Ceruloplasmin gene variants are associated with hyperferritinemia and increased liver iron in patients with NAFLD.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is a multifactorial disorder resulting from genetic and environmental factors. Hyperferritinemia has been associated with increased hepatic iron stores and worse outcomes in patients with NAFLD. The aim of this study was to evaluate the prevalence of variants of iron-related genes and their association with hyperferritinemia, hepatic iron stores and liver disease severity in patients with NAFLD. METHODS: From a cohort of 328 individuals with histological NAFLD, 23 patients with ferritin >750 ng/ml and positive iron staining, and 25 controls with normal ferritin and negative iron staining, were selected. Patients with increased transferrin saturation, anemia, inflammation, ß-thalassemia trait, HFE genotype at risk of iron overload and ferroportin mutations were excluded. A panel of 32 iron genes was re-sequenced. Literature and in silico predictions were employed for prioritization of pathogenic mutations. RESULTS: Patients with hyperferritinemia had a higher prevalence of potentially pathogenic rare variants (73.9% vs. 20%, p = 0.0002) associated with higher iron stores and more severe liver fibrosis (p <0.05). Ceruloplasmin was the most mutated gene and its variants were independently associated with hyperferritinemia, hepatic siderosis, and more severe liver fibrosis (p <0.05). In the overall cohort, ceruloplasmin variants were independently associated with hyperferritinemia (adjusted odds ratio 5.99; 95% CI 1.83-19.60; p = 0.0009). CONCLUSIONS: Variants in non-HFE iron genes, particularly ceruloplasmin, are associated with hyperferritinemia and increased hepatic iron stores in patients with NAFLD. Carriers of such variants have more severe liver fibrosis, suggesting that genetic predisposition to hepatic iron deposition may translate into liver disease. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) is a common disease which can progress to cirrhosis and liver cancer. Increased levels of serum ferritin are often detected in patients with NAFLD and have been associated with altered iron metabolism and worse patient outcomes. We found that variants of genes related to iron metabolism, particularly ceruloplasmin, are associated with high ferritin levels, hepatic iron deposition and more severe liver disease in an Italian cohort of patients with NAFLD.

Iron Overload in the Liver of 2 Children: Nonalcoholic Steatohepatitis and Juvenile Hemochromatosis.

BACKGROUND: Iron overload disorders are hereditary hemochromatosis and secondary etiologies other than hereditary hemochromatosis. We describe 2 boys presenting with iron overload. Juvenile hemochromatosis and nonalcoholic steatohepatitis (NASH) related iron overload are the genetic and secondary causes, respectively. OBSERVATIONS: Both patients benefited from phlebotomy even if they had different etiologies. CONCLUSIONS: In childhood, the diagnosis of iron overload syndromes is crucial because they do not confront us with obvious symptoms and findings. Early initiation of a phlebotomy program can prevent mortality. NASH might lead to iron overload and iron overload might aggravate the clinical course of NASH.

Human macrophage ferroportin biology and the basis for the ferroportin disease.

Ferroportin (FPN1) is the sole iron exporter in mammals, but its cell-specific function and regulation are still elusive. This study examined FPN1 expression in human macrophages, the cells that are primarily responsible on a daily basis for plasma iron turnover and are central in the pathogenesis of ferroportin disease (FD), the disease attributed to lack-of-function FPN1 mutations. We characterized FPN1 protein expression and traffic by confocal microscopy, western blotting, gel filtration, and immunoprecipitation studies in macrophages from control blood donors (donor) and patients with either FPN1 p.A77D, p.G80S, and p.Val162del lack-of-function or p.A69T gain-of-function mutations. We found that in normal macrophages, FPN1 cycles in the early endocytic compartment does not multimerize and is promptly degraded by hepcidin (Hepc), its physiological inhibitor, within 3-6 hours. In FD macrophages, endogenous FPN1 showed a similar localization, except for greater accumulation in lysosomes. However, in contrast with previous studies using overexpressed mutant protein in cell lines, FPN1 could still reach the cell surface and be normally internalized and degraded upon exposure to Hepc. However, when FD macrophages were exposed to large amounts of heme iron, in contrast to donor and p.A69T macrophages, FPN1 could no longer reach the cell surface, leading to intracellular iron retention. CONCLUSION: FPN1 cycles as a monomer within the endocytic/plasma membrane compartment and responds to its physiological inhibitor, Hepc, in both control and FD cells. However, in FD, FPN1 fails to reach the cell surface when cells undergo high iron turnover. Our findings provide a basis for the FD characterized by a preserved iron transfer in the enterocytes (i.e., cells with low iron turnover) and iron retention in cells exposed to high iron flux, such as liver and spleen macrophages. (Hepatology 2017;65:1512-1525).

Exome sequencing in a patient with Catel-Manzke-like syndrome excludes the involvement of the known genes and reveals a possible candidate.

In the present study we describe the exome sequencing and analysis of a patient with Catel-Manzke-like phenotype showing bilateral hyperphalangism of the second finger and thumb clinodactyly due to a unilateral delta phalanx, associated with growth, cardiac and vertebral defects. The exome sequencing analysis excluded pathogenetic mutations in the genes known to cause syndromes with hyperphalangism and did not identify any alteration in the X-chromosome or de novo mutations in likely candidate genes. Under the assumption of an autosomal recessive mode of inheritance and based on the frequency of the single nucleotide variants found in homozygous or double heterozygous states and the results of computer prediction programs, only one gene, DNAH10, emerged as a candidate in the pathogenesis of the disease in our patient. However, the differences among the known biological functions of DNAH10 and the genes involved in the other syndromes with hyperphalangism, suggest caution in the interpretation of the results.

Ferroportin-related haemochromatosis associated with novel Y64H mutation of the SCL40A1 gene.

In this paper we described the first Polish patient with ferroportin disease. Hereditary haemochromatosis (HH) is a condition associated with universal iron overload, and it is divided into four types, according to the Online Mendelian Inheritance in Man (OMIM) database. Ferroportin disease represented a rare type of HH, with autosomal dominant trait of inheritance. In our patient we detected a novel mutation in the ferroportin gene, with non-classical phenotype.

Juvenile Hemochromatosis, Genetic Study and Long-term Follow up after Therapy.

BACKGROUND Hereditary hemochromatosis (HH) is a very rare disease in Iran and reported cases are all negative for HFE mutation. We report a family affected by severe juvenile hemochromatosis (JH) with a detailed molecular study of the family members. METHODS We studied a pedigree with siblings affected by juvenile HH and followed them for 3 years. Microsatellite and gene sequencing analysis was performed for all family members. RESULTS Two siblings (the proband and his sister, aged 26 and 30 years, respectively) were found to have clinical findings of JH. The proband's brother, who presented with hyperpigmentation, died of probable JH at the age of 24 years. Gene sequencing analysis showed that the proband has a homozygote c.265T>C (p.C89R) HJV mutation + a heterozygote c.884T>C (p.V295A) mutation of HFE. The affected proband's sister presented with the same HJV c.265T>C (p.C89R) homozygote mutation. In addition, we found the HJV c.98-6C>G polymorphic variant in both the sister and proband (homozygote). Sequencing of hepcidin (HAMP), TfR2, and FPN revealed no mutation. CONCLUSION We have shown that molecular analysis of the HH related gene is a powerful tool for reliable diagnosis of JH and, in conjunction with magnetic resonance imaging (MRI) and noninvasive liver stiffness measurement by elastography, is adequate tool for management and follow up of HH.

Non-HFE hepatic iron overload.

Numerous clinical entities have now been identified to cause pathologic iron accumulation in the liver. Some are well described and have a verified hereditary basis; in others the genetic basis is still speculative, while in several cases nongenetic iron-loading factors are apparent. The non- HFE hemochromatosis syndromes identifies a subgroup of hereditary iron loading disorders that share with classic HFE-hemochromatosis, the autosomal recessive trait, the pathogenic basis (i.e., lack of hepcidin synthesis or activity), and key clinical features. Yet, they are caused by pathogenic mutations in other genes, such as transferrin receptor 2 ( TFR2), hepcidin ( HAMP), hemojuvelin ( HJV) , and ferroportin ( FPN), and, unlike HFE-hemochromatosis, are not restricted to Caucasians. Ferroportin disease, the most common non- HFE hereditary iron-loading disorder, is caused by a loss of iron export function of FPN resulting in early and preferential iron accumulation in Kupffer cells and macrophages with high ferritin levels and low-to-normal transferrin saturation. This autosomal dominant disorder has milder expressivity than hemochromatosis. Other much rarer genetic disorders are associated with hepatic iron load, but the clinical picture is usually dominated by symptoms and signs due to failure of other organs (e.g., anemia in atransferrinemia or neurologic defects in aceruloplasminemia). Finally, in the context of various necro-inflammatory or disease processes (i.e., chronic viral or metabolic liver diseases), regional or local iron accumulation may occur that aggravates the clinical course of the underlying disease or limits efficacy of therapy.

Keratin variants are overrepresented in primary biliary cirrhosis and associate with disease severity.

UNLABELLED: Keratins (K) 8 and 18 variants predispose carriers to the development of end-stage liver disease and patients with chronic hepatitis C to disease progression. Hepatocytes express K8/K18, whereas biliary epithelia express K8/K18/K19. K8-null mice, which are predisposed to liver injury, spontaneously develop anti-mitochondrial antibodies (AMA) and have altered hepatocyte mitochondrial size and function. There is no known association of K19 with human disease and no known association of K8/K18/K19 with human autoimmune liver disease. We tested the hypothesis that K8/K18/K19 variants associate with primary biliary cirrhosis (PBC), an autoimmune cholestatic liver disease characterized by the presence of serum AMA. In doing so, we analyzed the entire exonic regions of K8/K18/K19 in 201 Italian patients and 200 control blood bank donors. Five disease-associated keratin heterozygous variants were identified in patients versus controls (K8 G62C/R341H/V380I, K18 R411H, and K19 G17S). Four variants were novel and included K19 G17S/V229M/N184N and K18 R411H. Overall, heterozygous disease-associated keratin variants were found in 17 of 201 (8.5%) PBC patients and 4 of 200 (2%) blood bank donors (P < 0.004, odds ratio = 4.53, 95% confidence interval = 1.5-13.7). Of the K19 variants, K19 G17S was found in three patients but not in controls and all K8 R341H (eight patients and three controls) associated with concurrent presence of the previously described intronic K8 IVS7+10delC deletion. Notably, keratin variants associated with disease severity (12.4% variants in Ludwig stage III/IV versus 4.2% in stages I/II; P < 0.04, odds ratio = 3.25, 95% confidence interval = 1.02-10.40), but not with the presence of AMA. CONCLUSION: K8/K18/K19 variants are overrepresented in Italian PBC patients and associate with liver disease progression. Therefore, we hypothesize that K8/K18/K19 variants may serve as genetic modifiers in PBC.

Lack of enterocyte iron accumulation in the ferroportin disease.

Ferroportin-associated iron overload (also known as the ferroportin disease) is a common cause of hereditary hyperferritinemia. It was originally proposed that loss-of-protein function mutations account for iron overload in the FD. This hypothesis is consistent with the phenotype reported in most patients with FD of early iron accumulation in tissues, particularly in macrophages, in spite of relatively normal-low circulatory iron. It was still unclear, however, how FPN mutations would affect iron retention in enterocytes. We studied histologically the intestine of six patients with different FPN mutations as compared to other subjects with various iron disorders. We found that regardless of the underlying FPN mutation, no iron accumulation was found in absorbing enterocytes while, intestinal villi showed marked signs of iron accumulation in the cells of lamina propria. Not surprisingly, in the liver, iron excess was found mainly in Kupffer cells. These results indicate that FPN haploinsufficiency is not limiting for iron export from enterocytes.

Juvenile hemochromatosis associated with pathogenic mutations of adult hemochromatosis genes.

BACKGROUND AND AIMS: Juvenile hemochromatosis is a severe form of hereditary iron overload that has thus far been linked to pathogenic mutations of the gene coding for hemojuvelin (HJV), on chromosome 1, or, more rarely, that coding for hepcidin ( HAMP ), on chromosome 19. A milder adult-onset form is due to pathogenic mutations of HFE or, rarely, serum transferrin receptor 2. METHODS: We studied a pedigree with siblings affected by both juvenile and adult-onset hereditary hemochromatosis. Affected subjects underwent full clinical evaluation, as well as microsatellite and gene sequencing analysis. RESULTS: Two siblings (male and female, aged 24 and 25 years, respectively) were hospitalized for severe endocrinopathy and cardiomyopathy. At age 18 and 17 years, they had presented with impotence and amenorrhea, respectively, and increased serum iron levels. Hypogonadotropic hypogonadism was confirmed in both, and liver biopsy showed marked hepatic iron accumulation and micronodular cirrhosis. Iron levels were normalized after 24 months (female) and 36 months (male) of weekly phlebotomies. Microsatellite analysis showed no linkage with chromosome 1 and 19, and gene sequencing showed no hemojuvelin or hepcidin gene mutations. Instead, combined mutations of HFE (C282Y/H63D compound heterozygosity) and serum transferrin receptor 2 (Q317X homozygosity) were found. A 21-year-old brother with a milder phenotype resembling classic adult-onset hereditary hemochromatosis carried only the Q317X serum transferrin receptor 2 homozygote mutation. CONCLUSIONS: Juvenile hereditary hemochromatosis is not a distinct monogenic disorder invariably due to hemojuvelin or hepcidin mutations: it may be genetically linked to the adult-onset form of hereditary hemochromatosis.

Iron overload in Africans and African-Americans and a common mutation in the SCL40A1 (ferroportin 1) gene.

The product of the SLC40A1 gene, ferroportin 1, is a main iron export protein. Pathogenic mutations in ferroportin 1 lead to an autosomal dominant hereditary iron overload syndrome characterized by high serum ferritin concentration, normal transferrin saturation, iron accumulation predominantly in macrophages, and marginal anemia. Iron overload occurs in both the African and the African-American populations, but a possible genetic basis has not been established. We analyzed the ferroportin 1 gene in 19 unrelated patients from southern Africa (N = 15) and the United States (N = 4) presenting with primary iron overload. We found a new c. 744 C-->T (Q248H) mutation in the SLC40A1 gene in 4 of these patients (3 Africans and 1 African-American). Among 22 first degree family members, 10 of whom were Q248H heterozygotes, the mutation was associated with a trend to higher serum ferritin to amino aspartate transferase ratios (means of 14.8 versus 4.3 microg/U; P = 0.1) and lower hemoglobin concentrations (means of 11.8 versus 13.2 g/dL; P = 0.1). The ratio corrects serum ferritin concentration for alcohol-induced hepatocellular damage. We also found heterozygosity for the Q248H mutation in 7 of 51 (14%) southern African community control participants selected because they had a serum ferritin concentration below 400 microg/L and in 5 of 100 (5%) anonymous African-Americans, but we did not find the change in 300 Caucasians with normal iron status and 25 Caucasians with non-HFE iron overload. The hemoglobin concentration was significantly lower in the African community controls with the Q248H mutation than in those without it. We conclude that the Q248H mutation is a common polymorphism in the ferroportin 1 gene in African populations that may be associated with mild anemia and a tendency to iron loading.