Multiparametric renal function assessment in cirrhotic patients shows high prevalence of medically actionable changes in multiple modules.

AIM: Renal dysfunction is a common complication of cirrhosis, occurring either as part of multiorgan involvement in acute illness or secondary to advanced liver disease. To date, no study has comprehensively assessed multiple renal function parameters in hospitalized patients with cirrhosis through a multiparametric analysis of renal biochemistry markers. METHODS: We conducted a retrospective, observational study including all consecutive patients hospitalized with cirrhosis who underwent a 43-multiparametric renal function assessment between January 1, 2021, and June 30, 2023. RESULTS: All patients showed at least one of the following renal abnormalities: Kidney Disease: Improving Global Outcomes stage G2 or higher, sodium and/or chloride excretion fraction <1%, electrolyte-free water clearance <0.4 mL/min, or tubular maximum phosphate reabsorption capacity <0.8 mmol/L. The estimated glomerular filtration rate equations significantly overestimated the measured creatinine clearance with median differences of +14 mL/min/1.73 m2 (95% CI 6-29) and +9 mL/min/1.73 m2 (95% CI 2-15) for European Kidney Function Consortium equations, respectively. Notably, 54% and 39% of patients demonstrated estimated glomerular filtration rates exceeding 30% of the measured creatinine clearance when the Chronic Kidney Disease - Epidemiology Collaboration and European Kidney Function Consortium formulas were employed, respectively. Substantial discrepancies in Kidney Disease: Improving Global Outcomes stage assignments were observed between the estimated glomerular filtration rate- and measured creatinine clearance-based assessments. CONCLUSIONS: This study underscores the value of a multiparametric renal function assessment as a routine tool for evaluating renal function in patients with cirrhosis. A high prevalence of medically actionable renal abnormalities spanning multiple renal function modules, including alterations in glomerular function, salt and solute-free water excretion, and proximal tubule phosphate reabsorption, has been demonstrated in hospitalized patients with cirrhosis.

Blood phenylalanine fluctuation in phenylketonuric children treated by BH4 or low-phenylalanine diet from birth.

The prognosis of phenylketonuria (PKU) is related to the quality of metabolic control all life-long. PKU treatment is based on a low-Phe diet, 6R-tetrahydrobiopterin (BH4) treatment for the BH4-responsive PKU patients or enzyme replacement therapy. Fluctuations in blood phenylalanine (Phe) concentrations may be an important determinant of intellectual outcome in patients with early and continuously treated phenylketonuria (PKU). The aim of this work is to study the fluctuation of Blood Phe in patients treated by BH4 from birth in comparison with patients treated by low-Phe diet. We conducted a retrospective study in a national reference center for PKU management. We compared mean phenylalanine blood concentration and its fluctuation in 10 BH4-responder patients (BH4R) and in 10 BH4 non-responder patients (BH4NR) treated from birth. The mean blood Phe concentration is similar between the two groups before 10 years of age (290 ± 135 (BH4R) vs. 329 ± 187 µmol/L, p = 0.066 (BH4NR)) while it is lower in the BH4R group after 10 years of age. (209 ± 69 vs. 579 ± 136 µmol/L, p = 0.0008). Blood Phe fluctuation is significantly lower in the BH4R group compared to the BH4NR group (70.2 ± 75.6 vs. 104.4 ± 111.6 µmol/L, p < 0.01) before 6 years of age. There are no significant differences observed on nutritional status, growth, and neuropsychological tests between the two groups. BH4 introduced in the neonatal period is associated with less blood Phe fluctuation before 6 years. Additional time and patients are required to determine if the decrease in Phe fluctuation would positively impact the long-term outcome of PKU patients.

Predictors of the utility of clinical exome sequencing as a first-tier genetic test in patients with Mendelian phenotypes: results from a referral center study on 603 consecutive cases.

BACKGROUND: Clinical exome sequencing (CES) provides a comprehensive and effective analysis of relevant disease-associated genes in a cost-effective manner compared to whole exome sequencing. Although several studies have focused on the diagnostic yield of CES, no study has assessed predictors of CES utility among patients with various Mendelian phenotypes. We assessed the effectiveness of CES as a first-level genetic test for molecular diagnosis in patients with a Mendelian phenotype and explored independent predictors of the clinical utility of CES. RESULTS: Between January 2016 and December 2019, 603 patients (426 probands and 177 siblings) underwent CES at the Department of Molecular Medicine of the University Hospital of Nancy. The median age of the probands was 34 years (IQR, 12-48), and the proportion of males was 46.9% (200/426). Adults and children represented 64.8% (276/426) and 35.2% (150/426), respectively. The median test-to-report time was 5.6 months (IQR, 4.1-7.2). CES revealed 203 pathogenic or likely pathogenic variants in 160 patients, corresponding to a diagnostic yield of 37.6% (160/426). Independent predictors of CES utility were criteria strongly suggestive of an extreme phenotype, including pediatric presentation and patient phenotypes associated with an increased risk of a priori probability of a monogenic disorder, the inclusion of at least one family member in addition to the proband, and a CES prescription performed by an expert in the field of rare genetic disorders. CONCLUSIONS: Based on a large dataset of consecutive patients with various Mendelian phenotypes referred for CES as a first-tier genetic test, we report a diagnostic yield of ~ 40% and several independent predictors of CES utility that might improve CES diagnostic efficiency.

Stemness of Normal and Cancer Cells: The Influence of Methionine Needs and SIRT1/PGC-1α/PPAR-α Players.

Stem cells are a population of undifferentiated cells with self-renewal and differentiation capacities. Normal and cancer stem cells share similar characteristics in relation to their stemness properties. One-carbon metabolism (OCM), a network of interconnected reactions, plays an important role in this dependence through its role in the endogenous synthesis of methionine and S-adenosylmethionine (SAM), the universal donor of methyl groups in eukaryotic cells. OCM genes are differentially expressed in stem cells, compared to their differentiated counterparts. Furthermore, cultivating stem cells in methionine-restricted conditions hinders their stemness capacities through decreased SAM levels with a subsequent decrease in histone methylation, notably H3K4me3, with a decrease in stem cell markers. Stem cells' reliance on methionine is linked to several mechanisms, including high methionine flux or low endogenous methionine biosynthesis. In this review, we provide an overview of the recent discoveries concerning this metabolic dependence and we discuss the mechanisms behind them. We highlight the influence of SIRT1 on SAM synthesis and suggest a role of PGC-1α/PPAR-α in impaired stemness produced by methionine deprivation. In addition, we discuss the potential interest of methionine restriction in regenerative medicine and cancer treatment.

ALDH1L2 Knockout in U251 Glioblastoma Cells Reduces Tumor Sphere Formation by Increasing Oxidative Stress and Suppressing Methionine Dependency.

Previously, the in vitro growth of cancer stem cells in the form of tumor spheres from five different brain cancer cell lines was found to be methionine-dependent. As this earlier work indicated that ALDH1L2, a folate-dependent mitochondria aldehyde dehydrogenase gene, is upregulated in glioblastoma stem cells, we invalidated this gene using CRISPR-cas 9 technique in this present work. We reported here that this invalidation was effective in U251 glioblastoma cells, and no cas9 off target site could be detected by genome sequencing of the two independent knockout targeting either exon I or exon III. The knockout of ALDH1L2 gene in U251 cells rendered the growth of the cancer stem cells of U251 methionine independent. In addition, a much higher ROS (reactive oxygen radicals) level can be detected in the knockout cells compared to the wild type cells. Our evidence here linked the excessive ROS level of the knockout cells to reduced total cellular NADPH. Our evidence suggested also that the cause of the slower growth of the knockout turmor sphere may be related to its partial differentiation.

Diagnostic yield of clinical exome sequencing as a first-tier genetic test for the diagnosis of genetic disorders in pediatric patients: results from a referral center study.

The emergence of next-generation sequencing enabled a cost-effective and straightforward diagnostic approach to genetic disorders using clinical exome sequencing (CES) panels. We performed a retrospective observational study to assess the diagnostic yield of CES as a first-tier genetic test in 128 consecutive pediatric patients addressed to a referral center in the North-East of France for a suspected genetic disorder, mainly an inborn error of metabolism between January 2016 and August 2020. CES was performed using the TruSight One (4811 genes) or the TruSight One expanded (6699 genes) panel on an Illumina sequencing platform. The median age was 6.5 years (IQR 2.0-12.0) with 43% of males (55/128), and the median disease duration was 7 months (IQR 1-47). In the whole analysis, the CES diagnostic yield was 55% (70/128). The median test-to-report time was 5 months (IQR 4-7). According to CES indications, the CES diagnostic yields were 81% (21/26) for hyperlipidemia, 75% (6/8) for osteogenesis imperfecta, 64% (25/39) for metabolic disorders, 39% (10/26) for neurological disorders, and 28% (8/29) for the subgroup of patients with miscellaneous conditions. Our results demonstrate the usefulness of a CES-based diagnosis as a first-tier genetic test to establish a molecular diagnosis in pediatric patients with a suspected genetic disorder with a median test-to-report time of 5 months. It highlights the importance of a close interaction between the pediatrician with expertise in genetic disorders and the molecular medicine physician to optimize both CES indication and interpretation. Diagnostic yield of clinical exome sequencing (CES) as a first-tier genetic test for diagnosing genetic disorders in 128 consecutive pediatric patients referred to a reference center in the North-East of France for a suspected genetic disorder, mainly an inborn error of metabolism between January 2016 and August 2020. The CES diagnostic yields are reported in the whole population and patients' subgroups (hyperlipidemia, osteogenesis imperfecta, metabolic diseases, neurological disorders, miscellaneous conditions) (Icons made by Flaticon, flaticon.com; CC-BY-3.0).

A noncoding RNA modulator potentiates phenylalanine metabolism in mice.

The functional role of long noncoding RNAs (lncRNAs) in inherited metabolic disorders, including phenylketonuria (PKU), is unknown. Here, we demonstrate that the mouse lncRNA Pair and human HULC associate with phenylalanine hydroxylase (PAH). Pair-knockout mice exhibited excessive blood phenylalanine (Phe), musty odor, hypopigmentation, growth retardation, and progressive neurological symptoms including seizures, which faithfully models human PKU. HULC depletion led to reduced PAH enzymatic activities in human induced pluripotent stem cell-differentiated hepatocytes. Mechanistically, HULC modulated the enzymatic activities of PAH by facilitating PAH-substrate and PAH-cofactor interactions. To develop a therapeutic strategy for restoring liver lncRNAs, we designed GalNAc-tagged lncRNA mimics that exhibit liver enrichment. Treatment with GalNAc-HULC mimics reduced excessive Phe in Pair -/- and Pah R408W/R408W mice and improved the Phe tolerance of these mice.

The spectrum of biochemical alterations associated with organ dysfunction and inflammatory status and their association with disease outcomes in severe COVID-19: A longitudinal cohort and time-series design study.

BACKGROUND: In patients with severe COVID-19, no data are available on the longitudinal evolution of biochemical abnormalities and their ability to predict disease outcomes. METHODS: Using a retrospective, longitudinal cohort study design on consecutive patients with severe COVID-19, we used an extensive biochemical dataset of serial data and time-series design to estimate the occurrence of organ dysfunction and the severity of the inflammatory reaction and their association with acute respiratory failure (ARF) and death. FINDINGS: On the 162 studied patients, 1151 biochemical explorations were carried out for up to 59 biochemical markers, totaling 15,260 biochemical values. The spectrum of biochemical abnormalities and their kinetics were consistent with a multi-organ involvement, including lung, kidney, heart, liver, muscle, and pancreas, along with a severe inflammatory syndrome. The proportion of patients who developed an acute kidney injury (AKI) stage 3, increased significantly during follow-up (0·9%, day 0; 21·4%, day 14; P<0·001). On the 20 more representative biochemical markers (>250 iterations), only CRP >90 mg/L (odds ratio [OR] 6·87, 95% CI, 2·36-20·01) and urea nitrogen >0·36 g/L (OR 3·91, 95% CI, 1·15-13·29) were independently associated with the risk of ARF. Urea nitrogen >0·42 g/L was the only marker associated with the risk of COVID-19 related death. INTERPRETATION: Our results point out the lack of the association between the inflammatory markers and the risk of death but rather highlight a significant association between renal dysfunction and the risk of COVID-19 related acute respiratory failure and death.

Long-term ACE Inhibitor/ARB Use Is Associated With Severe Renal Dysfunction and Acute Kidney Injury in Patients With Severe COVID-19: Results From a Referral Center Cohort in the Northeast of France.

BACKGROUND: In patients with severe coronavirus disease 2019 (COVID-19), data are scarce and conflicting regarding whether chronic use of angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) influences disease outcomes. In patients with severe COVID-19, we assessed the association between chronic ACEI/ARB use and the occurrence of kidney, lung, heart, and liver dysfunctions and the severity of the inflammatory reaction as evaluated by biomarkers kinetics, and their association with disease outcomes. METHODS: We performed a retrospective longitudinal cohort study on consecutive patients with newly diagnosed severe COVID-19. Independent predictors were assessed through receiver operating characteristic analysis, time-series analysis, logistic regression analysis, and multilevel modeling for repeated measures. RESULTS: On the 149 patients included in the study 30% (44/149) were treated with ACEI/ARB. ACEI/ARB use was independently associated with the following biochemical variations: phosphorus >40 mg/L (odds ratio [OR], 3.35, 95% confidence interval [CI], 1.83-6.14), creatinine >10.1 mg/L (OR, 3.22, 2.28-4.54), and urea nitrogen (UN) >0.52 g/L (OR, 2.65, 95% CI, 1.89-3.73). ACEI/ARB use was independently associated with acute kidney injury stage ≥1 (OR, 3.28, 95% CI, 2.17-4.94). The daily dose of ACEI/ARB was independently associated with altered kidney markers with an increased risk of +25 to +31% per each 10 mg increment of lisinopril-dose equivalent. In multivariable multilevel modeling, UN >0.52 g/L was independently associated with the risk of acute respiratory failure (OR, 3.54, 95% CI, 1.05-11.96). CONCLUSIONS: Patients chronically treated with ACEI/ARB who have severe COVID-19 are at increased risk of acute kidney injury. In these patients, the increase in UN associated with ACEI/ARB use could predict the development of acute respiratory failure.

Coronavirus disease 2019: acute Fanconi syndrome precedes acute kidney injury.

BACKGROUND: Recent data have shown that severe acute respiratory syndrome coronavirus 2 can infect renal proximal tubular cells via Angiotensin Converting Enzyme 2 (ACE2) . Our objective was to determine whether Fanconi syndrome is a frequent clinical feature in coronavirus disease 2019 (COVID-19) patients. METHODS: A retrospective cohort of 42 laboratory-confirmed COVID-19 patients without history of kidney disease hospitalized in University Hospital of Nancy was investigated. Patients were admitted to the intensive care unit (ICU) (n = 28) or the Medical department (n = 14) and were screened at least once for four markers of proximal tubulopathy. RESULTS: The mean (standard deviation) follow-up was 19.7 (±12.2) days. Of the patients, 75% (30/40) showed at least two proximal tubule abnormalities (incomplete Fanconi syndrome). The main disorders were proteinuria (88%, n = 35), renal phosphate leak defined by renal phosphate threshold/glomerular filtration rate (TmPi/GFR) <0.77 (55%, n = 22), hyperuricosuria (43%, n = 17) and normoglycaemic glycosuria (30%, n = 12). At the time of the first renal evaluation, ICU patients presented more frequent (96 versus 62%, P = 0.0095) and more severe (844 ± 343 versus 350 ± 221 mg/g, P = 0.0001) proteinuria, and a trend for an increased number of proximal tubule abnormalities (P = 0.038). During follow-up, they presented a lower nadir of serum phosphate [median (interquartile range) 0.68 (0.43-0.76) versus 0.77 (0.66-1.07) mmol/L, P = 0.044] and Acute kidney Injury (AKI) during the hospitalization (P = 0.045). Fanconi syndrome preceded severe AKI KDIGO Stages 2 and 3 in 88% (7/8) of patients. Proximal tubular abnormalities (such as proteinuria, TmPi/GFR and glycosuria in five, two and two patients, respectively) were not detected anymore in recovering patients before hospital discharge. CONCLUSION: Incomplete Fanconi syndrome is highly frequent in COVID-19 patients and precedes AKI or disappears during the recovery phase.

The Genetic Landscape and Epidemiology of Phenylketonuria.

Phenylketonuria (PKU), caused by variants in the phenylalanine hydroxylase (PAH) gene, is the most common autosomal-recessive Mendelian phenotype of amino acid metabolism. We estimated that globally 0.45 million individuals have PKU, with global prevalence 1:23,930 live births (range 1:4,500 [Italy]-1:125,000 [Japan]). Comparing genotypes and metabolic phenotypes from 16,092 affected subjects revealed differences in disease severity in 51 countries from 17 world regions, with the global phenotype distribution of 62% classic PKU, 22% mild PKU, and 16% mild hyperphenylalaninemia. A gradient in genotype and phenotype distribution exists across Europe, from classic PKU in the east to mild PKU in the southwest and mild hyperphenylalaninemia in the south. The c.1241A>G (p.Tyr414Cys)-associated genotype can be traced from Northern to Western Europe, from Sweden via Norway, to Denmark, to the Netherlands. The frequency of classic PKU increases from Europe (56%) via Middle East (71%) to Australia (80%). Of 758 PAH variants, c.1222C>T (p.Arg408Trp) (22.2%), c.1066-11G>A (IVS10-11G>A) (6.4%), and c.782G>A (p.Arg261Gln) (5.5%) were most common and responsible for two prevalent genotypes: p.[Arg408Trp];[Arg408Trp] (11.4%) and c.[1066-11G>A];[1066-11G>A] (2.6%). Most genotypes (73%) were compound heterozygous, 27% were homozygous, and 55% of 3,659 different genotypes occurred in only a single individual. PAH variants were scored using an allelic phenotype value and correlated with pre-treatment blood phenylalanine concentrations (n = 6,115) and tetrahydrobiopterin loading test results (n = 4,381), enabling prediction of both a genotype-based phenotype (88%) and tetrahydrobiopterin responsiveness (83%). This study shows that large genotype databases enable accurate phenotype prediction, allowing appropriate targeting of therapies to optimize clinical outcome.

Genetic, epigenetic and genomic mechanisms of methionine dependency of cancer and tumor-initiating cells: What could we learn from folate and methionine cycles.

Methionine-dependency is a common feature of cancer cells, which cannot proliferate without constant inputs of exogenous methionine even in the presence of its precursor, homocysteine. The endogenous synthesis of methionine is catalyzed by methionine synthase, which transfers the methyl group of 5-methyltetrahydrofolate (5-methylTHF) to homocysteine in the presence of vitamin B12 (cobalamin, cbl). Diverse mechanisms can produce it, including somatic mutations, aberrant DNA methylation (epimutations) and altered expression of genes. Around twenty somatic mutations have been reported as a cause of methionine dependency. Some of them are contributors but not sufficient on their own to cause methionine dependency. Epigenetic invalidation of MMACHC gene expression triggers methionine dependency of the MeWo-LC1 melanoma cancer cell line. This epimutation is generated by aberrant antisense transcription of the adjacent gene PRDX1. Methionine dependency involves the abnormal expression of 1-CM genes in cancer stem cells. It is related to an increased demand for methionine and SAM, which is not compensated by the increased production of formate by glycine decarboxylase pathway in lung cancer tumor spheres. Tumor spheres of glioblastoma U251 are methionine-dependent through disruption of folate metabolism. The rescue of the growth of glioblastoma stem cells by folate shows the considerable importance to evaluate the influence of supplements and dietary intake of folate on the risk of tumor development, in particular in countries subjected to mandatory food fortification in folic acid. Dietary methionine restriction or the use of methioninase represent promising anticancer therapeutic strategies that deserve to be explored in combination with chemotherapy.

Population and evolutionary genetics of the PAH locus to uncover overdominance and adaptive mechanisms in phenylketonuria: Results from a multiethnic study.

BACKGROUND: Phenylketonuria (PKU) is the most common inborn error of amino acid metabolism in Europe. The reasons underlying the high prevalence of heterozygous carriers are not clearly understood. We aimed to look for pathogenic PAH variant enrichment according to geographical areas and patients' ethnicity using a multiethnic nationwide cohort of patients with PKU in France. We subsequently appraised the population differentiation, balancing selection and the molecular evolutionary history of the PAH locus. METHODS: The French nationwide PKU study included patients who have been referred at the national level to the University Hospital of Nancy, and for whom a molecular diagnosis of phenylketonuria was made by Sanger sequencing. We performed enrichment analyses by comparing alternative allele frequencies using Fisher's exact test with Bonferroni adjustment. We estimated the amount of genetic differentiation among populations using Wright's fixation index (Fst). To estimate the molecular evolutionary history of the PAH gene, we performed phylogenetic and evolutionary analyses using whole-genome and exome-sequencing data from healthy individuals and non-PKU patients, respectively. Finally, we used exome-wide association study to decipher potential genetic loci associated with population divergence on PAH. FINDINGS: The study included 696 patients and revealed 132 pathogenic PAH variants. Three geographical areas showed significant enrichment for a pathogenic PAH variant: North of France (p.Arg243Leu), North-West of France (p.Leu348Val), and Mediterranean coast (p.Ala403Val). One PAH variant (p.Glu280Gln) was significantly enriched among North-Africans (OR = 23·23; 95% CI: 9·75-55·38). PAH variants exhibiting a strong genetic differentiation were significantly enriched in the 'Biopterin_H' domain (OR = 6·45; 95% CI: 1·99-20·84), suggesting a balancing selection pressure on the biopterin function of PAH. Phylogenetic and timetree analyses were consistent with population differentiation events on European-, African-, and Asian-ancestry populations. The five PAH variants most strongly associated with a high selection pressure were phylogenetically close and were located within the biopterin domain coding region of PAH or in its vicinity. Among the non-PAH loci potentially associated with population divergence, two reached exome-wide significance: SSPO (SCO-spondin) and DBH (dopamine beta-hydroxylase), involved in neuroprotection and metabolic adaptation, respectively. INTERPRETATION: Our data provide evidence on the combination of evolutionary and adaptive events in populations with distinct ancestries, which may explain the overdominance of some genetic variants on PAH. FUNDING: French National Institute of Health and Medical Research (INSERM) UMR_S 1256.

Folate can promote the methionine-dependent reprogramming of glioblastoma cells towards pluripotency.

Methionine dependency of tumor growth, although not well-understood, is detectable by 11C-methionine positron emission tomography and may contribute to the aggressivity of glioblastomas (GBM) and meningiomas. Cytosolic folate cycle is required for methionine synthesis. Its dysregulation may influence cell reprogramming towards pluripotency. We evaluated methionine-dependent growth of monolayer (ML) cells and stem cell-like tumor spheres (TS) derived from 4 GBM (U251, U87, LN299, T98G) and 1 meningioma (IOMM-LEE) cell lines. Our data showed that for all cell lines studied, exogenous methionine is required for TS formation but not for ML cells proliferation. Furthermore, for GBM cell lines, regardless of the addition of folate cycle substrates (folic acid and formate), the level of 3 folate isoforms, 5-methytetrahydrofolate, 5,10-methenyltetrahydrofolate, and 10-formyltetrahydrofolate, were all downregulated in TS relative to ML cells. Unlike GBM cell lines, in IOMM-LEE cells, 5-methyltetrahydrofolate was actually more elevated in TS than ML, and only 5,10-methenyltetrahydrofolate and 10-formyltetrahydrofolate were downregulated. The functional significance of this variation in folate cycle repression was revealed by the finding that Folic Acid and 5-methyltetrahydrofolate promote the growth of U251 TS but not IOMM-LEE TS. Transcriptome-wide sequencing of U251 cells revealed that DHFR, SHMT1, and MTHFD1 were downregulated in TS vs ML, in concordance with the low activity cytosolic folate cycle observed in U251 TS. In conclusion, we found that a repressed cytosolic folate cycle underlies the methionine dependency of GBM and meningioma cell lines and that 5-methyltetrahydrofolate is a key metabolic switch for glioblastoma TS formation. The finding that folic acid facilitates TS formation, although requiring further validation in diseased human tissues, incites to investigate whether excessive folate intake could promote cancer stem cells formation in GBM patients.

Plasma mSEPT9: A Novel Circulating Cell-free DNA-Based Epigenetic Biomarker to Diagnose Hepatocellular Carcinoma.

BACKGROUND: Patients with cirrhosis are at high risk of hepatocellular carcinoma (HCC). The SEPT9 gene is a key regulator of cell division and tumor suppressor whose hypermethylation is associated with liver carcinogenesis. The primary aim of this study was to evaluate the diagnostic accuracy of a PCR-based assay for the analysis of SEPT9 promoter methylation in circulating cell-free DNA (mSEPT9) for diagnosing HCC among cirrhotic patients. METHODS: We report two phase II biomarker studies that included cirrhotic patients with or without HCC from France (initial study) and Germany (replication study). All patients received clinical and biological evaluations, and liver imaging according to current recommendations. The primary outcome was defined as the presence of HCC according to guidelines from the American Association for the Study of Liver Diseases. The diagnosis of HCC was confirmed by abdominal contrast-enhanced computed tomography scan and systematically discussed in a multidisciplinary consultation meeting. HCC-free cirrhotic patients were recruited if the screening abdominal ultrasound showed no evidence of HCC at the time of blood sampling for the mSEPT9 test and on the next visit six months later. The adjudicating physicians were blinded to patient results associated with the mSEPT9 test. FINDINGS: We included 289 patients with cirrhosis (initial: 186; replication: 103), among whom 98 had HCC (initial: 51; replication: 47). The mSEPT9 test exhibited high diagnostic accuracy for HCC diagnosis, with an area under the receiver operating characteristic curve (AUROC) of 0.944 (0.900-0.970, p<0.0001) in the initial study (replication: 0.930 [0.862-0.971, p<0.0001]; meta-analysis: AUROC=0.940 [0.910-0.970, p<0.0001], no heterogeneity: I2=0%, p=0.67; and no publication bias). In multivariate logistic regression analysis, the number of positive mSEPT9 triplicates was the only independent variable significantly associated with HCC diagnosis (initial: OR=6.30, for each mSEPT9 positive triplicate [2.92-13.61, p<0.0001]; replication: OR=6.07 [3.25-11.35, p<0.0001]; meta-analysis: OR=6.15 [2.93-9.38, p<0.0001], no heterogeneity: I2=0%, p=0.95; no publication bias). AUROC associated with the discrimination of the logistic regression models in initial and validation studies were 0.969 (0.930-0.989) and 0.942 (0.878-0.978), respectively, with a pooled AUROC of 0.962 ([0.937-0.987, p<0.0001], no heterogeneity: I2=0%, p=0.36; and no publication bias). INTERPRETATION: Among patients with cirrhosis, the mSEPT9 test constitutes a promising circulating epigenetic biomarker for HCC diagnosis at the individual patient level. Future prospective studies should assess the mSEPT9 test in the screening algorithm for cirrhotic patients to improve risk prediction and personalized therapeutic management of HCC.

Association of TCN2 rs1801198 c.776G>C polymorphism with markers of one-carbon metabolism and related diseases: a systematic review and meta-analysis of genetic association studies.

Background: Vitamin B-12 (cobalamin) deficiency may produce severe neurologic and hematologic manifestations. Approximately 20-25% of circulating cobalamin binds to transcobalamin 2 (TCN2), which is referred to as active vitamin B-12. The G allele of the TCN2 c.776G>C (rs1801198) polymorphism has been associated with a lower plasma concentration of holotranscobalamin. However, genotype association studies on rs1801198 have led to conflicting results regarding its influence on one-carbon metabolism (OCM) markers or its association with pathologic conditions.Objective: We assessed the association of rs1801198 genotypes with OCM marker concentrations and primary risks of congenital abnormalities, cancer, and Alzheimer disease.Design: We conducted a systematic review of the literature that was published from January 1966 to February 2017 and included all studies that assessed the association between rs1801198 and OCM markers or a pathologic condition.Results: Thirty-four studies met the inclusion criteria. Subjects with the rs1801198 GG genotype had significantly lower concentrations of holotranscobalamin [standardized mean difference (SMD): -0.445 (95% CI: -0.673, -0.217; P < 0.001); I2 = 48.16% (95% CI: 0.00%, 78.10%; P = 0.07)] and higher concentrations of homocysteine (European descent only) [SMD: 0.070 (95% CI: 0.020, 0.120; P = 0.01); I2 = 0.00% (95% CI: 0.00%, 49.59%; P = 0.73)] than did subjects with the rs1801198 CC genotype. The meta-analysis on the association between rs1801198 and methylmalonic acid (MMA) lacked statistical power. No significant difference was observed regarding cobalamin, folate, and red blood cell folate. No significant association was observed between rs1801198 and primary risks of congenital abnormalities, cancer, or Alzheimer disease.Conclusions: Meta-analysis results indicate an influence of rs1801198 on holotranscobalamin and homocysteine concentrations in European-descent subjects. In addition, well-designed and -powered studies should be conducted for assessing the association between rs1801198 and MMA and clinical manifestations that are linked to a decreased availability of cobalamin. This review was registered at www.crd.york.ac.uk/prospero as CRD42017058504.

Genotype-phenotype associations in French patients with phenylketonuria and importance of genotype for full assessment of tetrahydrobiopterin responsiveness.

BACKGROUND: Mutations in Phenylalanine Hydroxylase (PAH) gene cause phenylketonuria. Sapropterin (BH4), the enzyme cofactor, is an important therapeutical strategy in phenylketonuria. However, PAH is a highly polymorphic gene and it is difficult to identify BH4-responsive genotypes. We seek here to improve prediction of BH4-responsiveness through comparison of genotypes, BH4-loading test, predictions of responsiveness according to the literature and types and locations of mutations. METHODS: A total of 364 French patients among which, 9 % had mild hyperphenylalaninemia, 17.7 % mild phenylketonuria and 73.1 % classical phenylketonuria, benefited from a 24-hour BH4-loading test and had the PAH gene sequenced and analyzed by Multiplex Ligation Probe Amplification. RESULTS: Overall, 31.6 % of patients were BH4-responsive. The number of different mutations found was 127, including 26 new mutations. The mutations c.434A > T, c.500A > T, c.529G > C, c.1045 T > G and c.1196 T > C were newly classified as being BH4-responsive. We identified 261 genotypes, among which 46 were newly recognized as being BH4-responsive. Even though patients carry 2 responsive alleles, BH4-responsiveness cannot be predicted with certainty unless they present mild hyperphenylalaninemia. BH4-responsiveness cannot be predicted in patients carrying one responsive mutation only. In general, the milder the phenotype is, the stronger the BH4-response is. Almost exclusively missense mutations, particularly in exons 12, 11 and 8, are associated with BH4-responsiveness and any other type of mutation predicts a negative response. CONCLUSIONS: This study is the first of its kind, in a French population, to identify the phenotype associated with several combinations of PAH mutations. As others, it highlights the necessity of performing simultaneously BH4 loading test and molecular analysis in monitoring phenylketonuria patients.

Diagnostic Accuracy of Procalcitonin for Predicting Blood Culture Results in Patients With Suspected Bloodstream Infection: An Observational Study of 35,343 Consecutive Patients (A STROBE-Compliant Article).

Previous studies have suggested that procalcitonin is a reliable marker for predicting bacteremia. However, these studies have had relatively small sample sizes or focused on a single clinical entity. The primary endpoint of this study was to investigate the diagnostic accuracy of procalcitonin for predicting or excluding clinically relevant pathogen categories in patients with suspected bloodstream infections. The secondary endpoint was to look for organisms significantly associated with internationally validated procalcitonin intervals. We performed a cross-sectional study that included 35,343 consecutive patients who underwent concomitant procalcitonin assays and blood cultures for suspected bloodstream infections. Biochemical and microbiological data were systematically collected in an electronic database and extracted for purposes of this study. Depending on blood culture results, patients were classified into 1 of the 5 following groups: negative blood culture, Gram-positive bacteremia, Gram-negative bacteremia, fungi, and potential contaminants found in blood cultures (PCBCs). The highest procalcitonin concentration was observed in patients with blood cultures growing Gram-negative bacteria (median 2.2 ng/mL [IQR 0.6-12.2]), and the lowest procalcitonin concentration was observed in patients with negative blood cultures (median 0.3 ng/mL [IQR 0.1-1.1]). With optimal thresholds ranging from ≤0.4 to ≤0.75 ng/mL, procalcitonin had a high diagnostic accuracy for excluding all pathogen categories with the following negative predictive values: Gram-negative bacteria (98.9%) (including enterobacteria [99.2%], nonfermenting Gram-negative bacilli [99.7%], and anaerobic bacteria [99.9%]), Gram-positive bacteria (98.4%), and fungi (99.6%). A procalcitonin concentration ≥10 ng/mL was associated with a high risk of Gram-negative (odds ratio 5.98; 95% CI, 5.20-6.88) or Gram-positive (odds ratio 3.64; 95% CI, 3.11-4.26) bacteremia but dramatically reduced the risk of PCBCs or fungemia. In this large real-life setting experience with more than 35,000 patients, procalcitonin was highly effective at excluding bloodstream infections regardless of pathogen categories. The results from our study are limited by its cross-sectional design and deserve to be validated in prospective longitudinal studies.

CARM1 and PRMT1 are dysregulated in lung cancer without hierarchical features.

CARM1 and PRMT1 are 2 Protein Arginine Methyl Transferases (PRMT) dysregulated in cancer. CARM1 function is contradictory and depicted as facilitating proliferation or differentiation. PRMT1 is required for cell proliferation. CARM1 and PRMT1 cooperate for gene regulation. We report that CARM1 and PRMT1 are significantly overexpressed in 60 patients with Non-Small Cell Lung Carcinomas (NSCLC). CARM1 and PRMT1 correlated in healthy but not tumor tissue. Their levels of expression in tumor tissue were proportional to their levels of expression in the counterpart healthy tissue. Only CARM1 expression was found to be correlated with tumor differentiation and neither CARM1 nor PRMT1 expression was correlated with survival. Accordingly, CARM1 and PRMT1 are overexpressed in 2 NSCLC cell lines, A549 and H1299. Targeting PRMT1 with siRNA reduced proliferation, by decreasing cell growth and inhibiting soft agar colony formation, and promoted differentiation, by increasing the epithelial markers cytokeratin 7 and 8 and decreasing Neuromedin B receptor, which binds a mitogenic factor. siCARM1 yielded similar consequences but the conditions with siCARM1 reflected inhibition of both CARM1 and PRMT1. Together these results suggest that CARM1 and PRMT1 are involved in proliferation in lung cancer with no hierarchy of one protein over the other. The fact that CARM1 targeting suppresses PRMT1 in addition to CARM1 reinforces the functional importance of CARM1/PRMT1 interaction.

Folate and fetal programming: a play in epigenomics?

Folate plays a key role in the interactions between nutrition, fetal programming, and epigenomics. Maternal folate status influences DNA methylation, inheritance of the agouti phenotype, expression of imprinting genes, and the effects of mycotoxin FB1 on heterochromatin assembly in rodent offspring. Deficiency in folate and other methyl donors increases birth defects and produces visceral manifestations of fetal programming, including liver and heart steatosis, through imbalanced methylation and acetylation of PGC1-α and decreased SIRT1 expression, and produces persistent cognitive and learning disabilities through impaired plasticity and hippocampal atrophy. Maternal folate supplementation also produces long-term epigenomic effects in offspring, some beneficial and others negative. Deciphering these mechanisms will help understanding the discordances between experimental models and population studies of folate deficiency and supplementation.