Metabolomics Discloses a New Non-invasive Method for the Diagnosis and Prognosis of Patients with Alcoholic Hepatitis.

INTRODUCTION AND AIMS: Alcoholic hepatitis is the most severe manifestation of alcoholic liver disease. Unfortunately, there are still some unresolved issues in the diagnosis and management of this disease, such as the need of histological diagnosis, an accurate prognostic stratification, and the development of novel targeted therapies. The present study aimed at addressing these issues by means of metabolomics, a novel high-throughput approach useful in other liver diseases. MATERIAL AND METHODS: 64 patients with biopsy-proven alcoholic hepatitis were included and compared with 26 patients with decompensated alcoholic cirrhosis without superimposed alcoholic hepatitis, which was ruled out by liver biopsy. RESULTS: The comparison of the metabolic profiles of patients with alcoholic hepatitis and decompensated cirrhosis showed marked differences between both groups. Importantly, metabolic differences were found among alcoholic hepatitis patients when subjects were stratified according to 90-day survival. Based on these findings, two non-invasive signatures were developed. The first one allowed an accurate non-invasive diagnosis of alcoholic hepatitis (AUROC 0.932; 95% CI 0.901-0.963). The second signature showed a good performance in the prognostic stratification of patients with alcoholic hepatitis (AUROC 0.963; 95% CI 0.895-1.000). CONCLUSIONS: Signatures based on metabolomics allowed an accurate non-invasive diagnosis and prognostic stratification of alcoholic hepatitis. The differences observed in the metabolic profile of the patients according to the presence and severity of alcoholic hepatitis are related with different mechanisms involved in the pathophysiology of alcoholic hepatitis such as peroxisomal activity, synthesis of inflammatory mediators or oxidation. This information could be useful for the development of novel targeted therapies.

Metabolomics as a diagnostic tool for idiopathic non-cirrhotic portal hypertension.

BACKGROUND: Idiopathic non-cirrhotic portal hypertension (INCPH) is a rare life-threatening liver disease that lacks a specific diagnostic test being frequently misdiagnosed as cryptogenic cirrhosis. Preliminary data from our group identified a plasma metabolomic profile able to differentiate INCPH from patients with cirrhosis (CH) and healthy volunteers (HV). However, the untargeted methodology applied was unable to identify all the specific metabolites, hampering the possibility of building-up diagnostic models. This study applies a wide-coverage of previously identified metabolites through a high-throughput metabolomics technology, evaluating if there is a metabolomic profile that allows a non-invasive diagnosis of INCPH. METHODS: We included 34 patients with INCPH, 34 with CH and 34 HV. We performed a targeted metabolomic analysis of serum samples using UPLC-MS. The best combination of a set of specific metabolites was obtained using stepwise logistic regression (LR) and recursive partitioning analysis (RPA). RESULTS: After internal cross-validation, LR analysis identified a subset of 5-metabolites that clearly differentiate INCPH patients from CH and HV (average corrected optimism AUROC = 0.8871 [0.838-0.924]). Using high and low cut-off values the model has an excellent capacity to respectively diagnose or exclude INCPH. The RPA analysis strategy used the 3-metabolites signature differentiating INCPH from CH and the 2-metabolites signature differentiating INCPH from HV. A decision tree applying sequentially these metabolic profiles diagnosed 88% of INCPH patients. CONCLUSIONS: Different metabolomic profiles allow the diagnosis of INCPH with high specificity and sensibility and may represent excellent clinical tools for its diagnosis avoiding multiple and invasive tests.

Solute carrier family 2 member 1 is involved in the development of nonalcoholic fatty liver disease.

UNLABELLED: Susceptibility to develop nonalcoholic fatty liver disease (NAFLD) has genetic bases, but the associated variants are uncertain. The aim of the present study was to identify genetic variants that could help to prognose and further understand the genetics and development of NAFLD. Allele frequencies of 3,072 single-nucleotide polymorphisms (SNPs) in 92 genes were characterized in 69 NAFLD patients and 217 healthy individuals. The markers that showed significant allele-frequency differences in the pilot groups were subsequently studied in 451 NAFLD patients and 304 healthy controls. Besides this, 4,414 type 2 diabetes mellitus (T2DM) cases and 4,567 controls were genotyped. Liver expression of the associated gene was measured and the effect of its potential role was studied by silencing the gene in vitro. Whole genome expression, oxidative stress (OS), and the consequences of oleic acid (OA)-enriched medium on lipid accumulation in siSLC2A1-THLE2 cells were studied by gene-expression analysis, dihydroethidium staining, BODIPY, and quantification of intracellular triglyceride content, respectively. Several SNPs of SLC2A1 (solute carrier family 2 [facilitated glucose transporter] member 1) showed association with NAFLD, but not with T2DM, being the haplotype containing the minor allele of SLC2A1 sequence related to the susceptibility to develop NAFLD. Gene-expression analysis demonstrated a significant down-regulation of SLC2A1 in NAFLD livers. Enrichment functional analyses of transcriptome profiles drove us to demonstrate that in vitro silencing of SLC2A1 induces an increased OS activity and a higher lipid accumulation under OA treatment. CONCLUSIONS: Genetic variants of SLC2A1 are associated with NAFLD, and in vitro down-regulation of this gene promotes lipid accumulation. Moreover, the oxidative response detected in siSLC2A1-THLE2 cells corroborated the antioxidant properties previously related to this gene and linked the most representative clinical characteristics of NAFLD patients: oxidative injury and increased lipid storage.

Metabolomics discloses potential biomarkers for the noninvasive diagnosis of idiopathic portal hypertension.

OBJECTIVES: Idiopathic portal hypertension (IPH) is a rare cause of portal hypertension that lacks a specific diagnostic test. Requiring ruling-out other causes of portal hypertension it is frequently misdiagnosed. This study evaluates whether using high-throughput techniques there is a metabolomic profile allowing a noninvasive diagnosis of IPH. METHODS: Thirty-three IPH patients were included. Matched patients with cirrhosis (CH) and healthy volunteers (HV) were included as controls. Metabolomic analysis of plasma samples was performed using UPLC-time-of-flight-mass spectrometry. We computed Student's P-values, corrected by multiple comparison and VIP score (Variable Importance in the Projection). The metabolites were selected with an adjusted Benjamini Hochberg P value <0.05. We use markers with a greater VIP score, to build partial least squares projection to latent structures regression with discriminant analysis (PLS-DA) representative models to discriminate IPH from CH and from HV. The performance of the PLS-DA model was evaluated using R(2) and Q(2) parameter. An additional internal cross-validation was done. RESULTS: PLS-DA analysis showed a clear separation of IPH from CH with a model involving 28 metabolites (Q(2)=0.67, area under the curve (AUC)=0.99) and a clear separation of IPH from healthy subjects with a model including 31 metabolites (Q(2)=0.75, AUC=0.98). After cross-validation, both models showed high rates of sensitivity (94.8 and 97.5), specificity (89.1 and 89.7), and AUC (0.98 and 0.98), reinforcing the strength of our findings. CONCLUSIONS: A metabolomic profile clearly differentiating patients with IPH from CH and healthy subjects has been identified using subsets of 28 and 31 metabolites, respectively. Therefore, metabolomic analysis appears to be a valuable tool for the noninvasive diagnosis of IPH.

Impaired Function of Solute Carrier Family 19 Leads to Low Folate Levels and Lipid Droplet Accumulation in Hepatocytes.

Low serum folate levels are inversely related to metabolic associated fatty liver disease (MAFLD). The role of the folate transporter gene (SLC19A1) was assessed to clarify its involvement in lipid accumulation during the onset of MAFLD in humans and in liver cells by genomic, transcriptomic, and metabolomic techniques. Genotypes of 3 SNPs in a case-control cohort were initially correlated to clinical and serum MAFLD markers. Subsequently, the expression of 84 key genes in response to the loss of SLC19A1 was evaluated with the aid of an RT2 profiler-array. After shRNA-silencing of SLC19A1 in THLE2 cells, folate and lipid levels were measured by ELISA and staining techniques, respectively. In addition, up to 482 amino acids and lipid metabolites were semi-quantified in SLC19A1-knockdown (KD) cells through ultra-high-performance liquid chromatography coupled with mass spectrometry. SNPs, rs1051266 and rs3788200, were significantly associated with the development of fatty liver for the single-marker allelic test. The minor alleles of these SNPs were associated with a 0.6/-1.67-fold decreased risk of developing MAFLD. When SLC19A1 was KD in THLE2 cells, intracellular folate content was four times lower than in wild-type cells. The lack of functional SLC19A1 provoked significant changes in the regulation of genes associated with lipid droplet accumulation within the cell and the onset of NAFLD. Metabolomic analyses showed a highly altered profile, where most of the species that accumulated in SLC19A1-KD-cells belong to the chemical groups of triacylglycerols, diacylglycerols, polyunsaturated fatty acids, and long chain, highly unsaturated cholesterol esters. In conclusion, the lack of SLC19A1 gene expression in hepatocytes affects the regulation of key genes for normal liver function, reduces intracellular folate levels, and impairs lipid metabolism, which entails lipid droplet accumulation in hepatocytes.

Liquid chromatography-mass spectrometry-based parallel metabolic profiling of human and mouse model serum reveals putative biomarkers associated with the progression of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in most western countries. Current NAFLD diagnosis methods (e.g., liver biopsy analysis or imaging techniques) are poorly suited as tests for such a prevalent condition, from both a clinical and financial point of view. The present work aims to demonstrate the potential utility of serum metabolic profiling in defining phenotypic biomarkers that could be useful in NAFLD management. A parallel animal model/human NAFLD exploratory metabolomics approach was employed, using ultra performance liquid chromatography-mass spectrometry (UPLC-MS) to analyze 42 serum samples collected from nondiabetic, morbidly obese, biopsy-proven NAFLD patients, and 17 animals belonging to the glycine N-methyltransferase knockout (GNMT-KO) NAFLD mouse model. Multivariate statistical analysis of the data revealed a series of common biomarkers that were significantly altered in the NAFLD (GNMT-KO) subjects in comparison to their normal liver counterparts (WT). Many of the compounds observed could be associated with biochemical perturbations associated with liver dysfunction (e.g., reduced Creatine) and inflammation (e.g., eicosanoid signaling). This differential metabolic phenotyping approach may have a future role as a supplement for clinical decision making in NAFLD and in the adaption to more individualized treatment protocols.

Targeting Hepatic Glutaminase 1 Ameliorates Non-alcoholic Steatohepatitis by Restoring Very-Low-Density Lipoprotein Triglyceride Assembly.

Non-alcoholic steatohepatitis (NASH) is characterized by the accumulation of hepatic fat in an inflammatory/fibrotic background. Herein, we show that the hepatic high-activity glutaminase 1 isoform (GLS1) is overexpressed in NASH. Importantly, GLS1 inhibition reduces lipid content in choline and/or methionine deprivation-induced steatotic mouse primary hepatocytes, in human hepatocyte cell lines, and in NASH mouse livers. We suggest that under these circumstances, defective glutamine fueling of anaplerotic mitochondrial metabolism and concomitant reduction of oxidative stress promotes a reprogramming of serine metabolism, wherein serine is shifted from the generation of the antioxidant glutathione and channeled to provide one-carbon units to regenerate the methionine cycle. The restored methionine cycle can induce phosphatidylcholine synthesis from the phosphatidylethanolamine N-methyltransferase-mediated and CDP-choline pathways as well as by base-exchange reactions between phospholipids, thereby restoring hepatic phosphatidylcholine content and very-low-density lipoprotein export. Overall, we provide evidence that hepatic GLS1 targeting is a valuable therapeutic approach in NASH.

A Slow-Digesting Carbohydrate Diet during Rat Pregnancy Protects Offspring from Non-Alcoholic Fatty Liver Disease Risk through the Modulation of the Carbohydrate-Response Element and Sterol Regulatory Element Binding Proteins.

High-fat (HF) and rapid digestive (RD) carbohydrate diets during pregnancy promote excessive adipogenesis in offspring. This effect can be corrected by diets with similar glycemic loads, but low rates of carbohydrate digestion. However, the effects of these diets on metabolic programming in the livers of offspring, and the liver metabolism contributions to adipogenesis, remain to be addressed. In this study, pregnant insulin-resistant rats were fed high-fat diets with similar glycemic loads but different rates of carbohydrate digestion, High Fat-Rapid Digestive (HF-RD) diet or High Fat-Slow Digestive (HF-SD) diet. Offspring were fed a standard diet for 10 weeks, and the impact of these diets on the metabolic and signaling pathways involved in liver fat synthesis and storage of offspring were analyzed, including liver lipidomics, glycogen and carbohydrate and lipid metabolism key enzymes and signaling pathways. Livers from animals whose mothers were fed an HF-RD diet showed higher saturated triacylglycerol deposits with lower carbon numbers and double bond contents compared with the HF-SD group. Moreover, the HF-RD group exhibited enhanced glucose transporter 2, pyruvate kinase (PK), acetyl coenzyme A carboxylase (ACC) and fatty acid (FA) synthase expression, and a decrease in pyruvate carboxylase (PyC) expression leading to an altered liver lipid profile. These parameters were normalized in the HF-SD group. The changes in lipogenic enzyme expression were parallel to changes in AktPKB phosphorylation status and nuclear expression in carbohydrate-response element and sterol regulatory element binding proteins. In conclusion, an HF-RD diet during pregnancy translates to changes in liver signaling and metabolic pathways in offspring, enhancing liver lipid storage and synthesis, and therefore non-alcoholic fatty liver disease (NAFLD) risk. These changes can be corrected by feeding an HF-SD diet during pregnancy.

Feeding a slowly digestible carbohydrate diet during pregnancy of insulin-resistant rats prevents the excess of adipogenesis in their offspring.

An obesogenic environment during pregnancy has been shown to increase the risk of dysregulation on adipogenesis and insulin resistance in the offspring. Being essential for the growing fetus, glucose supply is guaranteed by a number of modifications in the mother's metabolism, and thus, glucose control during pregnancy especially among obese or diabetic women is paramount to prevent adverse consequences in their children. Besides the election of low-glycemic-index carbohydrates, the rate of carbohydrate digestion could be relevant to keep a good glucose control. In the present study, we compared the effects of two high-fat diets with similar glycemic load but different rates of carbohydrate digestion given to pregnant insulin-resistant rats. After birth, all animals were fed a standard diet until age 14 weeks. We analyzed offspring body composition, plasma and adipocyte lipidomics, lipid metabolism in adipose tissue and insulin sensitivity. Those animals whose mothers were fed the rapid-digesting carbohydrate diet exhibited an excessive adipogenesis. Thus, these animals showed a marked lipidemia, increased lipid synthesis in the adipose tissue and reduced glucose transporter amount in the adipose. On the contrary, those animals whose mothers were fed the slow-digesting carbohydrate diet showed a profile in the measured parameters closer to that of the offspring of healthy mothers. These results support the hypothesis that not only glycemic index but the rate of carbohydrate digestion during gestation may be critical to regulate the programming of adipogenesis in the offspring.

Metabolic alterations in urine extracellular vesicles are associated to prostate cancer pathogenesis and progression.

Urine contains extracellular vesicles (EVs) that concentrate molecules and protect them from degradation. Thus, isolation and characterisation of urinary EVs could increase the efficiency of biomarker discovery. We have previously identified proteins and RNAs with differential abundance in urinary EVs from prostate cancer (PCa) patients compared to benign prostate hyperplasia (BPH). Here, we focused on the analysis of the metabolites contained in urinary EVs collected from patients with PCa and BPH. Targeted metabolomics analysis of EVs was performed by ultra-high-performance liquid chromatography-mass spectrometry. The correlation between metabolites and clinical parameters was studied, and metabolites with differential abundance in PCa urinary EVs were detected and mapped into cellular pathways. We detected 248 metabolites belonging to different chemical families including amino acids and various lipid species. Among these metabolites, 76 exhibited significant differential abundance between PCa and BPH. Interestingly, urine EVs recapitulated many of the metabolic alterations reported in PCa, including phosphathidylcholines, acyl carnitines, citrate and kynurenine. Importantly, we found elevated levels of the steroid hormone, 3beta-hydroxyandros-5-en-17-one-3-sulphate (dehydroepiandrosterone sulphate) in PCa urinary EVs, in line with the potential elevation of androgen synthesis in this type of cancer. This work supports urinary EVs as a non-invasive source to infer metabolic changes in PCa.

Metabolomic-based noninvasive serum test to diagnose nonalcoholic steatohepatitis: Results from discovery and validation cohorts.

Nonalcoholic fatty liver disease (NAFLD) is the most common type of chronic liver disease worldwide and includes a broad spectrum of histologic phenotypes, ranging from simple hepatic steatosis or nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). While liver biopsy is the reference gold standard for NAFLD diagnosis and staging, it has limitations due to its sampling variability, invasive nature, and high cost. Thus, there is a need for noninvasive biomarkers that are robust, reliable, and cost effective. In this study, we measured 540 lipids and amino acids in serum samples from biopsy-proven subjects with normal liver (NL), NAFL, and NASH. Using logistic regression analysis, we identified two panels of triglycerides that could first discriminate between NAFLD and NL and second between NASH and NAFL. These noninvasive tests were compared to blinded histology as a reference standard. We performed these tests in an original cohort of 467 patients with NAFLD (90 NL, 246 NAFL, and 131 NASH) that was subsequently validated in a separate cohort of 192 patients (7 NL, 109 NAFL, 76 NASH). The diagnostic performances of the validated tests showed an area under the receiver operating characteristic curve, sensitivity, and specificity of 0.88 ± 0.05, 0.94, and 0.57, respectively, for the discrimination between NAFLD and NL and 0.79 ± 0.04, 0.70, and 0.81, respectively, for the discrimination between NASH and NAFL. When the analysis was performed excluding patients with glucose levels >136 mg/dL, the area under the receiver operating characteristic curve for the discrimination between NASH and NAFL increased to 0.81 ± 0.04 with sensitivity and specificity of 0.73 and 0.80, respectively. Conclusion: The assessed noninvasive lipidomic serum tests distinguish between NAFLD and NL and between NASH and NAFL with high accuracy. (Hepatology Communications 2018;2:807-820).

Metabolically active extracellular vesicles released from hepatocytes under drug-induced liver-damaging conditions modify serum metabolome and might affect different pathophysiological processes.

Hepatocytes are involved in the endogenous and drug metabolism; many of the enzymes involved in those processes are incorporated into extracellular vesicles and secreted into the bloodstream. Liver-damaging conditions modify the molecular cargo of those vesicles significantly. However, no information about the effect of these hepatic vesicles on the extracellular environment is available. Drug-induced liver damage increases the number of circulating extracellular vesicles and affects the release and content of hepatocyte-derived vesicles. In this work, we evaluated the metabolic effect of these vesicles on the composition of the serum. We performed a targeted ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) metabolomics analysis of serum samples. The samples had been first incubated with hepatic extracellular vesicles from hepatocytes challenged with acetaminophen or diclofenac. The incubation affected the serum levels of 67 metabolites, such as amino acids and different species of lipids. The metabolites included various species of phosphatidylcholines and phosphatidylethanolamines. These compounds are the components of biological membranes; our observations suggest that the vesicles might take part in remodelling and maintenance of the membranes. Alterations in the levels of some other serum metabolites might have deleterious consequences, for example, the tetracosanoic acid with its cardiovascular effects. However, some of the metabolites whose levels were increased, including alpha-linoleic and tauroursodeoxycholic acids, have been reported to have a protective effect. Our targeted metabolomics analysis indicated that the hepatic extracellular vesicles act as nano-metabolic machines supplying the extracellular environment with the means to integrate diverse tissue responses. In conclusion, we show that the hepatic extracellular vesicles are metabolically active and might play a role in the physiopathological response to hepatic insults, including drug-induced liver injury.

Metabolomic signatures associated with disease severity in multiple sclerosis.

OBJECTIVE: To identify differences in the metabolomic profile in the serum of patients with multiple sclerosis (MS) compared to controls and to identify biomarkers of disease severity. METHODS: We studied 2 cohorts of patients with MS: a retrospective longitudinal cohort of 238 patients and 74 controls and a prospective cohort of 61 patients and 41 controls with serial serum samples. Patients were stratified into active or stable disease based on 2 years of prospective assessment accounting for presence of clinical relapses or changes in disability measured with the Expanded Disability Status Scale (EDSS). Metabolomic profiling (lipids and amino acids) was performed by ultra-high-performance liquid chromatography coupled to mass spectrometry in serum samples. Data analysis was performed using parametric methods, principal component analysis, and partial least square discriminant analysis for assessing the differences between cases and controls and for subgroups based on disease severity. RESULTS: We identified metabolomics signatures with high accuracy for classifying patients vs controls as well as for classifying patients with medium to high disability (EDSS >3.0). Among them, sphingomyelin and lysophosphatidylethanolamine were the metabolites that showed a more robust pattern in the time series analysis for discriminating between patients and controls. Moreover, levels of hydrocortisone, glutamic acid, tryptophan, eicosapentaenoic acid, 13S-hydroxyoctadecadienoic acid, lysophosphatidylcholines, and lysophosphatidylethanolamines were associated with more severe disease (non-relapse-free or increase in EDSS). CONCLUSIONS: We identified metabolomic signatures composed of hormones, lipids, and amino acids associated with MS and with a more severe course.

A Metabolomics Signature Linked To Liver Fibrosis In The Serum Of Transplanted Hepatitis C Patients.

Liver fibrosis must be evaluated in patients with hepatitis C virus (HCV) after liver transplantation because its severity affects their prognosis and the recurrence of HCV. Since invasive biopsy is still the gold standard to identify patients at risk of graft loss from rapid fibrosis progression, it becomes crucial the development of new accurate, non-invasive methods that allow repetitive examination of the patients. Therefore, we have developed a non-invasive, accurate model to distinguish those patients with different liver fibrosis stages. Two hundred and three patients with HCV were histologically classified (METAVIR) into five categories of fibrosis one year after liver transplantation. In this cross-sectional study, patients at fibrosis stages F0-F1 (n = 134) were categorised as "slow fibrosers" and F2-F4 (n = 69) as "rapid fibrosers". Chloroform/methanol serum extracts were analysed by reverse ultra-high performance liquid chromatography coupled to mass spectrometry. A diagnostic model was built through linear discriminant analyses. An algorithm consisting of two sphingomyelins and two phosphatidylcholines accurately classifies rapid and slow fibrosers after transplantation. The proposed model yielded an AUROC of 0.92, 71% sensitivity, 85% specificity, and 84% accuracy. Moreover, specific bile acids and sphingomyelins increased notably along with liver fibrosis severity, differentiating between rapid and slow fibrosers.

Metabolic Characterization of Advanced Liver Fibrosis in HCV Patients as Studied by Serum 1H-NMR Spectroscopy.

Several etiologies result in chronic liver diseases including chronic hepatitis C virus infection (HCV). Despite its high incidence and the severe economic and medical consequences, liver disease is still commonly overlooked due to the lack of efficient non-invasive diagnostic methods. While several techniques have been tested for the detection of fibrosis, the available biomarkers still present severe limitations that preclude their use in clinical diagnostics. Liver diseases have also been the subject of metabolomic analysis. Here, we demonstrate the suitability of 1H NMR spectroscopy for characterizing the metabolism of liver fibrosis induced by HCV. Serum samples from HCV patients without fibrosis or with liver cirrhosis were analyzed by NMR spectroscopy and the results were submitted to multivariate and univariate statistical analysis. PLS-DA test was able to discriminate between advanced fibrotic and non-fibrotic patients and several metabolites were found to be up or downregulated in patients with cirrhosis. The suitability of the most significantly regulated metabolites was validated by ROC analysis. Our study reveals that choline, acetoacetate and low-density lipoproteins are the most informative biomarkers for predicting cirrhosis in HCV patients. Our results demonstrate that statistical analysis of 1H-NMR spectra is able to distinguish between fibrotic and non-fibrotic patients suffering from HCV, representing a novel diagnostic application for NMR spectroscopy.

Stabilization of LKB1 and Akt by neddylation regulates energy metabolism in liver cancer.

The current view of cancer progression highlights that cancer cells must undergo through a post-translational regulation and metabolic reprogramming to progress in an unfriendly environment. In here, the importance of neddylation modification in liver cancer was investigated. We found that hepatic neddylation was specifically enriched in liver cancer patients with bad prognosis. In addition, the treatment with the neddylation inhibitor MLN4924 in Phb1-KO mice, an animal model of hepatocellular carcinoma showing elevated neddylation, reverted the malignant phenotype. Tumor cell death in vivo translating into liver tumor regression was associated with augmented phosphatidylcholine synthesis by the PEMT pathway, known as a liver-specific tumor suppressor, and restored mitochondrial function and TCA cycle flux. Otherwise, in protumoral hepatocytes, neddylation inhibition resulted in metabolic reprogramming rendering a decrease in oxidative phosphorylation and concomitant tumor cell apoptosis. Moreover, Akt and LKB1, hallmarks of proliferative metabolism, were altered in liver cancer being new targets of neddylation. Importantly, we show that neddylation-induced metabolic reprogramming and apoptosis were dependent on LKB1 and Akt stabilization. Overall, our results implicate neddylation/signaling/metabolism, partly mediated by LKB1 and Akt, in the development of liver cancer, paving the way for novel therapeutic approaches targeting neddylation in hepatocellular carcinoma.

Results of the 1999-2000 collaborative exercise and proficiency testing program on mitochondrial DNA of the GEP-ISFG: an inter-laboratory study of the observed variability in the heteroplasmy level of hair from the same donor.

The Spanish and Portuguese working group (GEP) of international society for forensic genetics (ISFG) 1999-2000 collaborative exercise on mitochondrial DNA (mtDNA) included the analysis of four bloodstain samples and one hair shaft sample by 19 participating laboratories from Spain, Portugal and several Latin-American countries. A wide range of sequence results at position 16,093 of the HV1 (from T or C homoplasmy to different levels of heteroplasmy) were submitted by the different participating laboratories from the hair shaft sample during the first phase of this exercise. During the discussion of these results in the Annual GEP-ISFG 2000 Conference a second phase of this exercise was established with two main objectives: (i) to evaluate the incidence of the HV1 sequence heteroplasmy detected in Phase I across different sample types from the same donor including blood, saliva, and hair shafts, (ii) to perform a technical review of the electropherograms to evaluate the relative levels of heteroplasmies obtained by the different laboratories and also to examine the source of possible errors detected in Phase I. Anonymous review of the raw sequence data permitted the detection of three transcription errors and three errors due to methodological problems. Highly variable levels of heteroplasmy were found in the hair shaft and more stability in blood and saliva. Three laboratories found variable levels of heteroplasmy at position 16,093 across adjacent fragments from the same hair shaft. Two laboratories also described more than one heteroplasmic position from a single hair. The relevance of these findings for the interpretation of mtDNA data in the forensic context is also discussed.

Results of the GEP-ISFG collaborative study on two Y-STRs tetraplexes: GEPY I (DYS461, GATA C4, DYS437 and DYS438) and GEPY II (DYS460, GATA A10, GATA H4 and DYS439).

A collaborative exercise was carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG) in order to evaluate the performance of two Y-chromosome STR PCR tetraplexes, which include the loci DYS461, GATA C4, DYS437 and DYS438 (GEPY I), and DYS460, GATA A10, GATA H4 and DYS439 (GEPY II). The participating laboratories were asked to type three samples for the eight markers, using a specific amplification protocol. In addition, two control samples, with known haplotypes, were provided. The results obtained by the 13 different participating laboratories were identical, except for two laboratories that failed to type correctly the same two samples for GATA C4. By sequence analyses, two different GATA C4 allele structures were found. One control sample (allele 21) and two questioned samples (allele 22, correctly typed by all the laboratories, and allele 25) presented the following repeat structure: (TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)n, but different from the one found for allele 26 in one sample included in this exercise, as well as in the second control sample (allele 23), namely (TCTA)4(TGTA)2(TCTA)2(TGTA)2(TCTA)2(TGTA)2(TCTA)n. The collaborative exercise results proved that both Y-tetraplexes produce good amplification results, with the advantage of being efficiently typed using different separation and detection methodologies. However, since GATA C4 repeat presents a complex structure, with alleles differing in sequence structure, efficient denaturing conditions should be followed in order to avoid typing errors due to sizing problems.

Sequence polymorphisms of the mtDNA control region in a human isolate: the Georgians from Swanetia.

In this work, we analyzed the sequence diversity of the mtDNA control region (HVI and HVII) in a sample of 48 individuals from Swanetia (Georgia), using direct fluorescent-based sequencing methods. We identified 43 different mtDNA haplotypes resulting from 78 polymorphic sites (46 in HVI and 32 in HVII). Most of the variable positions identified in both HVI and HVII were transitions (82.6 and 71.9%, respectively). The frequency of length heteroplasmy in the homopolymeric C-stretch regions was the same for both segments (10.4%). The sequence diversity increased markedly when both hypervariable regions were analyzed jointly (HVI: 0.985, HVII: 0.975, HVI+HVII: 0.994). Accordingly, the probability of two randomly selected sequences matching (random match probability, RMP) decreased from 3.4% (HVI) to 2.6% (HVI+HVII), despite which the RMP values in Georgians remained higher than estimated in most Europeans. This suggests that the variability of maternal lineages tends to be lower in traditional human isolates and, therefore, the potential of discrimination of mtDNA in forensic analysis is more limited in this type of population. The incorporation of HVII data also contributed to the refinement of results regarding the genetic relationships among the samples included in the analyses, which stress the importance of considering HVII in both population and forensic genetics.

Ancestral origins of the prion protein gene D178N mutation in the Basque Country.

Fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD) are familial prion diseases with autosomal dominant inheritance of the D178N mutation. FFI has been reported in at least 27 pedigrees around the world. Twelve apparently unrelated FFI and fCJD pedigrees with the characteristic D178N mutation have been reported in the Prion Diseases Registry of the Basque Country since 1993. The high incidence of familial prion diseases in this region may reflect a unique ancestral origin of the chromosome carrying this mutation. In order to investigate this putative founder effect, we developed "happy typing", a new approach to the happy mapping method, which consists of the physical isolation of large haploid genomic DNA fragments and their analysis by the Polymerase Chain Reaction in order to perform haplotypic analysis instead of pedigree analysis. Six novel microsatellite markers, located in a 150-kb genomic segment flanking the PRNP gene were characterized for typing haploid DNA fragments of 285 kb in size. A common haplotype was found in patients from the Basque region, strongly suggesting a founder effect. We propose that "happy typing" constitutes an efficient method for determining disease-associated haplotypes, since the analysis of a single affected individual per pedigree should provide sufficient evidence.