The mitochondrial aspartate/glutamate carrier does not transport GABA.

É£-aminobutyric acid (GABA) is a four­carbon amino acid acting as the main inhibitory transmitter in the invertebrate and vertebrate nervous systems. The metabolism of GABA is well compartmentalized in the cell and the uptake of cytosolic GABA into the mitochondrial matrix is required for its degradation. A previous study carried out in the fruit fly Drosophila melanogaster indicated that the mitochondrial aspartate/glutamate carrier (AGC) is responsible for mitochondrial GABA accumulation. Here, we investigated the transport of GABA catalysed by the human and D. melanogaster AGC proteins through a well-established method for the study of the substrate specificity and the kinetic parameters of the mitochondrial carriers. In this experimental system, the D. melanogaster spliced AGC isoforms (Aralar1-PA and Aralar1-PE) and the human AGC isoforms (AGC1/aralar1 and AGC2/citrin) are unable to transport GABA both in homo- and in hetero-exchange with either glutamate or aspartate, i.e. the canonical substrates of AGC. Moreover, GABA has no inhibitory effect on the exchange activities catalysed by the investigated AGCs. Our data demonstrate that AGC does not transport GABA and the molecular identity of the GABA transporter in human and D. melanogaster mitochondria remains unknown.

Transcriptional and metabolic effects of aspartate-glutamate carrier isoform 1 (AGC1) downregulation in mouse oligodendrocyte precursor cells (OPCs).

Aspartate-glutamate carrier isoform 1 (AGC1) is a carrier responsible for the export of mitochondrial aspartate in exchange for cytosolic glutamate and is part of the malate-aspartate shuttle, essential for the balance of reducing equivalents in the cells. In the brain, mutations in SLC25A12 gene, encoding for AGC1, cause an ultra-rare genetic disease, reported as a neurodevelopmental encephalopathy, whose symptoms include global hypomyelination, arrested psychomotor development, hypotonia and seizures. Among the biological components most affected by AGC1 deficiency are oligodendrocytes, glial cells responsible for myelination processes, and their precursors [oligodendrocyte progenitor cells (OPCs)]. The AGC1 silencing in an in vitro model of OPCs was documented to cause defects of proliferation and differentiation, mediated by alterations of histone acetylation/deacetylation. Disrupting AGC1 activity could possibly reduce the availability of acetyl groups, leading to perturbation of many biological pathways, such as histone modifications and fatty acids formation for myelin production. Here, we explore the transcriptome of mouse OPCs partially silenced for AGC1, reporting results of canonical analyses (differential expression) and pathway enrichment analyses, which highlight a disruption in fatty acids synthesis from both a regulatory and enzymatic stand. We further investigate the cellular effects of AGC1 deficiency through the identification of most affected transcriptional networks and altered alternative splicing. Transcriptional data were integrated with differential metabolite abundance analysis, showing downregulation of several amino acids, including glutamine and aspartate. Taken together, our results provide a molecular foundation for the effects of AGC1 deficiency in OPCs, highlighting the molecular mechanisms affected and providing a list of actionable targets to mitigate the effects of this pathology.

Human mitochondrial uncoupling protein 3 functions as a metabolite transporter.

Since its discovery, a major debate about mitochondrial uncoupling protein 3 (UCP3) has been whether its metabolic actions result primarily from mitochondrial inner membrane proton transport, a process that decreases respiratory efficiency and ATP synthesis. However, UCP3 expression and activity are induced by conditions that would seem at odds with inefficient 'uncoupled' respiration, including fasting and exercise. Here, we demonstrate that the bacterially expressed human UCP3, reconstituted into liposomes, catalyses a strict exchange of aspartate, malate, sulphate and phosphate. The R282Q mutation abolishes the transport activity of the protein. Although the substrate specificity and inhibitor sensitivity of UCP3 display similarity with that of its close homolog UCP2, the two proteins significantly differ in their transport mode and kinetic constants.

Targeted quantitative metabolic profiling of brain-derived cell cultures by semi-automated MEPS and LC-MS/MS.

The accurate characterisation of metabolic profiles is an important prerequisite to determine the rate and the efficiency of the metabolic pathways taking place in the cells. Changes in the balance of metabolites involved in vital processes such as glycolysis, tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), as well as in the biochemical pathways related to amino acids, lipids, nucleotides, and their precursors reflect the physiological condition of the cells and may contribute to the development of various human diseases. The feasible and reliable measurement of a wide array of metabolites and biomarkers possesses great potential to elucidate physiological and pathological mechanisms, aid preclinical drug development and highlight potential therapeutic targets. An effective, straightforward, sensitive, and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach was developed for the simultaneous quali-quantitative analysis of 41 compounds in both cell pellet and cell growth medium obtained from brain-derived cell cultures. Sample pretreatment miniaturisation was achieved thanks to the development and optimisation of an original extraction/purification approach based on digitally programmed microextraction by packed sorbent (eVol®-MEPS). MEPS allows satisfactory and reproducible clean-up and preconcentration of both low-volume homogenate cell pellet lysate and cell growth medium with advantages including, but not limited to, minimal sample handling and method sustainability in terms of sample, solvents, and energy consumption. The MEPS-LC-MS/MS method showed good sensitivity, selectivity, linearity, and precision. As a proof of concept, the developed method was successfully applied to the analysis of both cell pellet and cell growth medium obtained from a line of mouse immortalised oligodendrocyte precursor cells (OPCs; Oli-neu cell line), leading to the unambiguous determination of all the considered target analytes. This method is thus expected to be suitable for targeted, quantitative metabolic profiling in most brain cell models, thus allowing accurate investigations on the biochemical pathways that can be altered in central nervous system (CNS) neuropathologies, including e.g., mitochondrial respiration and glycolysis, or use of specific nutrients for growth and proliferation, or lipid, amino acid and nucleotide metabolism.

Genetic inactivation of the Carnitine/Acetyl-Carnitine mitochondrial carrier of Yarrowia lipolytica leads to enhanced odd-chain fatty acid production.

BACKGROUND: Mitochondrial carriers (MCs) can deeply affect the intracellular flux distribution of metabolic pathways. The manipulation of their expression level, to redirect the flux toward the production of a molecule of interest, is an attractive target for the metabolic engineering of eukaryotic microorganisms. The non-conventional yeast Yarrowia lipolytica is able to use a wide range of substrates. As oleaginous yeast, it directs most of the acetyl-CoA therefrom generated towards the synthesis of lipids, which occurs in the cytoplasm. Among them, the odd-chain fatty acids (OCFAs) are promising microbial-based compounds with several applications in the medical, cosmetic, chemical and agricultural industries. RESULTS: In this study, we have identified the MC involved in the Carnitine/Acetyl-Carnitine shuttle in Y. lipolytica, YlCrc1. The Y. lipolytica Ylcrc1 knock-out strain failed to grow on ethanol, acetate and oleic acid, demonstrating the fundamental role of this MC in the transport of acetyl-CoA from peroxisomes and cytoplasm into mitochondria. A metabolic engineering strategy involving the deletion of YlCRC1, and the recombinant expression of propionyl-CoA transferase from Ralstonia eutropha (RePCT), improved propionate utilization and its conversion into OCFAs. These genetic modifications and a lipogenic medium supplemented with glucose and propionate as the sole carbon sources, led to enhanced accumulation of OCFAs in Y. lipolytica. CONCLUSIONS: The Carnitine/Acetyl-Carnitine shuttle of Y. lipolytica involving YlCrc1, is the sole pathway for transporting peroxisomal or cytosolic acetyl-CoA to mitochondria. Manipulation of this carrier can be a promising target for metabolic engineering approaches involving cytosolic acetyl-CoA, as demonstrated by the effect of YlCRC1 deletion on OCFAs synthesis.

In Vitro Effects of Low-energy Ultrasound Treatment on Healthy CD3/CD8+ Lymphocytes, Red blood cells, Acute Myeloid leukemia cells, and Jurkat cell line.

The study of the biological effects of low-energy ultrasound and its applications is a rapidly expanding research area. Low-energy ultrasound could be used as anti-tumoral therapy with or without the pharmacological combination even if the second situation has been scarcely investigated up to now. Very little information is available about the ultrasound effects on healthy red blood cells, CD3, and mainly CD8 subset lymphocytes which is the main subset cell having cytotoxic function towards cancer cells. In this study, we investigated in vitro the bioeffects of low energy ultrasound on red blood cells and PBMCs isolated from healthy donors as well as on two myeloid leukemia cell lines (OCI- AML-3 MOLM-13) and lymphoblastic Jurkat cell line. Using low-energy ultrasound (US), a study was conducted to determine how it affects CD3/CD8 lymphocytes and leukemia cells, as well as its potential role in treating blood cancers, by analyzing changes in mitochondrial membrane potential, phosphatidylserine asymmetry, morphological changes for myeloid AML cell lines, proliferation and cytotoxic activation of healthy lymphocytes, and apoptosis for RBCs after US exposure. Overall, we demonstrated that CD3/CD8 lymphocytes proliferation/activation and cytotoxic functions are fully preserved after ultrasound treatments, whereas leukemia cell lines undergo apoptosis and stop proliferating suggesting a potential method of treating blood cancer.

Histone Acetylation Defects in Brain Precursor Cells: A Potential Pathogenic Mechanism Causing Proliferation and Differentiation Dysfunctions in Mitochondrial Aspartate-Glutamate Carrier Isoform 1 Deficiency.

Mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) deficiency is an ultra-rare genetic disease characterized by global hypomyelination and brain atrophy, caused by mutations in the SLC25A12 gene leading to a reduction in AGC1 activity. In both neuronal precursor cells and oligodendrocytes precursor cells (NPCs and OPCs), the AGC1 determines reduced proliferation with an accelerated differentiation of OPCs, both associated with gene expression dysregulation. Epigenetic regulation of gene expression through histone acetylation plays a crucial role in the proliferation/differentiation of both NPCs and OPCs and is modulated by mitochondrial metabolism. In AGC1 deficiency models, both OPCs and NPCs show an altered expression of transcription factors involved in the proliferation/differentiation of brain precursor cells (BPCs) as well as a reduction in histone acetylation with a parallel alteration in the expression and activity of histone acetyltransferases (HATs) and histone deacetylases (HDACs). In this study, histone acetylation dysfunctions have been dissected in in vitro models of AGC1 deficiency OPCs (Oli-Neu cells) and NPCs (neurospheres), in physiological conditions and following pharmacological treatments. The inhibition of HATs by curcumin arrests the proliferation of OPCs leading to their differentiation, while the inhibition of HDACs by suberanilohydroxamic acid (SAHA) has only a limited effect on proliferation, but it significantly stimulates the differentiation of OPCs. In NPCs, both treatments determine an alteration in the commitment toward glial cells. These data contribute to clarifying the molecular and epigenetic mechanisms regulating the proliferation/differentiation of OPCs and NPCs. This will help to identify potential targets for new therapeutic approaches that are able to increase the OPCs pool and to sustain their differentiation toward oligodendrocytes and to myelination/remyelination processes in AGC1 deficiency, as well as in other white matter neuropathologies.

KRAS-regulated glutamine metabolism requires UCP2-mediated aspartate transport to support pancreatic cancer growth.

The oncogenic KRAS mutation has a critical role in the initiation of human pancreatic ductal adenocarcinoma (PDAC) since it rewires glutamine metabolism to increase reduced nicotinamide adenine dinucleotide phosphate (NADPH) production, balancing cellular redox homeostasis with macromolecular synthesis1,2. Mitochondrial glutamine-derived aspartate must be transported into the cytosol to generate metabolic precursors for NADPH production2. The mitochondrial transporter responsible for this aspartate efflux has remained elusive. Here, we show that mitochondrial uncoupling protein 2 (UCP2) catalyses this transport and promotes tumour growth. UCP2-silenced KRASmut cell lines display decreased glutaminolysis, lower NADPH/NADP+ and glutathione/glutathione disulfide ratios and higher reactive oxygen species levels compared to wild-type counterparts. UCP2 silencing reduces glutaminolysis also in KRASWT PDAC cells but does not affect their redox homeostasis or proliferation rates. In vitro and in vivo, UCP2 silencing strongly suppresses KRASmut PDAC cell growth. Collectively, these results demonstrate that UCP2 plays a vital role in PDAC, since its aspartate transport activity connects the mitochondrial and cytosolic reactions necessary for KRASmut rewired glutamine metabolism2, and thus it should be considered a key metabolic target for the treatment of this refractory tumour.

Statins Reduce Intratumor Cholesterol Affecting Adrenocortical Cancer Growth.

Mitotane causes hypercholesterolemia in patients with adrenocortical carcinoma (ACC). We suppose that cholesterol increases within the tumor and can be used to activate proliferative pathways. In this study, we used statins to decrease intratumor cholesterol and investigated the effects on ACC growth related to estrogen receptor α (ERα) action at the nuclear and mitochondrial levels. We first used microarray to investigate mitotane effect on genes involved in cholesterol homeostasis and evaluated their relationship with patients' survival in ACC TCGA. We then blocked cholesterol synthesis with simvastatin and determined the effects on H295R cell proliferation, estradiol production, and ERα activity in vitro and in xenograft tumors. We found that mitotane increases intratumor cholesterol content and expression of genes involved in cholesterol homeostasis, among them INSIG, whose expression affects patients' survival. Treatment of H295R cells with simvastatin to block cholesterol synthesis decreased cellular cholesterol content, and this affected cell viability. Simvastatin reduced estradiol production and decreased nuclear and mitochondrial ERα function. A mitochondrial target of ERα, the respiratory complex IV (COXIV), was reduced after simvastatin treatment, which profoundly affected mitochondrial respiration activating apoptosis. Additionally, simvastatin reduced tumor volume and weight of grafted H295R cells, intratumor cholesterol content, Ki-67 and ERα, COXIV expression and activity and increase terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. Collectively, these data demonstrate that a reduction in intratumor cholesterol content prevents estradiol production and inhibits mitochondrial respiratory chain-inducing apoptosis in ACC cells. Inhibition of mitochondrial respiration by simvastatin represents a novel strategy to counteract ACC growth.

Deficiency of Mitochondrial Aspartate-Glutamate Carrier 1 Leads to Oligodendrocyte Precursor Cell Proliferation Defects Both In Vitro and In Vivo.

Aspartate-Glutamate Carrier 1 (AGC1) deficiency is a rare neurological disease caused by mutations in the solute carrier family 25, member 12 (SLC25A12) gene, encoding for the mitochondrial aspartate-glutamate carrier isoform 1 (AGC1), a component of the malate-aspartate NADH shuttle (MAS), expressed in excitable tissues only. AGC1 deficiency patients are children showing severe hypotonia, arrested psychomotor development, seizures and global hypomyelination. While the effect of AGC1 deficiency in neurons and neuronal function has been deeply studied, little is known about oligodendrocytes and their precursors, the brain cells involved in myelination. Here we studied the effect of AGC1 down-regulation on oligodendrocyte precursor cells (OPCs), using both in vitro and in vivo mouse disease models. In the cell model, we showed that a reduced expression of AGC1 induces a deficit of OPC proliferation leading to their spontaneous and precocious differentiation into oligodendrocytes. Interestingly, this effect seems to be related to a dysregulation in the expression of trophic factors and receptors involved in OPC proliferation/differentiation, such as Platelet-Derived Growth Factor α (PDGFα) and Transforming Growth Factor ßs (TGFßs). We also confirmed the OPC reduction in vivo in AGC1-deficent mice, as well as a proliferation deficit in neurospheres from the Subventricular Zone (SVZ) of these animals, thus indicating that AGC1 reduction could affect the proliferation of different brain precursor cells. These data clearly show that AGC1 impairment alters myelination not only by acting on N-acetyl-aspartate production in neurons but also on OPC proliferation and suggest new potential therapeutic targets for the treatment of AGC1 deficiency.

Increased Levels of cAMP by the Calcium-Dependent Activation of Soluble Adenylyl Cyclase in Parkin-Mutant Fibroblasts.

Almost half of autosomal recessive early-onset parkinsonism has been associated with mutations in PARK2, coding for parkin, which plays an important role in mitochondria function and calcium homeostasis. Cyclic adenosine monophosphate (cAMP) is a major second messenger regulating mitochondrial metabolism, and it is strictly interlocked with calcium homeostasis. Parkin-mutant (Pt) fibroblasts, exhibiting defective mitochondrial respiratory/OxPhos activity, showed a significant higher value of basal intracellular level of cAMP, as compared with normal fibroblasts (CTRL). Specific pharmacological inhibition/activation of members of the adenylyl cyclase- and of the phosphodiesterase-families, respectively, as well as quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis, indicate that the higher level of cAMP observed in Pt fibroblasts can contribute to a higher level of activity/expression by soluble adenylyl cyclase (sAC) and to low activity/expression of the phosphodiesterase isoform 4 (PDE4). As Ca2+ regulates sAC, we performed quantitative calcium-fluorimetric analysis, showing a higher level of Ca2+ in the both cytosol and mitochondria of Pt fibroblasts as compared with CTRL. Most notably, inhibition of the mitochondrial Ca2+ uniporter decreased, specifically the cAMP level in PD fibroblasts. All together, these findings support the occurrence of an altered mitochondrial Ca2+-mediated cAMP homeostasis in fibroblasts with the parkin mutation.

Functional screening of antibiotic resistance genes from a representative metagenomic library of food fermenting microbiota.

Lactic acid bacteria (LAB) represent the predominant microbiota in fermented foods. Foodborne LAB have received increasing attention as potential reservoir of antibiotic resistance (AR) determinants, which may be horizontally transferred to opportunistic pathogens. We have previously reported isolation of AR LAB from the raw ingredients of a fermented cheese, while AR genes could be detected in the final, marketed product only by PCR amplification, thus pointing at the need for more sensitive microbial isolation techniques. We turned therefore to construction of a metagenomic library containing microbial DNA extracted directly from the food matrix. To maximize yield and purity and to ensure that genomic complexity of the library was representative of the original bacterial population, we defined a suitable protocol for total DNA extraction from cheese which can also be applied to other lipid-rich foods. Functional library screening on different antibiotics allowed recovery of ampicillin and kanamycin resistant clones originating from Streptococcus salivarius subsp. thermophilus and Lactobacillus helveticus genomes. We report molecular characterization of the cloned inserts, which were fully sequenced and shown to confer AR phenotype to recipient bacteria. We also show that metagenomics can be applied to food microbiota to identify underrepresented species carrying specific genes of interest.

Molecular characterization of a novel mosaic tet(S/M) gene encoding tetracycline resistance in foodborne strains of Streptococcus bovis.

The presence of antibiotic-resistance (AR) genes in foodborne bacteria of enteric origin represents a relevant threat to human health in the case of opportunistic pathogens, which can reach the human gut through the food chain. Streptococcus bovis is a human opportunistic pathogen often associated with infections in immune-compromised or cancer patients, and it can also be detected in the environment, including fermented foods. We have focused on the molecular characterization of a tetracycline (Tet)-resistance gene present in 39 foodborne isolates of S. bovis phenotypically resistant to this drug. The gene was identified as a novel tet(S/M) fusion, encoding a mosaic protein composed of the N-terminal 33 amino acids of Tet(S), in-frame with the Tet(M) coding sequence. Heterologous expression of the mosaic gene was found to confer Tet resistance upon Escherichia coli recipients. Moreover, the tet(S/M) gene was found to be transcriptionally inducible by Tet under the endogenous tet(S) promoter in both S. bovis and E. coli. Nucleotide sequencing of the surrounding genomic region of 16.2 kb revealed large blocks of homology with the genomes of Streptococcus infantarius and Lactococcus lactis. A subregion of about 4 kb containing mosaic tet(S/M) was flanked by two copies of the IS1216 mobile element. PCR amplification with primers directed outwards from the tet(S/M) gene identified the presence of a 4.3 kb circular form corresponding to the intervening chromosomal region between the two IS1216 elements, but lacking a replication origin. The circular element shared extensive overall homology with a region of the multidrug-resistance plasmid pK214 from Lc. lactis, containing tet(S), as well as the IS1216 transposase-containing element and intervening non-coding sequences. Linear reconstruction of the insertion events likely to have occurred within this genomic region, inferred from sequence homology, provides further evidence of the chromosomal rearrangements that drive genomic evolution in complex bacterial communities such as the gut and food microbiota.

Antibiotic resistance determinants in the interplay between food and gut microbiota.

A complex and heterogeneous microflora performs sugar and lactic acid fermentations in food products. Depending on the fermentable food matrix (dairy, meat, vegetable etc.) as well as on the species composition of the microbiota, specific combinations of molecules are produced that confer unique flavor, texture, and taste to each product. Bacterial populations within such "fermented food microbiota" are often of environmental origin, they persist alive in foods ready for consumption, eventually reaching the gastro-intestinal tract where they can interact with the resident gut microbiota of the host. Although this interaction is mostly of transient nature, it can greatly contribute to human health, as several species within the food microbiota also display probiotic properties. Such an interplay between food and gut microbiota underlines the importance of the microbiological quality of fermented foods, as the crowded environment of the gut is also an ideal site for genetic exchanges among bacteria. Selection and spreading of antibiotic resistance genes in foodborne bacteria has gained increasing interest in the past decade, especially in light of the potential transferability of antibiotic resistance determinants to opportunistic pathogens, natural inhabitants of the human gut but capable of acquiring virulence in immunocompromised individuals. This review aims at describing major findings and future prospects in the field, especially after the use of antibiotics as growth promoters was totally banned in Europe, with special emphasis on the application of genomic technologies to improve quality and safety of fermented foods.

Characterization of the Tn916 conjugative transposon in a food-borne strain of Lactobacillus paracasei.

Food-borne antibiotic-resistant lactic acid bacteria have received growing attention in the past few years. We have recently identified tetracycline-resistant Lactobacillus paracasei in samples of milk and natural whey starter cultures employed in the manufacturing process of a typical Italian fermented dairy product, Mozzarella di Bufala Campana. In the present study, we have characterized at the molecular level the genetic context of tetracycline resistance determinants in these natural strains, which we have identified as tet(M). This gene was present in 21 independent isolates, whose fingerprinting profiles were distributed into eight different repetitive extragenic palindromic groups by cluster analysis. We provide evidence that the gene is associated with the broad-host, conjugative transposon Tn916, which had never before been described to occur in L. paracasei. PCR analysis of four independent isolates by use of specifically designed primer pairs detected the presence of a circular intermediate form of the transposon, carrying a coupling sequence (GGCAAA) located between the two termini of Tn916. This novel coupling sequence conferred low conjugation frequency in mating experiments with the recipient strain JH2-2 of Enterococcus faecalis.

Antibiotic resistance and microbial composition along the manufacturing process of Mozzarella di Bufala Campana.

The use of antibiotics as growth promoters in livestock, banned in all EU member states in January 2006, has led to selection of antibiotic resistant strains within environmental bacteria, including gram-positive, non pathogenic bacteria that colonize the GI tract of humans and animals. In Italy and in other Mediterranean countries, fermented foods employing environmental bacteria pre-existing in the raw substrates, rather than industrial starters of defined genotype, represent a significant proportion of cheese and meat products carrying the official PDO designation (Protected Designation of Origin). Our study focused on the microbiological and molecular analysis of lactobacilli and of other lactic acid bacteria (LABs) isolated from the Italian PDO product water buffalo Mozzarella cheese, with the aim of identifying genes responsible for tetracycline, erythromycin and kanamycin resistance. We isolated over 500 LAB colonies from retail products, as well as from raw milk and natural whey starters employed in their production. Microbiological analysis showed that about 50% of these isolates were represented by lactobacilli, which were further characterized in terms of species and strain composition, as well as by determining phenotypic and genotypic antibiotic resistance. To overcome the limits of culture-dependent approaches that select only cultivable species, we have also extracted total DNA from the whole microbiome present in the cheese and investigated the presence of specific antibiotic resistance genes with molecular approaches. Genetic determinants of antibiotic resistance were identified almost exclusively in bacteria isolated from the raw, unprocessed substrates, while the final, marketed products did not contain phenotypically resistant lactobacilli, i.e. displaying MIC values above the microbiological breakpoint. Overall, our results suggest that the traditional procedures necessary for manufacturing of this typical cheese, such as high temperature treatments, lead to a final product with low bacterial counts, lower biodiversity and lack of significant presence of antibiotic resistant lactobacilli.

Genetic analysis of variegate porphyria (VP) in Italy: identification of six novel mutations in the protoporphyrinogen oxidase (PPOX) gene.

Variegate Porphyria (VP) is one of the acute hepatic porphyrias, and is clinically characterised by skin lesions and acute neuropsychiatric/visceral attacks that occur separately or together. The disorder is caused by a partial deficiency of protoporphyrinogen oxidase, the penultimate enzyme in the heme biosynthetic pathway, and a number of mutations have been described for the corresponding gene (PPOX). Here we report a genetic analysis of VP in Italy, and the identification of six novel and three previously characterised mutations from nine affected individuals and families. Among those newly identified, two mutations were small deletions (c.418_419delAA; c.759delA), leading to the formation of premature stop codons, two were splicing defects (IVS10+2T>G; IVS12+1G>C), one was a nonsense (c.384G>A=p.W128X) and one a missense mutation (c.848T>A=I283N). This is the first study of the molecular genetics of Variegate Porphyria in patients of Italian origin, and the finding of six novel mutations out of nine identified confirms the genetic heterogeneity observed for this disorder.