Ferritin is a ubiquitous protein capable of storing iron in the cell cytosol. Stored iron is released and made available for cellular needs by the degradation of ferritin itself. Small amounts of ferritin are present in the blood and consist of ferritin L, a glycosylated form of L called ferritin G, and trace amounts of ferritin H. It is secreted mainly by macrophages, hepatocytes, and lymphoid cells, but most aspects of its secretion remain not fully elucidated. Serum ferritin has broad clinical utility primarily as an indicator of iron stores, so low values of serum ferritin are indicative of a deficient state and high values of iron overload. However, the causes of increased serum ferritin are numerous, in many cases serum ferritin is increased disproportionately to iron stores such as in acute and chronic liver disease, infectious and inflammatory states, metabolic disorders, and high alcohol intake that are frequently observed in the clinical setting. Therefore, the diagnosis of hyperferritinemia requires a careful strategy including personal and family history, biochemical, instrumental, and targeted genetic testing. In fact, there are rare forms of genetically determined hyperferritinemia not associated with iron overload, such as hereditary cataract hyperferritinemia syndrome (HHCS) due to mutations in the Iron responsive Element (IRE) located in the 5' untranslated region of the FTL gene. More recently, a second dominant form of genetic hyperferritinemia without iron overload or cataract (benign hyperferritinemia) has been identified.

Preliminary results obtained so far have made it possible, through WES analysis, to identify the involvement of the STAB1 gene, which was found to be mutated in the studied subjects in whom reduced serum ferritin glycosylation and reduced plasma concentration of the protein itself were observed. It is therefore deemed necessary to proceed with the assay of glycosylated ferritin and the protein encoded by the gene to assess its sensitivity and specificity as a predictive test before performing the genetic analysis of STAB1. To achieve this goal, patients with undefined hyperferritinemia afferent to the SSD Rare Diseases of the IRCCS San Gerardo Foundation in whom to perform glycosylated ferritin and STAB1 protein assay in parallel with STAB1 sequencing will be evaluated. Similar investigations will be performed in a control group consisting of cases of hyperferritinemia due to genetically determined iron overload.

Ferritin is a ubiquitous protein capable of storing iron in the cell cytosol. Cytosolic ferritin consists of two subunits, the light chain (L-ferritin) and the heavy chain (H-ferritin) that assemble in different proportions to form apo-ferritin shells. Ferritin nanoshells consist of 24 subunits of the L- and H-chains that create a cavity in which intracellular iron is collected. The stored iron is released and made available for cellular needs by the degradation of ferritin. Ferritin can bind, oxidize, and store up to 4500 Fe(II) atoms, preventing iron-mediated oxidative stress. This is achieved by regulating ferritin synthesis according to cellular iron content and oxidative stress at both the post-transcriptional (via the Iron Responsive Element (IRE)-regulatory protein (IRP) system) and transcriptional levels. Small amounts of ferritin are present in the blood and consist of ferritin L, a glycosylated form of L called ferritin G, and traces of ferritin H. It is secreted primarily by macrophages, hepatocytes, and lymphoid cells, but most aspects of its secretion remain unknown. Serum ferritin has broad clinical utility primarily as an indicator of intracellular iron stores.

The causes of increased serum ferritin are numerous, including primary and secondary iron overload disorders, but also conditions in which serum ferritin is increased disproportionately to the body's iron stores such as chronic liver disease, inflammatory and metabolic disorders that are frequently observed in the clinical setting. Therefore, the diagnosis of hyperferritinemia requires a systematic strategy including personal and family history, biochemical and instrumental tests. In addition, alterations in the regulation of ferritin synthesis due to mutations in the iron-sensitive element L-ferritin (IRE) cause hereditary hyperferritinemia cataract syndrome (HHCS), an inherited disease characterized by elevated serum ferritin without iron overload and early onset of bilateral cataracts. Recently, a second dominant form of genetic hyperferritinemia without iron overload or cataract has been reported. Amino acid substitutions at positions 26, 27, and 30 in the heterozygous state in the L-ferritin A helix have been found in these patients. The resulting ferritin appears unusually susceptible to glycosylation, leading to serum glycosylated ferritin values consistently >90%. The reason for the development of hyperferritinemia and hyperglycosylation associated with these mutant forms of ferritin has not been established. It is probably related to increased secretion, but may also contribute to delayed clearance. Some cases of hyperferritinemia still remain unexplained, and the currently unknown candidate gene that we think we have identified among candidate genes needs to be validated in a larger population of subjects with the listed characteristics.

The primary objective of the study is to sequence the candidate gene that emerged from previous studies as mutated in patients with the same clinical features.

The secondary objective is to include the candidate gene in the routine genetic diagnosis of subjects with hyperferritinemia without tissue iron overload.

The study will last about 12 months the time needed for the recovery of patients with a genetic diagnosis not yet defined, analysis of medical records and research of the DNA sample stored at the Laboratory of Cytogenetics and Medical Genetics of San Gerardo Hospital and genetic analysis by Next Generation Sequencing. The study, although simple in its idea, requires a very careful organization of access and monitoring of selected patients that require the presence of a study manager dedicated to the project.

The study extension includes:glycosylated ferritin assay in patients with hyperferritinemia referred to the Rare Disease Center of IRCCS Foundation - San Gerardo dei Tintori selected according to the inclusion and exclusion criteria; soluble protein assay encoded by the STAB1 gene in patients in point A and patients in group D.

Inclusion Criteria:

Among patients referred to the Center for Rare Diseases of Monza will be enrolled only subjects with:

• ferritin > 1000 g / L in men and > 500 g / L,
• transferrin saturation <45%
• absence of hepatic iron overload, evaluated by liver biopsy or MRI, as indicated in the attached flow chart.

Exclusion Criteria:

Patients with hyperferritinemia attributable to:

• genetically determined causes [mutations in the HFE gene (homozygosity or heterozygosity for p.Cys282Tyr, homozygosity for p.His63Asp or compound heterozygosity for variants of p.Cys282Tyr and p. His63Asp), ferroportin and L-Ferritin gene mutations];
• presence of more than one component of metabolic syndrome (according to NCEP-ATPIII criteria: triglycerides >150 mg/dL, blood glucose >100 mg/dL, HDL <40 mg/dL in men and <50 mg/dL in women, waist circumference >102 cm in men and >88 cm in women; blood pressure ≥130/≥85 mm/Hg);
• alcohol intake >5 g/day chronic hepatitis,
• history of blood transfusion or parenteral iron treatment,
• late skin porphyria,
• hyperthyroidism,
• presence of cataracts or family history of early-onset cataracts
• acute or chronic inflammatory disorders.

Hyperferritinemia patients followed at the Rare Diseases Center in San Gerardo Hospital