Disturbed iron metabolism in erythropoietic protoporphyria and association of GDF15 and gender with disease severity.

Patients with erythropoietic protoporphyria (EPP) have reduced activity of the enzyme ferrochelatase that catalyzes the insertion of iron into protoporphyrin IX (PPIX) to form heme. As the result of ferrochelatase deficiency, PPIX accumulates and causes severe photosensitivity. Among different patients, the concentration of PPIX varies considerably. In addition to photosensitivity, patients frequently exhibit low serum iron and a microcytic hypochromic anemia. The aims of this study were to (1) search for factors related to PPIX concentration in EPP, and (2) characterize anemia in EPP, i.e., whether it is the result of an absolute iron deficiency or the anemia of chronic disease (ACD). Blood samples from 67 EPP patients (51 Italian and 16 Swiss) and 21 healthy volunteers were analyzed. EPP patients had lower ferritin (p = 0.021) and hepcidin (p = 0.031) concentrations and higher zinc-protoporphyrin (p < 0.0001) and soluble-transferrin-receptor (p = 0.0007) concentrations compared with controls. This indicated that anemia in EPP resulted from an absolute iron deficiency. Among EPP patients, PPIX concentrations correlated with both growth differentiation factor (GDF) 15 (p = 0.012) and male gender (p = 0.015). Among a subgroup of patients who were iron replete, hemoglobin levels were normal, which suggested that iron but not ferrochelatase is the limiting factor in heme synthesis of individuals with EPP.

Hereditary hemochromatosis type 1 phenotype modifiers in Italian patients. The controversial role of variants in HAMP, BMP2, FTL and SLC40A1 genes.

Hereditary hemochromatosis (HH) is a heterogeneous disorder of iron metabolism. The most common form of the disease is Classic or type 1 HH, mainly caused by a biallelic missense p.Cys282Tyr (c.845G>A) mutation in the HFE gene. However, the penetrance of p.Cys282Tyr/p.Cys282Tyr genotype is incomplete in terms of both biochemical and clinical expressivity. Lack of penetrance is thought to be caused by several genetic and environmental factors. Recently, a lot of evidences on HH genetic modifiers were produced, often without conclusive results. We investigated 6 polymorphisms (rs10421768 in HAMP gene, rs235756 in BMP2 gene, rs2230267 in FTL gene, rs1439816 in SLC40A1 gene, rs41295942 in TFR2 gene and rs2111833 in TMPRSS6 gene) with uncertain function in order to further evaluate their role in an independent cohort of 109 HH type 1 patients. Our results make it likely the role of rs10421768, rs235756, rs2230267 and rs1439816 polymorphisms, respectively in HAMP, BMP2, FTL and SLC40A1 genes in HH expressivity. In addition, previous and our findings support a hypothetical multifactorial model of HH, characterized by a principal gene (HFE in HH type 1) and minor genetic and environmental factors that still have to be fully elucidated.

Evaluation of Hedgehog Pathway Inhibition on Nevoid Basal Cell Carcinoma Syndrome Fibroblasts and Basal Cell Carcinoma-Associated Fibroblasts: Are Vismodegib and Sonidegib Useful to Target Cancer-Prone Fibroblasts?

Activating mutations in the Hh pathway underlies the development of sporadic and familial skin BCC. For these oncogenic proliferations displaying ligand-independent activation of the intracellular pathway, two molecules have been approved for therapeutic purposes: vismodegib and sonidegib. Improper Hh signalling occurs in many human tumours also via a paracrine mechanism (ligand-dependent) in which the secretion of Hh ligands by stromal cells support tumour growth. On the other hand, the mobilization of neoplastic stroma by cancer cells is sustained by the activation of Hh signalling in surrounding fibroblasts suggesting a central role of this bidirectional crosstalk in carcinogenesis. Additionally, loss-of-function mutations in the PTCH1 gene in the context of NBCCS, an autosomal dominant disorder predisposing to multiple BCCs, determine tumour permissive phenotypes in dermal fibroblasts. Here, profiling syndromic and BCC-associated fibroblasts unveiled an extraordinary similarity characterized by overexpression of several Hh target genes and a marked pro-inflammatory outline. Both cell types exposed to Hh inhibitors displayed reversion of the tumour-prone phenotype. Under vismodegib and sonidegib treatment, the Wnt/ß-catenin pathway, frequently over-active in tumour stroma, resulted down-regulated by pAKT-GSK3ß axis and consequent increase of ß-catenin turnover. Overall, this study demonstrated that vismodegib and sonidegib impacting on fibroblast tumour supportive functions might be considered in therapy for BCC independently to the mutation status of Hh components in neoplastic cells.

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. OBJECTIVE: To provide epidemiological data of EPP in Italy. MATERIALS & 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 ≤10 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 + 5G > A [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 α-melanotide implant. Three cases of severe acute liver involvement, requiring OLT, were observed. CONCLUSION: These data define, for the first time, the clinical and molecular epidemiology of EPP in Italy.

Novel mutation of PPOX gene in a patient with abdominal pain and syndrome of inappropriate antidiuresis.

PURPOSE: Acute porphyrias are metabolic disorders of heme biosynthesis characterized by acute life-threatening attacks. The diagnosis is often missed since clinical presentation is aspecific mimicking other medical and surgical conditions. Variegate porphyria (VP) is an autosomal dominant inherited disease with incomplete penetrance due to decreased activity of the Protoporphyrinogen Oxydase (PPOX) gene; most VP mutations are family specific. We report the case of a 40 year-old woman who presented many times to the emergency department complaining of unexplained abdominal pain and laboratory investigations showed repeatedly hyponatremia. Syndrome of inappropriate antidiuresis (SIAD) was confirmed and measurement of urine porphobilinogen and delta-aminolevulinic acid disclosed the diagnosis of acute porphyria. The genetic analysis of PPOX gene was performed. METHODS: The entire coding sequence and exon/intron boundaries of PPOX gene were amplified in 5 different Polymerase Chain Reaction (PCR) fragments. In silico prediction of the pathogenicity of the mutation was determined by using different tools, Polyphen2, SNPs&GO, SNPs3D. RESULTS: The genetic analysis of PPOX gene revealed a novel missense variant c.1376 G > A (p.Cys459Tyr) in heterozygous state. The same variant was later found in one of her cousins with skin lesions and other three younger asymptomatic relatives. We provided evidence that this novel mutation is likely to be pathogenetic. CONCLUSIONS: Our case highlights the importance of considering VP in the differential diagnosis of SIAD and underlines the role of genetic screening in the management of such patients. The finding of a novel mutation of PPOX gene in our index case has allowed to recognize an affected family.

Phototherapy for vitiligo, what's new?

Vitiligo is a disorder characterized by the development of depigmented macules and patches. Existing treatments include topical and systemic immunosuppressants, topical vitamin D analogues in monotherapy or in association with phototherapy, phototherapy and surgical techniques, which together may serve to halt disease progression, stabilize depigmented lesions, and encourage repigmentation. Narrow-band UVB (NB-UVB 310-315 nm) radiation is now considered as the "gold standard" for the treatment of diffuse vitiligo. This article provides a brief overview of the different phototherapy based treatments in vitiligo.

Twelve novel HGD gene variants identified in 99 alkaptonuria patients: focus on 'black bone disease' in Italy.

Alkaptonuria (AKU) is an autosomal recessive disorder caused by mutations in homogentisate-1,2-dioxygenase (HGD) gene leading to the deficiency of HGD enzyme activity. The DevelopAKUre project is underway to test nitisinone as a specific treatment to counteract this derangement of the phenylalanine-tyrosine catabolic pathway. We analysed DNA of 40 AKU patients enrolled for SONIA1, the first study in DevelopAKUre, and of 59 other AKU patients sent to our laboratory for molecular diagnostics. We identified 12 novel DNA variants: one was identified in patients from Brazil (c.557T>A), Slovakia (c.500C>T) and France (c.440T>C), three in patients from India (c.469+6T>C, c.650-85A>G, c.158G>A), and six in patients from Italy (c.742A>G, c.614G>A, c.1057A>C, c.752G>A, c.119A>C, c.926G>T). Thus, the total number of potential AKU-causing variants found in 380 patients reported in the HGD mutation database is now 129. Using mCSM and DUET, computational approaches based on the protein 3D structure, the novel missense variants are predicted to affect the activity of the enzyme by three mechanisms: decrease of stability of individual protomers, disruption of protomer-protomer interactions or modification of residues in the region of the active site. We also present an overview of AKU in Italy, where so far about 60 AKU cases are known and DNA analysis has been reported for 34 of them. In this rather small group, 26 different HGD variants affecting function were described, indicating rather high heterogeneity. Twelve of these variants seem to be specific for Italy.

Heterogeneity of mutations in the ferrochelatase gene in Italian patients with erythropoietic protoporphyria.

Mutations and a low-expressed allele IVS3-48c (in trans to the mutation) of the ferrochelatase (FECH) gene are responsible for erythropoietic protoporphyria (EPP) which is characterized clinically by cutaneous photosensitivity. In this study of 15 Italian EPP families, we identified 10 different FECH gene mutations, six of them were novel mutations (Q32X; IVS2-2, a-->g; IVS3-67, g-->a; 488-501del 14bp; IVS5-3, c-->t and 757-761delAGAAG). Four were known mutations (213insT; R115X; S264L and 899-900delTG). Real-time PCR quantification of FECH mRNA from a patient carrying mutation IVS-67, g-->a revealed a 51% reduction in the normal FECH transcript. This mutation generated an abnormal mRNA species with a 63-bp intron 3 retention--the same mRNA species which the low-expressed FECH allele IVS3-48c also produces. In addition, a double intronic deletion (IVS8-61 del ag /IVS9-58 del a) was found in some patients as well as their healthy relatives of this cohort. Sequencing of 50 Italian control subjects revealed an 11% prevalence of the double deletion. However, the double intronic deletion is not a disease-causing mutation as evidenced by a normal amount of FECH mRNA measured from a non-porphyric individual who carried the double deletion. Another interesting finding of this study was a 1% prevalence of IVS3-48c allele in the white Italian population, comparing to 11% in the French population. Further epidemiological studies are needed to test whether the 1% frequency of the low-expressed IVS3-48c allele is associated with a low prevalence of EPP in the white Italian population.

Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase.

Glutamate decarboxylase is a vitamin B6-dependent enzyme, which catalyses the decarboxylation of glutamate to gamma-aminobutyrate. In Escherichia coli, expression of glutamate decarboxylase (GadB), a 330 kDa hexamer, is induced to maintain the physiological pH under acidic conditions, like those of the passage through the stomach en route to the intestine. GadB, together with the antiporter GadC, constitutes the gad acid resistance system, which confers the ability for bacterial survival for at least 2 h in a strongly acidic environment. GadB undergoes a pH-dependent conformational change and exhibits an activity optimum at low pH. We determined the crystal structures of GadB at acidic and neutral pH. They reveal the molecular details of the conformational change and the structural basis for the acidic pH optimum. We demonstrate that the enzyme is localized exclusively in the cytoplasm at neutral pH, but is recruited to the membrane when the pH falls. We show by structure-based site-directed mutagenesis that the triple helix bundle formed by the N-termini of the protein at acidic pH is the major determinant for this behaviour.