C282Y Mutation Research Articles

Prevalence of C282Y, H63D and S65C gene mutations in patients with hyperferritinemia undergoing therapeutic phlebotomy suspected of having hereditary hemochromatosis

Background: Hemochromatosis is an autosomal recessive disorder caused by excessive iron overload in parenchymal organs. In Brazil, the prevalence of hereditary hemochromatosis (HH) may vary compared to other countries due to different ethnical backgrounds according to the geographic region studied. The prevalence of C282Y mutation of the HFE gene ranges from three to eight times lower in Brazilians than in Caucasians of Northern European origin. Methods: A retrospective cross-sectional study was carried out in 222 patients submitted to therapeutic phlebotomy between March 2011 and July 2014 in a tertiary hospital blood center. The prevalence of HH was determined in 89 (47.3%) out of 188 (84.6%) patients presenting hyperferritinemia, and divided in two genotype groups: C282Y mutation and other mutations. The HH patients’ geographical and clinical characteristics, serum ferritin rate (SF) and transferrin saturation (TS) were analyzed. Results: The HH group consisted of 71 males (79.7%) and 18 (20.3%) females, with a median age of 51.5 ± 10.6 years. There were 36 (40.0%) symptomatic patients and 65 (73.0%) tested positive for the HFE mutation. The initial baseline TS values were significantly higher in primary HH patients (48.6%, IC 95% 43.2% - 55.7%) compared to those with secondary HH (35.0%, IC 95% 27.5% 41.8%, p=0.001). Conclusion: The prevalence of C282Y mutation was higher in this population from the South region compared to previous reports in the Brazilian population, yet inferior to that of Northern Europeans.

1153 Many Reasons for Restless Legs, Not to Rest

Abstract Introduction Restless leg syndrome (RLS) involves an urge to move the legs that is exacerbated by rest, relieved by movement, and most prominent in the evening. While RLS is commonly associated with low ferritin, here we present a complex case in a patient requiring phlebotomy for symptom control related to elevated ferritin levels. Report of case(s) A 52 year-old male presented with uncontrolled RLS diagnosed 12 years prior. Sleep history included obstructive sleep apnea (OSA) on CPAP and hypersomnolence. Diagnostic polysomnogram was notable for an AHI 36.4 events/hr with associated hypoxia. Ferritin was 374ng/mL. He presented to our clinic on melatonin and wearing socks with bars of soap atop his feet. On exam, he was obese and plethoric with significant hypersomnolence despite excellent CPAP compliance. He was previously treated unsuccessfully with a combination of phenobarbital, trazodone, and ropinirole, which exacerbated his hypersomnia. He drank 4-5 liters (L) of soda daily. In the 5 years after initial presentation, he went through periods of total cessation to drinking up to 12L a day. He eventually developed uncontrolled diabetes, hypertension, hyperlipidemia, and NASH with advanced fibrosis. He was referred to Hematology and underwent therapeutic phlebotomy every 2-4 months. He was found to have HFE-C282Y Heterozygous mutation and NASH fibrosis without iron overload on MRI. His RLS was eventually controlled with a combination of pramipexole 1.5 mg, gabapentin 1800 mg, alprazolam 0.5 mg along with therapeutic phlebotomy to maintain a ferritin level less than 250 ng/mL. His hypersomnia was managed with amphetamine-dextroamphetamine. Conclusion RLS has been associated with high serum ferritin, iron, and saturation transferrin index levels with a low transferrin level due to impaired mobilization of stored iron. Iron deposits can be seen on MRI in the globus pallidus, dentate, red nuclei and substantia nigra. In this patient, soda cessation would help improve his diabetes, thereby decreasing chronic inflammation and lowering ferritin levels. Chronic liver disease, HFE C282Y mutations, and hypoxia from OSA can also contribute to increased ferritin levels. Abstaining from caffeine can likewise improve symptoms of RLS as caffeine is known to heighten proprioceptive awareness and increase neuromuscular reactivity which may include myoclonus. Support (if any)

Hemocromatosis

Hemochromatosis (HC) is one of the most common hereditary diseases. It is characterized by an excessive accumulation of iron in the body that can cause structural and functional damage to various organs and death if left untreated. The most common form of HC is due to a homozygous C282Y mutation in the HFE gene. The diagnosis is based on a clinical evaluation and plasma iron parameters together with genetic and imaging tests. The proper and timely diagnosis of HC is essential, given that simple interventions, such as phlebotomy, can prevent or reverse organ damage caused by iron overload.

Serum Erythroferrone Levels in a Large General Population: Towards a Better Understanding of Connections between Erythropoiesis and Iron Homeostasis

Introduction: Iron is essential for various biological functions [ Ganz T., Physiol Rev 2013], especially for erythropoiesis which is the most iron-consumer process daily. Hepcidin is the key hormone of iron homeostasis, preventing both iron deficiency and excess, by modulating iron absorption, recycling, and mobilization [ Camaschella C. et al, Haematologica 2020]. Erythroferrone (ERFE) constitutes the principal erythroid regulator of hepcidin: in the presence of enhanced erythropoiesis, expanding erythroblasts produce ERFE which, by impeding bone morphogenetic proteins (BMPs) signaling in hepatocytes, suppress hepcidin, in turn increasing iron availability for the bone marrow [ Ganz T., Free Rad Biol Med 2019]. Increased ERFE levels have been demonstrated in expanded/ineffective erythropoiesis (IE) (e.g., in thalassemia intermedia), where chronic ERFE-mediated hepcidin suppression may cause progressive iron accumulation even in subjects not regularly transfused. In view of this, ERFE has been proposed as a potential biomarker of IE and an appealing target for treating iron overload deriving from IE [ Srole D.N. and Ganz T., J Cell Physiol 2020]. However, studies that fully elucidate the role of ERFE in the general population under physiological conditions are still lacking. Our project evaluated age- and sex-related variations in ERFE levels, and their correlations with hematological and iron status parameters, in a large subpopulation from the Cooperative Health Research In South Tyrol (CHRIS) study. Subjects and methods: The CHRIS study is a population-based study carried out in South Tyrol (Northern Italy) aimed at investigating acquired and genetic basis of common chronic conditions associated with aging [ Pattaro C. et al, J Transl Med 2015]. Blood samples were tested for several biochemical and molecular parameters, including those related to iron status, such as ferritin, transferrin saturation (TSat), soluble transferrin receptor (sTfR), hepcidin, and C282Y and H63D mutations. Serum ERFE was measured in stored aliquots by the Human Erythroferrone IETM ELISA kit (Intrinsic Lifesciences - The BioIron Company TM, USA), according to manufacturer instructions. Results: We measured ERFE in 3688 subjects aged >18 years (55% female). A total of 777 subjects (21% of the whole population; 57% females) had ERFE concentrations below the lower limit of detection for our method (we assigned them an arbitrary value of 0.16 for statistical analyses). Figure 1 illustrates age- and sex-dependent variations of ferritin, sTfR, hepcidin, and ERFE in the whole population. After stratification by hemoglobin (Hb) levels, we observed that ERFE was significantly higher in anemic (i.e., Hb<12 g/dl in females and <13 g/dl in males; n=100) than in non-anemic subjects (respectively 3.74 and 0.65 ng/ml; p<0.0001). In anemic subjects, ERFE showed a strong negative correlation with Hb (r = -0.5; p < 0.001), TSat (r = -0.58; p < 0.001), ferritin (r = -0.48; p < 0.001) and hepcidin (r = -0.32; p = 0.001), and a positive correlation with sTfR (r = 0.58; p < 0.001). Moreover, ERFE was higher in iron-deficient (ID) subjects (i.e., ferritin <30 mg/l; n=773) than in non-ID subjects (0.92 vs. 0.63 ng/ml; p<0.0001). Although ID subjects had lower Hb levels than non-ID subjects (13.7 vs 15 g/dl; p<0.0001), Hb levels were broadly within the normal range.To better explore the relationship between ERFE and iron status, we divided the population into 4 subgroups, according to sTfR and ERFE levels. Considering the skewed distribution of the variables, we used sTfR above or below the 3° tertile (1316 ng/ml), in order to identify subjects having higher or lower iron needs, respectively. Analogously, we further divided the population in subjects with ERFE above or below the 3° tertile (1.21 ng/ml). As illustrated in Figure 2, after stratification, we observed that hepcidin reduced, in subjects with high ERFE levels, when iron requirement was high (0.94 vs 2.10 nM; p < 0.0001), but it didn't when iron requirement was low (3.35 vs. 3.05 nM; p = 0.72), even in presence of normal Hb levels (Hb 14.3 g/dl). Discussion: our data confirmed an association among ERFE, Hb and iron status parameters under conditions of anemia (when erythropoiesis is stimulated); moreover, if confirmed in further studies, it could suggest an effect of ERFE on hepcidin regulation could be linked to depletion of iron stores, even before anemia develops.

Phenotypes Associated with HFE p.C282Y Homozygosity, the Main Hereditary Hemochromatosis Genotype, in Four Large Genetic Cohorts

Introduction Hereditary hemochromatosis (HH) is the most common known recessive hereditary disease in people of northern European descent. HH is most commonly caused by the HFE-C282Y homozygosity. Disease penetrance is highly variable (1-20%). We have examined the association between C282Y homozygosity and multiple phenotypes, both case-control and quantitative, across 4 international cohorts (deCODE genetics, UK Biobank, Denmark (Copenhagen Hospital Biobank / Danish Blood Donor Study) and Intermountain Healthcare) in an effort to shed a new light on the pathophysiology of this common hereditary disease. Methods We preformed a phenome wide association study meta-analyses for C282Y homozygosity across the 4 beforementioned cohorts, comprising around 970.000 individuals, of those 5928 were homozygous for the C282Y mutation (746 in deCODE/Iceland MAF 6.71%, 2865 in UK Biobank MAF 7.94%, 1703 in CHB MAF 5.6% and 614 in Intermountain Healthcare MAF 6.19%). The analyses comprised both case-control phenotypes (approximately 7000), cases being defined by ICD-10 codes, and continuous quantitative phenotypes (approximately 5500), both results from blood tests and proteome data from deCODE and UK Biobank. Results C282Y homozygotes had a higher prevalence of the following liver, hematological and rheumatic diseases compared to a control group with no copy of the C282Y variant. Diagnosed fibrosis and cirrhosis of liver (odds ratio 4.30, 95% confidence interval 3.35 to 5.51, p=1.77x10 -30), liver cell carcinoma (9.04, 6.01 to 13.58, p=3.24x10 -26) and fatty liver disease (1.75, 1.45 to 2.11, p=5.42x10 -09) were strikingly more common in C282Y homozygotes. Unsurprisingly C282Y homozygotes also had higher liver parameters. Notably C282Y homozygotes had a higher prevalence of diagnosed polycythemia vera (7.24, 4.93 to 10.62, p=4.37x10 -24) yet no association was found with clonal hematopoiesis (1.09, 0.88 to 1.34, p=4.47x10 -01). Strong association was found between C282Y homozygosity and increased hemoglobin (0.41 standard deviations, 95% confidence interval 0.38 to 0.45, p=6.84x10 -142), MCH (0.98, 0.93 to 1.03, p=<1.00x10 -300) and MCV (0.82, 0.78 to 0.86, p=<1.00x10 -300). Red blood cell count (RBC) was decreased (-0.27, -0.30 to -0.23, p=1.06x10 -50), while reticulocyte count (0.30, 0.26 to 0.34, p=1.42x10 -54) and percentage (0.36, 0.32 to 0.40, p=7.91x10 -79) were increased. Erythropoietin (EPO) measurements were markedly decreased in C282Y homozygotes (-0.31, -0.40 to -0.22, p=1.77x10 -11). Homozygous individuals had an increased risk of being diagnosed with osteoarthritis of hip (1.83, 1.63 to 2.07, p=4.30x10 -23) and were more likely to undergo total hip replacement (2.00, 1.69 to 2.29, p=5.81x10 -18). In contrast to these risk enhancing effects C282Y homozygotes had a lower prevalence of cardiovascular disease (CVD). C282Y homozygotes had decreased risk of coronary artery disease (CAD) (0.79, 0.72 to 0.86, p=4.26x10 -07) and angina pectoris (0.77, 0.69 to 0.86, p=4.28x10 -06) and were less likely to undergo coronary artery bypass graft (0.60, 0.49 to 0.74, p=2.39x10 -06). Pure hypercholesterolemia (0.76, 0.69 to 0.83, p=2.52x10 -09) and disorders of lipoprotein metabolism (0.75, 0.66 to 0.85, p=8.41x10 -06) was less commonly diagnosed. C282Y homozygotes were less likely to take cholesterol lowering statin drugs (0.74, 0.67 to 0.81, p=1.45x10 -09). Unsurprisingly with regard to these results strong association was found with lower cholesterol measurements in C282Y homozygotes, the effects being most pronounced on LDL cholesterol (-0.38, -0.42 to -0.35, p=1.61x10 -99). Total cholesterol was also markedly lowered (-0.35, -0.38 to -0.31, p=1.38x10 -91), as well as non HDL cholesterol (-0.34, -0.37 to -0.30, p=2.36x10 -81). No significant association was found with HDL cholesterol and triglyceride measurements. Conclusions C282Y homozygosity was associated with significantly increased risk of liver disease, liver cell carcinoma and osteoarthritis of hip. Homozygous individuals had increased risk of non-clonal primary polycythemia, association with polycythemia vera likely due to misdiagnosis or early diagnosis exacerbated by significantly increased hemoglobin. C282Y homozygotes had a significant decrease in LDL, non-HDL and total cholesterol measurements and were protected against cardiovascular disease.

Association of mutations in hemochromatosis genes with clinical severity of calcium pyrophosphate arthritis

Aims: To study factors associated with the development of calcium pyrophosphate (CPP) arthritis and the severity phenotype. Methods: Transversal case-control study. Cases had to be confirmed by both X-ray chondrocalcinosis and CPP crystals in synovial fluid. Controls had neither chondrocalcinosis nor CPP crystals in synovial fluid. Patients and controls with hemochromatosis or primary hyperparathyroidism were not included. Mutations of hemochromatosis genes (HFE), magnesium (Mg), calcium (Ca), phosphate, iron (Fe), transferrin saturation, ferritin, parathyroid hormone (PTH), and calcifediol levels were studied. Results: Three hundred patients and 300 sex and age matched controls were compared. Lower serum Mg (sMg) and higher ferritin levels were found among patients. Hypomagnesemia (HypoMg) and HFE mutations were more frequent among patients. Involvement of over one joint was observed in 199 (66.4%) patients whereas persistent joint inflammation was retrieved in 154 (51.4%) of the patients. Initial analysis showed that the frequency of polyarticular and inflammatory phenotypes seemed to be progressively overrepresented in patients with HFE mutations. Further bivariate and multivariate analysis adjusted for the time from onset disclosed that the presence of genotypes with C282Y mutations was associated with polyarticular disease (hazard risk 3.501, 95% confidence interval 1.862–6.581, P < 0.001). Although C282Y mutations also seemed to be associated with inflammatory patterns, the association did not reach statistical significance (P = 0.173). Conclusions: Low sMg and high ferritin levels are associated with CPP arthritis (CPPA). In patients without hemochromatosis, HFE mutations, and specifically C282Y mutations seem to associate with the polyarticular disease phenotype, and plausibly with the chronic inflammatory phenotype.

HFE hemochromatosis gene polymorphism and porphyria cutanea tarda

Purpose. To study the frequency of genotypes and alleles of C282Y and H63D mutations in the HFE gene in patients with porphyria cutanea tarde of the West Siberian region and their influence on the clinical and biochemical characteristics of the disease. Materials and methods. We observed 14 patients with porphyria cutanea tarde (PCT), who underwent a comprehensive general clinical and instrumental examination. The indicators of the excretory profile of porphyrin metabolism indicators were purposefully determined, a molecular genetic examination was carried out to determine the genotypes and alleles of the C282Y and H63D mutations of the hemochromatosis gene HFE. Results and discussion. Molecular genetic research found that the HFE gene mutation was found in 7 patients (50.0%). Polymorphism for the C282Y allele was found in 2 (14.3%) patients, and for the H63D allele - in 5 patients (35.7%). Risk factors for the manifestation of PCT included frequent alcohol consumption and chronic HCV infection. Genotype 1b was recorded more often (9 people), less often - genotype 3a (4 people). The excretory profile of porphyrin metabolism indices in patients of both groups exceeded the control values. Porphyrin metabolism parameters in patients without HFE gene mutations were significantly higher than those in patients without HFE gene mutations. Conclusions. Polymorphism of the hemochromatosis gene HFE in PCT was detected in 50% of patients. The most common mutation was the H63D allele. The level of porphyrin metabolism disorders in patients with HFE hemochromatosis gene mutations is significantly lower. The clinical picture of PCT in all observed patients did not differ. Chronic viral hepatitis C is assessed as a risk factor for the manifestation of PCT.

Hereditary hemochromatosis: An update vision of the laboratory diagnosis

Haemochromatosis (HC) is an inherited disorder of iron metabolism. The 85–90% of Hereditary hemochromatosis cases are caused by mutations in HFE gene (HC type 1). The remaining 10–15% of HC cases are caused by mutations in other non-HFE genes (HJV, HAMP, TRF2, SLC40A1, BMP6). The study of patients for the diagnosis of HC has an important laboratory approached: analysis of biochemical parameters and genetic studies. To confirm a case, it is necessary to carry out a genetic study of the C282Y and H63D mutations. The presence of C282Y mutation in homozygosis is compatible with the diagnosis of HC type 1. Due to the incomplete penetrance of this mutation and the variable phenotypic expression, the severe forms of the disease are relatively rare. The study of variants in non-HFE genes allows more detailed study of both non-classic HC cases and those with more severe clinical expression. The genotype characterization of a patient not always justified the phenotype expression of the symptoms in this disease. All laboratory clinicians must consider recommendation provide by the experts in the Materia.

Type 1 Hemochromatosis: A Review of the History, Inheritance and Treatment of Iron Overload Disorder

Hereditary hemochromatosis (also known as type 1 hemochromatosis, iron overload disorder, or the Celtic Curse) is a genetic disorder characterized by an autosomal recessive inheritance pattern. This review project focused on gathering comprehensive data about the history, causes, inheritance, diagnosis, and treatment of iron overload disorder. Through such research, it was discovered that type 1 hemochromatosis may occur due to one of two nucleotide sequence changes (either the C282Y mutation or the H63D mutation) in the HFE gene, which codes for the construction of the homeostatic iron regulator protein and regulates production of the hormone hepcidin. As a result of these mutations, individuals affected by hemochromatosis have elevated iron levels and excess deposits of iron in tissues and organs. Such deposits may cause a variety of symptoms and complications, including fatigue, skin discoloration, heart failure, cirrhosis, liver cancer, and diabetes. Hemochromatosis is one of the most common genetic disorders in the world, affecting one in every three hundred individuals. The majority of hemochromatosis patients are of northern European descent. Currently, there is no cure for type 1 hemochromatosis; however, the primary treatment method for this disorder is phlebotomy. Due to the widespread prevalence of the disorder, researchers continue to explore experimental treatment methods, including gene therapy and iron chelation therapy.

S3055 Late Manifestation of Heterozygous H63D Mutation as Hereditary Hemochromatosis in 89-Year-Old Man

Introduction: Hereditary hemochromatosis (HH) is an autosomal recessive that usually affects middle-aged people [1]. In most patients, homozygous C282Y mutations in the HFE gene express the disease. But in some occurrences, compound heterozygous, including C282Y and H63D mutations, were also witnessed. We present an atypical case where heterozygous H63D manifested as HH in an 89-year-old man. Case Description/Methods: An 89-year-old Caucasian man of Italian descent presented with confusion, left-sided facial droop, and slurring of speech. Past medical history included end-stage renal disease on hemodialysis and atrial fibrillation. For the past few months before presentation, the patient has been complaining of increasingly tired with intermittent episodes of slurring speech, mainly after he receives dialysis. During the initial physical examination, the patient had confusion but was alert & oriented to self and time. There was mild abdominal distension and positive for asterixis, and the skin was bronze in color. Initial labs showed in table 1. The patient had persistent hypoglycemia and required intravenous glucose administration. Brain imaging was normal. CT scan of the abdomen, pelvis, and chest showed severe cardiomegaly and a moderate amount of ascites. Iron studies were abnormal. Ascites fluid analysis was consistent with cirrhosis. Although, an ultrasound right upper quadrant was negative for any cirrhotic morphology. MRI showed hemosiderosis. 2D Echo showed a severely dilated right ventricle. The patient empirically underwent two sessions of phlebotomy. Genetic testing confirmed heterozygous H63D mutation. The patient followed up for the phlebotomy sessions and demonstrated significant improvement. Discussion: This case highlights the consideration of HH in differential regardless of age if a patient presents with symptoms suspected of HH. Standard genetic testing includes testing C282Y and H63D mutations in the HFE gene on chromosome 6. Less frequently tested S65C, HFE4, TFR2-HHC, and FTH1 gene mutations also have the expression of HH in sporadic cases [2-3]. Previously, heterozygous C282Y carriers with iron overload are assumed to have other genetic changes or influence by environmental factors, such as alcohol or liver disease, that increase disease expression [2]. However, no such literature is available for heterozygous H63D carriers. Given the unusual age for phenotype expression and atypical disease, the presentation makes this unique case for learning the pathophysiology and genetics of HH.Figure 1.: A: MRI of liver showing decreased signal of the liver and spleen on in phase imaging is likely due to hemosiderosis. B: Two-dimensional transthoracic echocardiography showing severely dilated right ventricle and both atria. Moderate pulmonary hypertension was also noticed. Table 1. - Day 1 Day 2 Day 3 Day 4 Day 15 Day 42 Hemoglobin (g/dL) 15.6 14.8 15.1 13.8 13.6 12.2 AST (U/L) 85 102 135 125 109 71 ALT (U/L) 56 57 70 74 72 46 Bilirubin (mg/dL) 3.0 2.9 2.6 2.9 2.6 2.1 INR 1.3 1.4 1.4 1.4 1.2 1.2 Ammonia (µmol/L) 99 45 46 42 31 Platelets (10ˆ3/µL) 103 86 82 63 59 91 Ferritin (ng/ml) 1283 725 Iron (µg/dL) 187 130 Transferrin Saturation (%) 94 74 Total Iron-binding Capacity (µg/dL) 200 276

DIFFERENCES IN THE FREQUENCY OF POLYMORPHISMS IN THE HFE GENE (H63D AND C282Y) IN A POPULATION GROUP FROM NORTHWEST SÃO PAULO, BRAZIL

Hereditary hemochromatosis (HH) is an autosomal recessive disease characterized by excess iron. People with polymorphisms in the HFE gene ( H63D and C282Y ) may develop inflammatory processes resulting from changes in iron metabolism. The ability of the HFE protein to make connections with transferrin receptors, located on cell membranes, allows it to help control the absorption of iron in the body. This work sought to investigate the frequency of HFE gene variants in a sample from the city of São José do Rio Preto, SP, Brazil. DNA samples from 105 people were subjected to molecular analysis to confirm mutations in the HFE gene ( H63D and C282Y ), using PCR-RFLP. The group was separated by male and female gender, age group of 10 in 10 years, and ethnic groups. Statistical analyzes were performed using the IBM SPSS Statistics 20 software and data were presented as percentages, with a significance level of 0.05. The associations between the wild-type homozygote, heterozygote, and mutant homozygote genotypes for each mutation were tested by the Chi-Square test. Then, the allele frequencies in the genotypes were calculated. Fisher's exact test was performed to verify the association between gender, age group, and ethnicity with mutations in the HFE gene. The sample included: 55 men (52.38%) and 50 women (47.62%), with ages ranging from 18 to 62 years, with an average of 30 years. A total of 210 alleles were evaluated and for the H63D mutation, 78 wild-type homozygotes, 26 heterozygotes, and 1 mutant homozygote were found. For the C282Y mutation, 99 wild-type homozygotes, 6 heterozygotes and no mutant homozygote were found. Allelic frequency analysis indicated the occurrence of significant differences between wild-type homozygous, heterozygous, and mutant homozygous genotypes for the mutations (p < 0.05). Fisher's exact test indicated no association between gender, age, and ethnicity for the H63D and C282Y polymorphisms (p > 0.05). The heterozygous genotype for H63D was more frequent in men (15-14%) than women 11 (10%). For C282Y equal frequencies (3-3%) were found for heterozygosity between men and women. H63D heterozygosity was more frequent in Caucasians (22-21%). In African-derived, only one individual was heterozygous for H63D . The frequency of HH genotypes varies among different populations. The low incidence or absence of homozygous alleles is due to the different degrees of miscegenation in the formation of the Brazilian population. The H63D mutation is an important marker for the Brazilian population, being frequent in Africa, although it has low phenotypic expression. The C282Y is predominantly associated with homozygosity for clinical cases, however, it is rare in African populations and frequent in Caucasians. Our results corroborate the data in the literature, with a predominance of the H63D mutation in Caucasians, African-derived, and Afro-descendants, and C282Y in Caucasians. The frequency of H63D and C282Y polymorphisms in the HFE gene showed a higher occurrence of heterozygotes for H63D in this population group. Studies to characterize HH are needed to devise strategies for effective support to patients and, therefore, improve quality of life.

In vivo adenine base editing reverts C282Y and improves iron metabolism in hemochromatosis mice

Hemochromatosis is one of the most common inherited metabolic diseases among white populations and predominantly originates from a homozygous C282Y mutation in the HFE gene. The G > A transition at position c.845 of the gene causes misfolding of the HFE protein, ultimately resulting in its absence at the cell membrane. Consequently, the lack of interaction with the transferrin receptors 1 and 2 leads to systemic iron overload. We screened potential gRNAs in a highly precise cell culture assay and applied an AAV8 split-vector expressing the adenine base editor ABE7.10 and our candidate gRNA in 129-Hfetm.1.1Nca mice. Here we show that a single injection of our therapeutic vector leads to a gene correction rate of >10% and improved iron metabolism in the liver. Our study presents a proof-of-concept for a targeted gene correction therapy for one of the most frequent hereditary diseases affecting humans.

C282Y/H63D Compound Heterozygosity Is a Low Penetrance Genotype for Iron Overload-related Disease

BackgroundHereditary hemochromatosis (HH) occurs due to mutations in the HFE gene. While the C282Y mutation is the most common genotype reported in HH, other genotypes are found less frequently, indicating variable degrees of penetrance. We studied the penetrance of the C282Y/H63D compound heterozygote genotype in developing clinically significant iron overload.MethodsWe have completed a retrospective analysis on every individual within Newfoundland & Labrador who were diagnosed as C282Y/H63D compound heterozygote between 1996 and 2009 through a molecular genetics study. We collected data for up to 10 years following the initial genotyping using electronic health records, including laboratory values, phlebotomy status, radiologic reports and clinic records. Iron overload status was classified based on the HealthIron study.ResultsBetween 1996 and 2009, 247 individuals with available health records tested positive for C282Y/H63D compound heterozygosity. Over the 10 years of our study, 5.3% of patients exhibited iron overload-related disease on the background of documented iron overload. Including these individuals, 10.1% of patients had documented iron overload, 23.1% of patients had a provisional iron overload and the remaining 66.8% of patients had no evidence of iron overload. Only 44 patients had documented phlebotomies, likely based on their severe phenotype at baseline. Despite phlebotomy, the prevalence of iron overload was higher among these patients. The penetrance of compound heterozygosity was also significantly higher among men (P < 0.01).ConclusionC282Y/H63D compound heterozygosity is a low penetrance genotype in HH. This is the largest reported cohort of C282Y/H63D compound heterozygotes in North America with an extended follow-up.

20-Year-Old Man With Elevated Liver Tests

20-Year-Old Man With Elevated Liver Tests

Hemochromatosis

A man in his mid-30s was found to have a markedly elevated ferritin level (>2500 μg/L) and a serum transferrin saturation of 95%. A liver biopsy was performed. A Perls iron stain demonstrated severe iron overload, with iron deposited in the hepatocytes. The patient then underwent genetic testing, which identified homozygous C282Y mutations in the HFE gene.

Hemochromatosis Gene Mutation in Persons Developing Erythrocytosis on Combined Testosterone and SGLT-2 Inhibitor Therapy.

In clinical trials, sodium-glucose cotransporter-2 inhibitors (SGLT-2i) use alone in persons with type 2 diabetes (T2D) or testosterone replacement therapy (TRT) prescription alone in men with hypogonadism was shown to lead to a modest but significant increase in red blood cell mass. Recent evidence indicates that combined use of TRT and SGLT-2i in persons with T2D may be associated with risk of erythrocytosis. However, factor(s) that may lead to the development of erythrocytosis in these patients is unknown. We describe here 5 consecutive patients with hypogonadism on chronic TRT who developed erythrocytosis following addition of SGLT-2i empagliflozin for optimization of T2D management. In addition to the careful review of medical history, all patients underwent genetic screening for hereditary hemochromatosis. We have found that none of the patients had C282Y mutation in the HFE (Homeostatic Iron Regulator) gene and 4 out of 5 patients had heterozygosity in the H63D allele. Upon TRT discontinuation or its dose reduction or referral for scheduled phlebotomy, patients showed resolution of erythrocytosis. Our study reaffirms that practitioners should monitor for changes in hematocrit following the initiation of SGLT-2i in persons with T2D and hypogonadism on chronic TRT. Also, for the first time, we showed that in some of the patients receiving combined TRT and SGLT-2i H63D heterozygosity in the HFE gene may mediate the development of new-onset erythrocytosis.

Screening for hereditary haemochromatosis in patients undergoing knee arthroplasty : a retrospective cohort study of 2,035 patients.

AimsHereditary haemochromatosis is a genetic disorder that is caused by several known mutations in the human homeostatic iron regulator protein (HFE) gene. Abnormal accumulation of iron causes a joint disease that resembles osteoarthritis (OA), but appears at a relatively younger age and is accompanied by cirrhosis, diabetes, and injury to other organs. Increased serum transferrin saturation and ferritin levels are known markers of haemochromatosis with high positive predictive values.MethodsWe have retrospectively analyzed the iron studies of a cohort of 2,035 patients undergoing knee joint arthroplasty due to OA.ResultsNo patients had HFE gene C282Y, S65C, or H63D mutations testing. In total, 18 patients (2.96%) of the male cohort and 51 (3.58%) of the female cohort had pathologically increased ferritin levels that may be indicative of haemochromatosis. Seven patients (0.34%) had serum transferrin saturation above 45%.ConclusionThe awareness for the diagnosis of this disorder in Orthopaedics is low and needs improvement. Osteoarthritic patients undergoing knee arthroplasty should be routinely screened for haemochromatosis by iron studies and referred to genetic testing when needed.Level of evidence: Level III - Retrospective cohort study.Cite this article: Bone Jt Open 2021;2(12):1062–1066.

HFE variants in colorectal cancer and their clinicopathological correlations

The study aimed to screen mutation of human homeostatic iron regulator (HFE) in colorectal carcinoma (CRC) and detect their associations with clinicopathological parameters. Expression of HFE was determined by quantitative polymerase chain reaction in matched CRC and non neoplastic colorectal mucosal tissue of 76 patients. Genomic DNA extracted were subjected to high high-resolution melt curve analysis and Sanger sequencing to detect mutations in HFE. The associations of the identified mutations with a variety of clinical features were determined. Approximately 60% of CRC showed low HFE expression. Of the ten 10 mutations identified in exons 2 and 4, c.187C>G (H63D), c845G>A (C282Y), c.193A>T (S65C), g.3828T>C, g.5795T>C, and g.5728G>A were known mutations. Four novel mutations were discovered; : c.184G>A, c.220T>G, c.322A>C, and c.324T>C. Heterozygous H63D and C282Y mutations were seen in 71% and 49% of cancer tissue, respectively. Tumour site (p=0.048) and gender (p=0.039) were significantly associated with H63D and C282Y mutation status, respectively. Local spread of cancer was significantly associated with C282Y mutations in CRC cancer and adjacent non-neoplastic tissue (p=0.029 & and p=0.004, respectively). There was a statistically significant association between H63D and C282Y negativity in matched non-neoplastic colorectal mucosa tissue and pathological staging of cancer (p=0.047 & and p=0.001, respectively). Patients with H63D and C282Y mutations in cancer tissue tend to have higher survival rates. Hence HFE mutations are common in CRC and are associated with clinicopathological parameters, implying the potential clinical significance of HFE mutations in colorectal carcinogenesis.

Appropriate Clinical Genetic Testing of Hemochromatosis Type 2-4, Including Ferroportin Disease.

Hereditary hemochromatosis (HH) is an inherited iron overload disorder due to a deficiency of hepcidin, or a failure of hepcidin to degrade ferroportin. The most common form of HH, Type 1 HH, is most commonly due to a homozygous C282Y mutation in HFE and is relatively well understood in significance and action; however, other rare forms of HH (Types 2–4) exist and are more difficult to identify and diagnose in clinical practice. In this review, we describe the clinical characteristics of HH Type 2–4 and the mutation patterns that have been described in these conditions. We also review the different methods for genetic testing available in clinical practice and a pragmatic approach to the patient with suspected non-HFE HH.

Factors associated with serum ferritin levels and iron excess: results from the EPIC-EurGast study.

Excess iron is involved in the development of non-communicable diseases such as cancer, type 2 diabetes and cardiovascular conditions. We aimed to describe the prevalence of excess iron and its determinants in healthy European adults. Sociodemographic, lifestyle, iron status, dietary information, and HFE genotyping were obtained from controls from the nested case-control study EPIC-EurGast study. High sensitivity C-reactive protein (hsCRP) was measured to address possible systemic inflammation. Descriptive and multivariate analyses were used to assess iron status and its determinants. Out of the 828 participants (median age: 58.7years), 43% were females. Median serum ferritin and prevalence of excess iron were 143.7µg/L and 35.2% in males, respectively, and 77µg/L and 20% in females, both increasing with latitude across Europe. Prevalence of HFE C282Y mutation was significantly higher in Northern and Central Europe (~ 11%) than in the South (5%). Overweight/obesity, age, and daily alcohol and heme iron intake were independent determinants for iron status, with sex differences even after excluding participants with hsCRP > 5mg/L. Obese males showed a greater consumption of alcohol, total and red meat, and heme iron, compared with those normal weight. Obesity, higher alcohol and heme iron consumption were the main risk factors for excess iron in males while only age was associated with iron overload in females. Weight control and promoting healthy lifestyle may help prevent iron overload, especially in obese people. Further research is needed to clarify determinants of excess iron in the healthy adult population, helping to reduce the associated comorbidities.