MRI-Based Iron Phenotyping and Patient Selection for Next-Generation Sequencing of Non-Homeostatic Iron Regulator Hemochromatosis Genes

Abstract

Background and aims: High serum ferritin is frequent among patients with chronic liver disease and commonly associated with hepatic iron overload. Genetic causes of high liver iron include homozygosity for the p.Cys282Tyr variant in homeostatic iron regulator (HFE) and rare variants in non-HFE genes. The aims of the present study were to describe the landscape and frequency of mutations in hemochromatosis genes and determine whether patient selection by noninvasive hepatic iron quantification using MRI improves the diagnostic yield of next-generation sequencing (NGS) in patients with hyperferritinemia.

Approach and results: A cohort of 410 unselected liver clinic patients with high serum ferritin (defined as ≥200 μg/L for women and ≥300 μg/L for men) was investigated by HFE genotyping and abdominal MRI R2*. Forty-one (10%) patients were homozygous for the p.Cys282Tyr variant in HFE. Of the remaining 369 patients, 256 (69%) had high transferrin saturation (TSAT; ≥45%) and 199 (53%) had confirmed hepatic iron overload (liver R2* ≥70 s^-1 ). NGS of hemochromatosis genes was carried out in 180 patients with hepatic iron overload, and likely pathogenic variants were identified in 68 of 180 (38%) patients, mainly in HFE (79%), ceruloplasmin (25%), and transferrin receptor 2 (19%). Low spleen iron (R2* <50 s^-1 ), but not TSAT, was significantly associated with the presence of mutations. In 167 patients (93%), no monogenic cause of hepatic iron overload could be identified.

Conclusions: In patients without homozygosity for p.Cys282Tyr, coincident pathogenic variants in HFE and non-HFE genes could explain hyperferritinemia with hepatic iron overload in a subset of patients. Unlike HFE hemochromatosis, this type of polygenic hepatic iron overload presents with variable TSAT. High ferritin in blood is an indicator of the iron storage disease, hemochromatosis. A simple genetic test establishes this diagnosis in the majority of patients affected. MRI of the abdomen can guide further genetic testing.

Fig. 1

Study flowchart. Number of patients in different iron phenotype/genotype subgroups. Dual colored boxes show the relative number of patients with TSAT ≥ and < 45%.

Fig. 2

Serum iron parameters, tissue iron concentrations and correlation analysis in individual patients stratified by HFE genotype. (A) Transferrin saturation is significantly higher in p.C282Y homozygous in comparison with all other HFE genotypes, defining the first step on the diagnostic algorithm for hemochromatosis. (B) Liver R2* as a surrogate of HIC is also significantly higher in p.C282Y homozygous in comparison with other HFE genotypes. (C, D) Patients homozygous for the p.C282Y mutation show a significantly stronger correlation between liver R2* and transferrin saturation (C) and between ferritin and hepcidin concentrations (D). (E, F) No significant differences where observed in thecorrelation between liver R2* (E) and spleen R2* (F) with hepcidin/log(ferritin).

Fig. 3

NGS results. Top: transferrin saturation, liver and spleen R2*, central heatmap: distribution of mutations in the selected genes, and percentages on the right side: overall mutation frequency for each gene.

Fig. 4

Spleen R2* as a selecting criterion for NGS. (A) In the subgroup of patients who were further studied through NGS (n = 180), spleen R2* values were significantly lower in patients where probably disease‐associated mutations were found. (B) ROC curve analysis showed an AUC of 0.64 (95% confidence interval between parentheses), with the highest Youdex index for a cut‐off of 50 s^‐1 (sensitivity 49%, specificity 77%).

Fig. 5

Prevalence of hepatic iron overload and hemochromatosis gene variants after exclusion of likely secondary causes of hyperferritinemia and according to TSAT. Other: alpha‐1antitrypsin deficiency, drug induced liver injury or Wilson disease.