Penetrance, cancer incidence and survival in HFE haemochromatosis—A population‐based cohort study

Haemochromatosis is characterized by progressive iron overload affecting the liver and can cause cirrhosis and hepatocellular carcinoma. Most haemochromatosis patients are homozygous for p.C282Y in HFE, but only a minority of individuals with this genotype will develop the disease. The aim was to assess the penetrance of iron overload, fibrosis, hepatocellular carcinoma and life expectancy.


| INTRODUC TI ON
Haemochromatosis is a genetic disease characterized by progressive iron overload primarily affecting the liver. 1,2Early disease manifestations of haemochromatosis include fatigue and other non-specific symptoms such as arthralgia.Advanced stages of the disease can be complicated by cirrhosis, hepatocellular carcinoma, diabetes and arthropathy. 3,4The most common genotype associated with haemochromatosis is homozygosity for p.C282Y in HFE, which is present in >80% of patients.Rarely, the disease is caused by pathogenic variants in genes encoding haemojuvelin, hepcidin, transferrin receptor-2 and ferroportin. 5,6The unifying pathogenesis is reduced functional hepcidin, which is the hormone that controls ingress of iron into the body and egress of iron from macrophages into the circulation. 7Hepcidin normally inhibits the iron exporter ferroportin, which limits the release of iron from absorptive intestinal epithelial cells and macrophages. 8mozygosity for p.C282Y is associated with inappropriately low hepcidin expression, which results in elevated transferrin saturation and iron overload as biochemical hallmarks of haemochromatosis. 9Adequate treatment of iron overload with therapeutic phlebotomy is effective in preventing clinical complications. 10 individuals homozygous for p.C282Y, the diagnosis haemochromatosis can be made when transferrin saturation and ferritin are elevated. 11Documentation of hepatic iron overload by liver biopsy or magnetic resonance imaging are only required for treatment initiation in haemochromatosis patients not homozygous for p.C282Y. 1,12,13Elevated transferrin saturation (TSAT) and high serum ferritin define penetrant disease in patients homozygous for the p.C282Y. 11,14,15[21][22] The present population-based cohort study was carried out to assess the age-and sex-dependent risk for the development of provisional iron overload in p.C282Y homozygotes.The long-term follow-up allowed us to also assess the life expectancy and cancer incidence in a real-life clinical setting and compare outcomes with a matched population-based control group.

| ME THODS
The present study was selected from patients referred to the Laboratory of Hepatology at the Department of Medicine, University Hospital of Innsbruck, for HFE genotyping between January 1997 and December 2021.Our Laboratory is the main diagnostic laboratory to perform this test in the county of Tyrol, Austria, during the study period and covers >95% of all HFE genotyping tests performed in this region.Of all 11 088 patients who were referred to our department for the evaluation of suspected liver disease or haemochromatosis between 1997 and 2021, 8839 met the inclusion criterion of residence in Tyrol as defined by the postal code of their primary address registered with the national health insurance at the time of genotyping.(Figure 1).Incident cancer diagnoses were assessed for our haemochromatosis cohort from the regional cancer registry (Tiroler Tumorregister, which is 'Gold certified' by the North American Association of Central Cancer Registries), where all incident cancer diagnoses are recorded. 24The database was queried for all individuals referred for genotyping and the results were compared with expected cancer incidences, calculated from reported, age-standardized incidence rates, in the propensity score-matched cohort.Genomic DNA extraction from peripheral blood and genotyping were carried out as previously described using a validated TaqMan allelic discrimination assay (TaqMan SNP Genotyping assay for reference SNP ID number rs1800562 and rs1799945, ThermoFisher Scientific, Vienna, Austria). 25netrant disease was either defined as the presence of provisional iron overload (ferritin >300 μg/L for men and postmenopausal women, >200 μg/L for premenopausal women in association with transferrin saturation >55% for men and >45% for women) as previously described. 11Advanced liver fibrosis was ruled out using  S1) and genotype frequencies from the KORA cohort (Tables S2-S4). 23Additional information can be found in the Supplementary Material.
Extracted data from electronic health records were collected in Microsoft Excel before performing statistical analyses in IBM Statistics SPSS v27.0 and R v4.0.3.Graphs were created using the package survminer and ggplot2.Alpha was set at 0.05 for statistical significance.This study complies with STROBE reporting guidelines and was approved by the local ethics committee of the Medical University of Innsbruck (protocol number: 1253/2019).

| RE SULTS
In this cohort of 8839 individuals referred for HFE genotyping to a Hepatology clinic, the frequency for p.C282Y homozygosity was 6.1% (542) and the allele frequency for p.C282Y was 15.8%, which is higher in this than in the non-Finish European control populations as reported in the gnomAD database (15.8% vs. 5.7%, p < .001). 27mographic data, clinical and biochemical findings of the p.C282Y homozygous patient cohort are summarized in Table 1.
When male and female patients were compared, male patients were found to be significantly younger and had significantly higher serum iron, ferritin and transferrin saturation as compared to females at time of genotyping.The majority of patients had high serum iron, hyperferritinaemia and elevated transferrin saturation.Fibrosis could be ruled-out by a FIB-4 < 1.3 score in more female than male patients, but this difference did not reach statistical significance (Table 1).
F I G U R E 2 Comparison of expected and observed cases of individuals homozygote for p.C282Y grouped by sex.The number of expected patients for each group was calculated from regional allele frequencies and census data (details see Supplementary Material).
Next, homozygous patients with available serum iron parameters at the time of genotyping were grouped by the presence or absence of provisional iron overload, as surrogate for haemochromatosis.HFE p.C282Y homozygotes with provisional iron overload, were more likely to be male, but not significantly older at genotyping when compared with individuals without Cumulative age and sex-specific penetrance of (A) provisional iron overload or (B) of FIB-4 score ≥1.3 in p.C282Y homozygotes.
provisional iron overload.During a median follow-up of >10 years in both groups, incident cancer diagnoses and in particular hepatocellular carcinoma incidence rates were not significantly different (Table 1 and Table S5).
Figure 2 shows the age-and gender-distribution of diagnosed p.C282Y homozygotes and the number of expected p.C282Y homozygotes calculated from population data.This comparison shows that a larger proportion of males with p.C282Y homozygosity was diagnosed at relatively younger ages than females.The majority of male p.C282Y homozygotes were diagnosed in the 30-69 years age groups.
Age-dependent penetrance estimates of haemochromatosis defined as p.C282Y homozygosity with provisional iron overload or Fib-4 ≥ 1.3 for both sexes are shown in Figure 3.
During a median follow-up of 12.5 years, 30 patients were diagnosed with cancer, of whom 8 patients (all male) developed hepatocellular carcinoma (Table S7).Age-specific life expectancy and cancer incidence among p.C282Y homozygotes were compared with a propensity score-matched control population.As shown in Figure 4A, In search for predictors of life expectancy in haemochromatosis patients, a Cox regression analysis was performed.As shown in Table 2, Tables S8 and S9, FIB-4 score as continuous variable, malignancy and cardiovascular risk factors were negatively associated with life expectancy.The strongest, independent predictor of survival was any cancer diagnosis.Younger age at diagnosis was a predictor of higher life expectancy in males after adjustment for FIB-4 and cancer.The causes of death could be assessed in 55 of 71 p.C282Y homozygote patients.In this cohort, cancer was the most common cause (45%) of death followed by cardiovascular disease (35%; Table S6).The most frequent comorbidities were steatosis (54.2%), smoking (39.3%) and arterial hypertension (37.2%) as detailed in Table S10.and definition of penetrant disease.For the present study, penetrant disease was defined as provisional iron overload.This definition has been adopted from previous studies and was chosen, because current guidelines are based on the same criteria for diagnosis and treatment indication. 1,11,15,28For the assessment of haemochromatosis penetrance defined as liver fibrosis, FIB-4 could also be used as a surrogate, where a threshold of <1.3 has been suggested to exclude significant fibrosis. 29,30Recent guidelines recommend that patients with haemochromatosis and a FIB-4 ≥ 1.3 should be investigated for the presence of liver disease.Survival status and cancer incidence could be reliably assessed from national health insurance and cancer registry.The median follow-up period of over 12 years is also sufficiently long to assess cancer incidence.In our study, hepatocellular cancer incidence was not increased in homozygous p.C282Y patients when compared with a propensity score-matched control cohort.6][37][38] Our study provides longitudinal TA B L E 2 Cox regression of life expectancy.

TA B L E 1
Baseline characteristics of patients homozygous for p.C282Y.resident registry (Zentrales Melderegister -Statistik Austria) -details see Supplementary Materials.This population was used to calculate the number of expected individuals homozygous for p.C282Y using the frequency of this genotype from the KORA cohort, which is a population-based study conducted in the nearby region of Augsburg, Germany.23 Results were also compared with allelic frequencies in non-Finish European population published in gnomAD.
a FIB-4 score >1.3 at the time of genotyping.Age of menopause was assumed with 54 years (median age of natural menopause in Europe). 26Age-dependent penetrance of haemochromatosis was calculated by dividing the cumulative number of C282Y homozygous patients with penetrant disease by the cumulative number of expected C282Y homozygous individuals of the same age or younger.This ratio was iteratively calculated for each diagnosed patient with penetrant disease and the results plotted against age at diagnosis.The number of expected C282Y homozygous individuals was calculated from demographic data of the Tyrolean population from the national resident registry (Table the life expectancy of individuals homozygous for p.C282Y was significantly reduced as compared with the matched control population.As shown in Figure 4B, median lifetime in p.C282Y homozygous patients who developed HCC was lowest with 69.7 years.Life expectancy in the group of patients with provisional iron overload or FIB-4 ≥ 1.3 at diagnosis was not significantly different from the group without provisional iron overload or FIB-4 < 1.3 at genotyping (Figure 4C,D).Next, the observed age-dependent HCC incidence was compared with the modelled HCC incidence in the propensity score-matched control population.As shown in Figure5A, no significant difference was noted.As all observed HCCs occurred in males, this analysis was repeated in p.C282Y homozygous patients as well as in modelled controls separated by sex.All p.C282Y homozygotes who developed HCC had a FIB4 > 1.3 at the time of genotyping and no HCCs were diagnosed in the group of patients with a FIB-4 < 1.3 at the time of genotyping.As shown in Figure5B, no difference in HCC incidence was found when the analysis was confined to males or females.
Life expectancy of p.C282Y homozygotes (B) with and without HCC (C) with and without provisional iron overload (pIOL) and (D) FIB-4 score <1.3 or ≥1.3 compared with a modelled propensity score-matched control population.

4
| DISCUSS ION The present study was conducted to investigate the epidemiology, penetrance, cancer incidence and life expectancy in haemochromatosis patients.The study shows that the highest diagnostic rate of p.C282Y homozygous individuals was observed in the 50-59 age group.In this age group, over 50% of male and 28% of female cases expected from modelling were diagnosed.The risk of developing iron overload in p.C282Y homozygotes is dependent on multiple factors including sex, age, study population F I G U R E 5 (A) Cumulative lifetime HCC risk for p.C282Y homozygotes compared with a modelled propensity score-matched control population.(B) Cumulative lifetime HCC risk for p.C282Y homozygotes compared with a modelled propensity score-matched control population stratified by sex.
Reduced survival and the observed age-dependent increase in penetrance among p.C282Y homozygotes call for earlier diagnosis of haemochromatosis to prevent complications.