Haemochromatosis.

Haemochromatosis is one of the most common genetic diseases affecting patients of northern European ancestry. It is overdiagnosed in patients without iron overload and is underdiagnosed in many patients. Early diagnosis by genetic testing and therapy by periodic phlebotomy can prevent the most serious complications, which include liver cirrhosis, liver cancer, and death. This Seminar includes an update on the origins of haemochromatosis; and an overview pathophysiology, genetics, natural history, signs and symptoms, differential diagnoses, treatment with phlebotomy, outcomes, and future directions.

Association of Hemochromatosis HFE p.C282Y Homozygosity With Hepatic Malignancy.

Importance: Hereditary hemochromatosis is predominantly caused by the HFE p.C282Y homozygous pathogenic variant. Liver carcinoma and mortality risks are increased in individuals with clinically diagnosed hereditary hemochromatosis, but risks are unclear in mostly undiagnosed p.C282Y homozygotes identified in community genotyping. Objective: To estimate the incidence of primary hepatic carcinoma and death by HFE variant status. Design, Setting, and Participants: Cohort study of 451 186 UK Biobank participants of European ancestry (aged 40-70 years), followed up from baseline assessment (2006-2010) until January 2018. Exposures: Men and women with HFE p.C282Y and p.H63D genotypes compared with those with neither HFE variants. Main Outcomes and Measures: Two linked co-primary outcomes (incident primary liver carcinoma and death from any cause) were ascertained from follow-up via hospital inpatient records, national cancer registry, and death certificate records, and from primary care data among a subset of participants for whom data were available. Associations between genotype and outcomes were tested using Cox regression adjusted for age, assessment center, genotyping array, and population genetics substructure. Kaplan-Meier lifetable probabilities of incident diagnoses were estimated from age 40 to 75 years by HFE genotype and sex. Results: A total of 451 186 participants (mean [SD] age, 56.8 [8.0] years; 54.3% women) were followed up for a median (interquartile range) of 8.9 (8.3-9.5) years. Among the 1294 male p.C282Y homozygotes, there were 21 incident hepatic malignancies, 10 of which were in participants without a diagnosis of hemochromatosis at baseline. p.C282Y homozygous men had a higher risk of hepatic malignancies (hazard ratio [HR], 10.5 [95% CI, 6.6-16.7]; P < .001) and all-cause mortality (n = 88; HR, 1.2 [95% CI, 1.0-1.5]; P = .046) compared with men with neither HFE variant. In lifetables projections for male p.C282Y homozygotes to age 75 years, the risk of primary hepatic malignancy was 7.2% (95% CI, 3.9%-13.1%), compared with 0.6% (95% CI, 0.4%-0.7%) for men with neither variant, and the risk of death was 19.5% (95% CI, 15.8%-24.0%), compared with 15.1% (95% CI, 14.7%-15.5%) among men with neither variant. Among female p.C282Y homozygotes (n = 1596), there were 3 incident hepatic malignancies and 60 deaths, but the associations between homozygosity and hepatic malignancy (HR, 2.1 [95% CI, 0.7-6.5]; P = .22) and death (HR, 1.2 [95% CI, 0.9-1.5]; P = .20) were not statistically significant. Conclusions and Relevance: Among men with HFE p.C282Y homozygosity, there was a significantly increased risk of incident primary hepatic malignancy and death compared with men without p.C282Y or p.H63D variants; there was not a significant association for women. Further research is needed to understand the effects of early diagnosis and treatment.

Prevalence and characteristics of anti-HCV positivity and chronic hepatitis C virus infection in HFE p.C282Y homozygotes.

INTRODUCTION AND AIM: Observations of hepatitis C virus (HCV) infection in adults with hemochromatosis are limited. MATERIALS AND METHODS: We determined associations of serum ferritin (SF) with anti-HCV in non-Hispanic white North American adults in a post-screening examination. Cases included p.C282Y homozygotes (regardless of screening transferrin saturation (TS) and SF) and participants (regardless of HFE genotype) with high screening TS/SF. Controls included participants without p.C282Y or p.H63D who had normal screening TS/SF. Participants with elevated alanine aminotransferase underwent anti-HCV testing. We determined prevalence of chronic HCV infection in consecutive Alabama and Ontario referred adults with HFE p.C282Y homozygosity. RESULTS: In post-screening participants, anti-HCV prevalence was 0.3% [95% CI: 0.02, 2.2] in 294 p.C282Y homozygotes, 9.5% [7.2, 12.3] in 560 Cases without p.C282Y homozygosity, and 0.7% [0.2, 2.3] in 403 Controls. Anti-HCV was detected in 7.2% of 745 participants with and 0.8% of 512 participants without elevated SF (odds ratio 9.9 [3.6, 27.6]; p<0.0001). Chronic HCV infection prevalence in 961 referred patients was 1.0% (10/961) [95% confidence interval (CI): 0.5, 2.0]. Ten patients with chronic HCV infection had median age 45y (range 29-67) and median SF 1163µg/L (range 303-2001). Five of eight (62.5%) patients had biopsy-proven cirrhosis. CONCLUSIONS: Odds ratio of anti-HCV was increased in post-screening participants with elevated SF. Prevalence of anti-HCV in post-screening participants with HFE p.C282Y homozygosity and chronic HCV infection in referred adults with HFE p.C282Y homozygosity in North America is similar to that of Control participants with HFE wt/wt and normal screening TS/SF.

Cirrhosis in Hemochromatosis: Independent Risk Factors in 368 HFE p.C282Y Homozygotes.

INTRODUCTION AND AIM: We sought to identify independent risk factors for cirrhosis in HFE p.C282Y homozygotes in a cross-sectional study. MATERIAL AND METHODS: We evaluated 368 p.C282Y homozygotes who underwent liver biopsy and compared characteristics of those with and without cirrhosis. We performed multivariable logistic regression on cirrhosis with: age; sex; race/ethnicity; diabetes; blood pints/units donated voluntarily; erythrocyte pints/units received; iron supplement use; alcohol intake, g/d; body mass index, kg/m2; swollen/tender 2nd/3rd metacarpophalangeal joints; elevated alanine aminotransferase; elevated aspartate aminotransferase; steatosis/fatty liver; iron removed by phlebotomy, g; and GNPAT p.D519G positivity. RESULTS: Mean age of 368 participants (73.6% men) was 47 ± 13 (standard deviation) y. Cirrhosis was diagnosed in 86 participants (23.4%). Participants with cirrhosis had significantly greater mean age, proportion of men, diabetes prevalence, mean daily alcohol intake, prevalence of swollen/ tender 2nd/3rd metacarpophalangeal joints, mean serum ferritin, elevated alanine aminotransferase, elevated aspartate aminotransferase, and mean iron removed; and significantly fewer mean blood pints/units donated. GNPAT p.D519G positivity was detected in 82 of 188 participants (43.6%). In a multivariable model for cirrhosis, there were four significant positive associations: age (10-y intervals) (odds ratio 2.2 [95% confidence interval 1.5, 3.3]); diabetes (3.3; [1.1, 9.7]); alcohol intake (14 g alcohol drinks/d) (1.5 [1.2, 1.8]); and iron removed, g (1.3 [1.2, 1.4]). There was no statistical evidence of two-way interactions between these variables. CONCLUSION: In conclusion, cirrhosis in HFE p.C282Y homozygotes is significantly associated with age, diabetes, daily alcohol intake, and iron removed by phlebotomy, taking into account the effect of other variables.

GNPAT p.D519G is independently associated with markedly increased iron stores in HFE p.C282Y homozygotes.

BACKGROUND: GNPAT p.D519G positivity is significantly increased in HFE p.C282Y homozygotes with markedly increased iron stores. We sought to determine associations of p.D519G and iron-related variables with iron stores in p.C282Y homozygotes. METHODS: We defined markedly increased iron stores as serum ferritin >2247pmol/L (>1000µg/L) and either hepatic iron >236µmol/g dry weight or iron >10g by induction phlebotomy (men and women). We defined normal or mildly elevated iron stores as serum ferritin <674.1pmol/L (<300µg/L) or either age≥40y with iron ≤2.5g iron by induction phlebotomy or age≥50y with ≤3.0g iron by induction phlebotomy (men only). We compared participant subgroups using univariate methods. Using multivariable logistic regression, we evaluated associations of markedly increased iron stores with these variables: age; iron supplement use (dichotomous); whole blood units donated; erythrocyte units received as transfusion; daily alcohol consumption, g; and p.D519G positivity (heterozygosity or homozygosity). RESULTS: The mean age of 56 participants (94.6% men) was 55±10 (SD) y; 41 had markedly increased iron stores. Prevalences of swollen/tender 2nd/3rd metacarpophalangeal joints and elevated aspartate or alanine aminotransferase were significantly greater in participants with markedly increased iron stores. Only participants with markedly increased iron stores had cirrhosis. In multivariable analyses, p.D519G positivity was the only exposure variable significantly associated with markedly increased iron stores (odds ratio 9.9, 95% CI [1.6, 60.3], p=0.0126). CONCLUSIONS: GNPAT p.D519G is strongly associated with markedly increased iron stores in p.C282Y homozygotes after correction for age, iron-related variables, and alcohol consumption.

Clinical evaluation of a hemochromatosis next-generation sequencing gene panel.

BACKGROUND: Next-generation sequencing of an iron metabolism gene panel could identify pathogenic mutations, improving on standard hemochromatosis genetic testing and providing a molecular diagnosis in patients with suspected iron overload. METHODS: A next-generation sequencing panel of 15 genes with known roles in iron metabolism was constructed. A total of 190 patients were sequenced: 94 from a tertiary hemochromatosis clinic and 96 submitted for HFE testing with biochemical evidence of iron overload [elevated ferritin (>450 µg/L) or transferrin saturation (>55%)] obtained from a chart review. RESULTS: From the hemochromatosis clinic cohort, six patients were diagnosed with non-HFE hemochromatosis due to homozygous hemojuvelin (HFE2) mutations. Ten additional heterozygous pathogenic mutations were observed. From the chart review cohort, a C-terminus ferritin light chain (FTL) frameshift mutation was observed consistent with neuroferritinopathy. Heterozygous deletion of HFE2 and four additional rare pathogenic or likely pathogenic heterozygous mutations were identified in seven patients. CONCLUSIONS: An iron metabolism gene panel provided a molecular diagnosis in six patients with non-HFE iron overload and is suitable for diagnostic purposes in exceptional cases in specialized clinics. Further research will be required to assess the modifier effect of rare heterozygous mutations in iron metabolism genes.

Clinical and Laboratory Associations with Persistent Hyperferritinemia in 373 Black Hemochromatosis and Iron Overload Screening Study Participants.

BACKGROUND: 373 black participants had elevated screening and post-screening serum ferritin (SF) (> 300 µg/L men; > 200 µg/L women). MATERIAL AND METHODS: We retrospectively studied SF and post-screening age; sex; body mass index; transferrin saturation (TS); ALT; AST; GGT; elevated C-reactive protein; ß-thalassemia; neutrophils; lymphocytes; monocytes; platelets; metacarpophalangeal joint hypertrophy; hepatomegaly; splenomegaly; diabetes; HFE H63D positivity; iron/alcohol intakes; and blood/erythrocyte transfusion units. Liver disease was defined as elevated ALT or AST. We computed correlations of SF and TS with: age; body mass index; ALT; AST; GGT; C-reactive protein; blood cell counts; and iron/alcohol. We compared participants with SF > 1,000 and ≤ 1,000 µg/L and performed regressions on SF. RESULTS: There were 237 men (63.5%). Mean age was 55 ± 13 (SD) y. 143 participants had liver disease (62 hepatitis B or C). There were significant correlations of SF: TS, ALT, AST, GGT, and monocytes (positive); and SF and TS with platelets (negative). 22 participants with SF > 1,000 µg/L had significantly higher median TS, ALT, and AST, and prevalences of anemia and transfusion > 10 units; and lower median platelets. Regression on SF revealed significant associations: TS; male sex; age; GGT; transfusion units (positive); and splenomegaly (negative) (p < 0.0001, 0.0016, 0.0281, 0.0025, 0.0001, and 0.0096, respectively). Five men with SF > 1,000 µg/L and elevated TS had presumed primary iron overload (hemochromatosis). Four participants had transfusion iron overload. CONCLUSION: Persistent hyperferritinemia in 373 black adults was associated with male sex, age, TS, GGT, and transfusion. 2.4% had primary iron overload (hemochromatosis) or transfusion iron overload.

A Fibroblast Growth Factor 21-Pregnane X Receptor Pathway Downregulates Hepatic CYP3A4 in Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) alters drug response. We previously reported that NAFLD is associated with reduced in vivo CYP3A drug-metabolism activity and hepatic CYP3A4 expression in humans as well as mouse and human hepatoma models of the disease. Here, we investigated the role of the lipid- and glucose-modulating hormone fibroblast growth factor 21 (FGF21) in the molecular mechanism regulating CYP3A4 expression in NAFLD. In human subjects, mouse and cellular NAFLD models with lower CYP3A4 expression, circulating FGF21, or hepatic FGF21 mRNA levels were elevated. Administration of recombinant FGF21 or transient hepatic overexpression of FGF21 resulted in reduced liver CYP3A4 luciferase reporter activity in mice and decreased CYP3A4 mRNA expression and activity in cultured Huh7 hepatoma cells. Blocking canonical FGF21 signaling by pharmacological inhibition of MEK1 kinase in Huh7 cells caused de-repression of CYP3A4 mRNA expression with FGF21 treatment. Mice with high-fat diet-induced simple hepatic steatosis and lipid-loaded Huh7 cells had reduced nuclear localization of the pregnane X receptor (PXR), a key transcriptional regulator of CYP3A4 Furthermore, decreased nuclear PXR was observed in mouse liver and Huh7 cells after FGF21 treatment or FGF21 overexpression. Decreased PXR binding to the CYP3A4 proximal promoter was found in FGF21-treated Huh7 cells. An FGF21-PXR signaling pathway may be involved in decreased hepatic CYP3A4 metabolic activity in NAFLD.

Exome sequencing in HFE C282Y homozygous men with extreme phenotypes identifies a GNPAT variant associated with severe iron overload.

UNLABELLED: To identify polymorphisms associated with variability of iron overload severity in HFE-associated hemochromatosis, we performed exome sequencing of DNA from 35 male HFE C282Y homozygotes with either markedly increased iron stores (n = 22; cases) or with normal or mildly increased iron stores (n = 13; controls). The 35 participants, residents of the United States, Canada, and Australia, reported no or light alcohol consumption. Sequencing data included 82,068 single-nucleotide variants, and 10,337 genes were tested for a difference between cases and controls. A variant in the GNPAT gene showed the most significant association with severe iron overload (P = 3 × 10(-6) ; P = 0.033 by the likelihood ratio test after correction for multiple comparisons). Sixteen of twenty-two participants with severe iron overload had glyceronephosphate O-acyltransferase (GNPAT) polymorphism p.D519G (rs11558492; 15 heterozygotes, one homozygote). No control participant had this polymorphism. To examine functional consequences of GNPAT deficiency, we performed small interfering RNA-based knockdown of GNPAT in the human liver-derived cell line, HepG2/C3A. This knockdown resulted in a >17-fold decrease in expression of the messenger RNA encoding the iron-regulatory hormone, hepcidin. CONCLUSION: GNPAT p.D519G is associated with a high-iron phenotype in HFE C282Y homozygotes and may participate in hepcidin regulation.

Movement Disorders Associated With Hemochromatosis.

BACKGROUND: Hereditary hemochromatosis (HH) is a genetic disorder causing pathological iron deposition and functional impairment of various organs, predominantly the liver. We assessed patients with HH for the presence of movement disorders. METHODS: We reviewed the charts of 616 patients with HH who attended hemochromatosis clinic at London Health Sciences Centre, London, ON, Canada, from 1988 to 2015. RESULTS: We found three HH patients with movement disorders, without any other major systemic manifestation. One had parkinsonism, another had chorea, and the third had tremor. All three patients had evidence of iron deposition in the brain, affecting the basal ganglia in the first two, and the dentate nucleus, red nucleus, and substantia nigra in the third patient. In addition to the C282Y homozygous mutation in the HFE gene, two of our patients had non-HFE gene mutations. CONCLUSION: HH should be considered in the differential diagnosis of movement disorders with pathological brain iron deposition. We report for the first time chorea in a patient with HH. Non-HFE gene mutations may predispose HH patients to iron deposition in the brain.

GNPAT variant (D519G) is not associated with an elevated serum ferritin or iron removed by phlebotomy in patients referred for C282Y-linked hemochromatosis.

BACKGROUND: Previous studies in high and low expressors has demonstrated that a variant in the GNPAT gene (D519G, Rs11558492, chromosome 1, exon 11) has been associated with severe iron overload in C282Y homozygotes for hemochromatosis. In this study, a GNPAT variant was assessed prospectively in patients referred for HFE testing over a range of serum ferritin levels. MATERIAL AND METHODS: Consecutive patients sent for HFE testing were studied for the GNPAT variant using a TaqMan kit assay (Life Technologies, Burlington, ON). Serum ferritin and iron removed by phlebotomy was compared in C282Y homozygotes with and without the GNPAT variant. The frequency of the GNPAT variant in referred patients was compared to a control population of voluntary blood donors without HFE mutations. RESULTS: There were 533 patients that had GNPAT analysis. The allele frequency for the GNPAT variant in C282Y homozygotes (n = 75) was 0.226 and in wild type control patients (n = 458) was 0.213 (p = .07). Forty-eight percent (of the C282Y homozygotes were heterozygous (n = 28) or homozygous (n = 8) for the GNPAT variant. The mean (log)ferritin and iron removed did not significantly differ between C282Y homozygous with GNPAT homozygotes, GNPAT heterozygotes, and without the GNPAT variant (p = 0.84). CONCLUSIONS: C282Y homozygotes referred for HFE testing commonly have a GNPAT variant. This GNPAT variant does not appear be a co-modifying gene affecting expression of HFE related hemochromatosis in this population. The GNPAT variant does not predict the severity of iron overload.

Serum ferritin is a biomarker for liver mortality in the Hemochromatosis and Iron Overload Screening Study.

BACKGROUND: We identified no reports of long-term follow-up of participants in hemochromatosis screening programs. We evaluated causes of death and survival in non-C282Y homozygous Canadian participants in the primary care-based hemochromatosis and iron overload screening (HEIRS) study. MATERIAL AND METHODS: Initial screening (IS) included transferrin saturation (TS), serum ferritin (SF), HFE genotyping (C282Y, H63D), and health questionnaire responses. By definition, participants without C282Y or H63D had HFE wt/wt. We linked 20,306 Canadian participants to the Ontario Death Registry for dates and causes of death 9 y after IS. We computed Cox proportional hazards to identify factors with increased death risks and Kaplan-Meier curves to estimate survival of non-C282Y homozygous participants with SF ≤ 1,000 µg/L and > 1,000 µg/dL. RESULTS: There were 19,052 evaluable participants (IS mean age 49 y; 60% women; 93 C282Y homozygotes). There were 988 deaths. Significantly increased hazard ratios for all-cause mortality were positively associated with TS, SF, men, and C282Y homozygosity, and liver disease, diabetes, and heart failure reports. Non-C282Y homozygous participants with SF > 1,000 µg/L had lower survival than those with SF ≤ 1,000 µg/L (p < 0.0001). CONCLUSIONS: Nine years after initial screening, non-C282Y homozygous participants and SF > 1,000 µg/L was associated with decreased survival.

Inflammation is not the cause of an elevated serum ferritin in non-alcoholic fatty liver disease.

BACKGROUND: In non-alcoholic fatty liver disease (NALFD), it has often been assumed that an elevation in serum ferritin is likely related to inflammation rather than iron overload. MATERIAL AND METHODS: Patients referred with NAFLD were entered into a clinical study of phlebotomy therapy. A liver biopsy with liver iron concentration was done at entry and 6 months after phlebotomy (n = 56) until the patient had a low serum ferritin or developed anemia. Serum ferritin was compared to liver iron concentration, ESR, CRP, BMI and grade of inflammation on liver biopsy. RESULTS: Iron removed by phlebotomy in NAFLD correlated with the decrease in serum ferritin (r = 0.57, p = 0.0014) and liver iron concentration (r = 0.57, p = 0.0013). There was no significant correlations between serum ferritin and ESR, CRP or grade of liver inflammation. CONCLUSIONS: Serum ferritin is related to liver iron storage in NAFLD and decreasing body iron stores by phlebotomy is reflected by an appropriate decrease in serum ferritin. Inflammation is not the cause of the elevated serum ferritin in fatty liver disease.

Association between celiac disease and iron deficiency in Caucasians, but not non-Caucasians.

BACKGROUND & AIMS: Celiac disease is an increasingly recognized disorder in Caucasian populations of European origin. Little is known about its prevalence in non-Caucasians. Although it is thought to be a cause of iron-deficiency anemia, little is known about the extent to which celiac disease contributes to iron deficiency in Caucasians, and especially non-Caucasians. We analyzed samples collected from participants in the Hemochromatosis and Iron Overload Screening study to identify individuals with iron deficiency and to assess the frequency of celiac disease. METHODS: We analyzed serum samples from white men (≥25 y) and women (≥50 y) who participated in the Hemochromatosis and Iron Overload Screening study; cases were defined as individuals with iron deficiency (serum ferritin level, ≤12 µg/L) and controls were those without (serum ferritin level, >100 µg/L in men and >50 µg/L in women). All samples also were analyzed for human recombinant tissue transglutaminase immunoglobulin A; positive results were confirmed by an assay for endomysial antibodies. Patients with positive results from both celiac disease tests were presumed to have untreated celiac disease, and those with a positive result from only 1 test were excluded from analysis. We analyzed HLA genotypes and frequencies of celiac disease between Caucasians and non-Caucasians with iron deficiency. RESULTS: Celiac disease occurred in 14 of 567 cases (2.5%) and in only 1 of 1136 controls (0.1%; Fisher exact test, P = 1.92 × 10(-6)). Celiac disease was more common in Caucasian cases (14 of 363; 4%) than non-Caucasian cases (0 of 204; P = .003). Only 1 Caucasian control and no non-Caucasian controls had celiac disease. The odds of celiac disease in individuals with iron deficiency was 28-fold (95% confidence interval, 3.7-212.8) that of controls; 13 of 14 cases with celiac disease carried the DQ2.5 variant of the HLA genotype. CONCLUSIONS: Celiac disease is associated with iron deficiency in Caucasians. Celiac disease is rare among non-Caucasians-even among individuals with features of celiac disease, such as iron deficiency. Celiac disease also is rare among individuals without iron deficiency. Men and postmenopausal women with iron deficiency should be tested for celiac disease.

Probability of C282Y homozygosity decreases as liver transaminase activities increase in participants with hyperferritinemia in the hemochromatosis and iron overload screening study.

UNLABELLED: Hemochromatosis is considered by many to be an uncommon disorder, although the prevalence of HFE (High Iron) 282 Cys → Tyr (C282Y) homozygosity is relatively high in Caucasians. Liver disease is one of the most consistent findings in advanced iron overload resulting from hemochromatosis. Liver clinics are often thought to be ideal venues for diagnosis of hemochromatosis, but diagnosis rates are often low. The Hemochromatosis and Iron Overload Screening (HEIRS) Study screened 99,711 primary care participants in North America for iron overload using serum ferritin and transferrin saturation measurements and HFE genotyping. In this HEIRS substudy, serum hepatic transaminases activities (e.g., alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) were compared between 162 C282Y homozygotes and 1,367 nonhomozygotes with serum ferritin levels >300 µg/L in men and >200 µg/L in women and transferrin saturation >45% in women and 50% in men. The probability of being a C282Y homozygote was determined for AST and ALT ranges. Mean ALT and AST activities were significantly lower in C282Y homozygotes than nonhomozygotes. The probability of being a C282Y homozygote increased as the ALT and AST activities decreased. CONCLUSION: Patients with hyperferritinemia are more likely to be C282Y homozygotes if they have normal liver transaminase activities. This paradox could explain the low yields of hemochromatosis screening reported by some liver clinics.