an elevated serum ferritin level and transferrin saturation (>45% in women, >50% in men).Determining the cause of elevated ferritin levels in participants without HFE mutations was beyond the scope of the present study.However, the HEIRS study has previously reported on liver disease (4) and diabetes (5) as potential causes of elevated ferritin levels.A potential iron loading genotype was considered in the present study to be C282Y homozygote, compound heterozygote (C282Y/ H63D) and H63D homozygote.
The post-test likelihood (PTL + ) of being a C282Y homozygote was calculated for men and women.The PTL + is the likelihood of the condition of interest given a positive test result (6).The estimated 95% CI for PTL + was based on binomial distribution with a normal approximation applied.
There were 437 of 3359 (13%) men and 311 of 2416 (12.8%) women with elevated serum ferritin level and transferrin saturation.In this subgroup, there was a higher proportion of participants who were C282Y homozygotes within each ferritin interval (Figure 1).In participants with an elevated transferrin saturation, the prevalence of a potential iron-loading HFE genotype increased to 30% in men and 42% in women.

dIsCussIon
In the present analysis, we demonstrated that most Caucasian participants in the HEIRS study with an elevated serum ferritin level <1000 µg/L were not C282Y homozygotes, compound heterozygotes or H63D homozygotes.The most likely causes of elevated ferritin levels in non-C282Y homozygotes include inflammation, obesity and alcohol consumption (1).Our study cannot exclude the possibility of non-C282Y genetically linked iron overload as the cause of the ferritin elevation in non-C282Y homozygotes; however, this has been a very rare diagnosis in North America (7).In the HEIRS study, approximately 20% of all the Caucasian men had an elevated serum ferritin level (>300 µg/L).In the HEIRS study, many participants were recruited from primary care clinics and clinical phlebotomy sites; therefore, they were already being tested for other reasons, which suggests that they were not representative of a normal healthy population.This could explain the higher ranges that were apparent in the present study.A low serum ferritin level is indicative of iron deficiency, but a serum ferritin level above the current recommended reference ranges is often not an indication of total body iron overload.Genetic testing for the C282Y mutation of the HFE gene is often used as a second-line confirmatory test for hemochromatosis in patients with an elevated ferritin level.In the present analysis, we assumed that individuals with an iron loading genotype and an elevated ferritin level had iron overload, but these patients may also have ferritin elevations for other reasons.In larger population-based studies, including the HEIRS study of compound heterozygotes and H63D homozygotes, <10% had iron overload (8).A clinician could improve the proportion of positive tests for C282Y homozygosity by the clinical interpretation of the clinical history, family history, physical examination and a careful review of other conditions causing elevated ferritin levels such as inflammation including liver disease, alcohol use and obesity.In clinical practice, it can be difficult to determine whether a patient with a mild elevation in ferritin level has iron overload.The use of invasive tests, such as liver biopsy, is not appealing to the patient.Magnetic resonance imaging has better sensitivity to detect higher levels of liver iron overload but is expensive and not widely available.A trial of phlebotomy is often performed but may lead to anemia and fatigue in patients without iron overload.A concomitant elevation in transferrin saturation has been demonstrated to increase the proportion of participants who are C282Y homozygotes.However, the biological variability of transferrin saturation is a limitation as a screening test (9).Predictors to assist in the identification of patients with iron overload in practice based on clinical conditions have been developed and validated (10).However, the restriction of HFE genotyping to highly selected cases could miss many patients with less classical presentations and often patients are referred to confirm whether hemochromatosis is the cause of the ferritin elevations.Genetic testing is less invasive than previous diagnostic tools, such as liver biopsy, and may be reassuring to patients and their families.Previous studies have suggested that only patients with a ferritin level ≥1000 µg/L should undergo further investigation for hemochromatosis and treatment (11).Previously undiagnosed C282Y homozygotes with serum ferritin values that remain <1000 µg/L are at low risk for developing hemochromatosis-related signs and symptoms at an age when the clinical manifestations would be expected to have developed (12).However, there is a fivefold increase in risk of death causally associated with iron overload in persons with hemochromatosis, C282Y homozygosity and serum ferritin level ≥1000 µg/L at diagnosis (13).This strongly suggests that it is preferable to diagnose hemochromatosis and perform phlebotomy therapy to achieve iron depletion well before seurm ferritin levels exceed 1000 µg/L.Therefore, the present study suggests that most patients with a modest elevation in ferritin level do not have C282Y-linked hemochromatosis and yet it is important to identify them.The clinical factors that increase the likelihood of being a C282Y homozygote are shown in Table 3.

Figure 1) The proportion of male and female participants with an elevated ferritin level and transferrin saturation who were C282Y homozygotes
An important clinical observation from the present study is that C282Y-linked hemochromatosis is not the most common cause of elevated serum ferritin levels in Caucasians, suggesting that patients should not be told that they have genetic hemochromatosis and iron overload before further assessment is performed.

TablE 2 Post-test likelihood of being a C282Y homozygote
*The estimated 95% CI for post-test likelihood was based on binomial distribution with a normal approximation applied