Heme Biosynthetic Gene Expression Analysis With dPCR in Erythropoietic Protoporphyria Patients

Background: The heme biosynthesis (HB) involves eight subsequent enzymatic steps. Erythropoietic protoporphyria (EPP) is caused by loss-of-function mutations in the ferrochelatase (FECH) gene, which in the last HB step inserts ferrous iron into protoporphyrin IX (PPIX) to form heme.Aim and method: The aim of this work was to for the first time analyze the mRNA expression of all HB genes in peripheral blood samples of patients with EPP having the same genotype FECH c.[215dupT]; [315-48T > C] as compared to healthy controls by highly sensitive and specific digital PCR assays (dPCR).Results: We confirmed a decreased FECH mRNA expression in patients with EPP. Further, we found increased ALAS2 and decreased ALAS1, CPOX, PPOX and HMBS mRNA expression in patients with EPP compared to healthy controls. ALAS2 correlated with FECH mRNA expression (EPP: r = 0.63, p = 0.03 and controls: r = 0.68, p = 0.02) and blood parameters like PPIX (EPP: r = 0.58 p = 0.06).Conclusion: Our method is the first that accurately quantifies HB mRNA from blood samples with potential applications in the monitoring of treatment effects of mRNA modifying therapies in vivo, or investigation of the HB pathway and its regulation. However, our findings should be studied in separated blood cell fractions and on the enzymatic level

Clinical and molecular epidemiology of erythropoietic protoporphyria in Italy

Background: Erythropoietic protoporphyria (EPP) is a rare inherited disease associated with heme metabolism, characterized by severe life-long photosensitivity and liver involvement. Objectives: To provide epidemiological data of EPP in Italy. Materials and Methods: Prospective/retrospective data of EPP patients were collected by an Italian network of porphyria specialist centres (Gruppo Italiano Porfiria, GrIP) over a 20-year period (1996-2017). Results: In total, 179 patients (79 females) with a clinical and biochemical diagnosis of EPP were assessed, revealing a prevalence of 3.15 cases per million persons and an incidence of 0.13 cases per million persons/year. Incidence significantly increased after 2009 (due to the availability of alfa-melanotide, which effectively limits skin photosensitivity). Mean age at diagnosis was 28 years, with only 22 patients (12.2%) diagnosed 6410 years old. Gene mutations were assessed in 173 (96.6%) patients; most (164; 91.3%) were FECH mutations on one allele in association with the hypomorphic variant, c.315-48C, on the other (classic EPP), and nine (5.2%) were ALAS2 mutations (X-linked EPP). Only one case of autosomal recessive EPP was observed. Of the 42 different FECH mutations, 15 are novel, three mutations collectively accounted for 45.9% (75/164) of the mutations (c.215dupT [27.2%], c.901_902delTG [11.5%] and c.67\u2009+\u20095G\u2009>\u2009A [7.2%]), and frameshift mutations were prevalent (33.3%). A form of light protection was used by 109/179 (60.8%) patients, and 100 (56%) had at least one \u3b1-melanotide implant. Three cases of severe acute liver involvement, requiring OLT, were observed. Conclusions: These data define, for the first time, the clinical and molecular epidemiology of EPP in Italy

Associated Effect of SLC40A1 and TMPRSS6 Polymorphisms on Iron Overload

Mutations in the ferroportin (FPN) gene SLC40A1 alter iron recycling and cause disturbances in iron homeostasis. The variants of TMPRSS6 contribute to the development of iron deficiencies. In this study, we determined the role of FPN and TMPRSS6 gene polymorphisms in the modulation of iron homeostasis based on biochemical parameters. PCR analysis and sequencing were performed to determine the single nucleotide polymorphisms (SNPs) SLC40A1 c.44–24G>C (rs1439816), SLC40A1 c.663T>C (rs2304704), and TMPRSS6 c.2207T>C (rs855791). Hemoglobin concentration and iron status were determined by standard procedures. We studied 79 iron-loaded individuals for SLC40A1 polymorphisms. Interestingly, 35/79 individuals with SLC40A1 SNPs also carried a TMPRSS6 c.2207T>C polymorphism. The biochemical values of the iron overloaded individuals were compared to those of the individuals carrying TMPRSS6 SNPs and the healthy individuals (wild-type group). The ferritin concentration, transferrin saturation % (TS%), and hemoglobin concentration were significantly higher in the participants with FPN SNPs than in the other three groups. The ferritin concentration and TS% were higher in participants with both SLC40A1 and TMPRSS6 SNPs than in the TMPRSS6 and wild-type groups, while hemoglobin concentration was significantly higher than that in the TMPRSS6 SNP group only. The participants with TMPRSS6 SNPs had significantly lower ferritin concentration, TS%, and hemoglobin concentration than all the other groups. SLC40A1 and TMPRSS6 SNPs might act in the opposite direction, preventing the development of severe iron overload, and the modulation of the iron status by TMPRSS6 SNPs might provide protection

Congenital erythropoietic porphyria linked to GATA1-R216W mutation: challenges for diagnosis

Congenital erythropoietic porphyria (CEP) is a rare genetic disease that is characterized by a severe cutaneous photosensitivity causing unrecoverable deformities, chronic hemolytic anemia requiring blood transfusion program, and by fatal systemic complications. A correct and early diagnosis is required to develop a management plan that is appropriate to the patient's needs. Recently only one case of X-linked CEP had been reported, describing the trans-acting GATA1-R216W mutation. Here, we have characterized two novel X-linked CEP patients, both with misleading hematological phenotypes that include dyserythropoietic anemia, thrombocytopenia, and hereditary persistence of fetal hemoglobin. We compare the previously reported case to ours and propose a diagnostic paradigm for this variant of CEP. Finally, a correlation between phenotype variability and the presence of modifier mutations in loci related to disease-causing gene is described

Non-contrast CT markers of intracerebral hemorrhage expansion: The influence of onset-to-CT time

Background: Hematoma expansion (HE) is an appealing therapeutic target in intracerebral hemorrhage (ICH) and non-contrast computed tomography (NCCT) features are promising predictors of HE. Aims: We investigated whether onset-to-CT time influences the diagnostic performance of NCCT markers for HE. Methods: Retrospective multicentre analysis of patients with primary ICH. The following NCCT markers were analyzed: hypodensities, heterogeneous density, blend sign, and irregular shape. HE was defined as growth > 6 mL and/or > 33%. We calculated the sensitivity, specificity, positive, and negative predictive values (PPVs and NPVs) of NCCT markers for HE, stratified by onset-to-CT time (<2 h, 2-4 h, 4-6 h, >6 h). Results: We included 1135 patients (median age 69, 53% males), of whom 307 (27%) experienced HE. Overall hypodensities had the highest sensitivity (0.68) and blend sign the highest specificity (0.87) for HE. Hypodensities were more common and had higher sensitivity (0.80) in patients with imaging within 2 h. The same result was observed for heterogeneous density, whereas irregular shape had a similar prevalence across time strata and higher sensitivity (0.79) beyond 6 h from onset. The frequency of blend sign increased with longer onset-to-CT time, whereas its specificity declined after 6 h from onset. Conclusion: The diagnostic performance of NCCT markers is influenced by imaging time. Hypodensities identified four out of five patients with HE within 2 h from onset, whereas irregular shape performed better in late presenters. Our findings may improve the use of NCCT markers in future studies and trials targeting HE

Risk factors for intracerebral hemorrhage in patients with atrial fibrillation on NOACs for stroke prevention

Background and Purpose: Clinical trials on stroke prevention in patients with atrial fibrillation have consistently shown clinical benefit from either warfarin or non–vitamin K antagonist oral anticoagulants (NOACs). NOAC-treated patients have consistently reported to be at lower risk for intracerebral hemorrhage (ICH) than warfarin-treated patients. The aims of this prospective, multicenter, multinational, unmatched, case-control study were (1) to investigate for risk factors that could predict ICH occurring in patients with atrial fibrillation during NOAC treatment and (2) to evaluate the role of CHA2DS2-VASc and HAS-BLED scores in the same setting. Methods: Cases were consecutive patients with atrial fibrillation who had ICH during NOAC treatment. Controls were consecutive patients with atrial fibrillation who did not have ICH during NOAC treatment. As within the CHA2DS2-VASc and HAS-BLED scores there are some risk factors in common, several multivariable logistic regression models were performed to identify independent prespecified predictors for ICH events. Results: Four hundred nineteen cases (mean age, 78.8±8.1 years) and 1526 controls (mean age, 76.0±10.3 years) were included in the study. From the different models performed, independent predictors of ICH were increasing age, concomitant use of antiplatelet agents, active malignancy, high risk of fall, hyperlipidemia, low clearance of creatinine, peripheral artery disease, and white matter changes. Low doses of NOACs (given according to label or not) and congestive heart failure were inversely associated with the risk of ICH. HAS-BLED and CHA2DS2-VASc scores performed poorly in predicting ICH with areas under the curves of 0.496 (95% CI, 0.468-0.525) and 0.530 (95% CI, 0.500-0.560), respectively. Conclusions: Several risk factors were associated to ICH in patients treated with NOACs for stroke prevention but not HAS-BLED and CHA2DS2-VASc scores