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.

Folate- and vitamin B12-deficient diet during gestation and lactation alters cerebellar synapsin expression via impaired influence of estrogen nuclear receptor α.

Deficiency in the methyl donors vitamin B12 and folate during pregnancy and postnatal life impairs proper brain development. We studied the consequences of this combined deficiency on cerebellum plasticity in offspring from rat mothers subjected to deficient diet during gestation and lactation and in rat neuroprogenitor cells expressing cerebellum markers. The major proteomic change in cerebellum of 21-d-old deprived females was a 2.2-fold lower expression of synapsins, which was confirmed in neuroprogenitors cultivated in the deficient condition. A pathway analysis suggested that these proteomic changes were related to estrogen receptor α (ER-α)/Src tyrosine kinase. The influence of impaired ER-α pathway was confirmed by abnormal negative geotaxis test at d 19-20 and decreased phsophorylation of synapsins in deprived females treated by ER-α antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP). This effect was consistent with 2-fold decreased expression and methylation of ER-α and subsequent decreased ER-α/PPAR-γ coactivator 1 α (PGC-1α) interaction in deficiency condition. The impaired ER-α pathway led to decreased expression of synapsins through 2-fold decreased EGR-1/Zif-268 transcription factor and to 1.7-fold reduced Src-dependent phosphorylation of synapsins. The treatment of neuroprogenitors with either MPP or PP1 (4-(4'-phenoxyanilino)-6,7-dimethoxyquinazoline, 6,7-dimethoxy-N-(4-phenoxyphenyl)-4-quinazolinamine, SKI-1, Src-l1) Src inhibitor produced similar effects. In conclusion, the deficiency during pregnancy and lactation impairs the expression of synapsins through a deregulation of ER-α pathway.

Association of IL1B polymorphism with left ventricular systolic dysfunction: a relation with the release of interleukin-1ß in stress condition.

OBJECTIVE: Proinflammatory cytokines play a major role in the pathomechanisms of heart failure. Besides this, the influence of mental stress on heart failure is poorly documented despite its effects on sympathetic stimulation of interleukin-1ß (IL-1ß) secretion. We examined whether the polymorphisms of proinflammatory cytokines are predictors of left ventricular systolic dysfunction (LVSD) and if so, whether such associations are related to the secretion of these cytokines, in 572 consecutive patients under mental stress produced by coronary angiography. METHODS: We examined IL-1RN (VNTR), IL1A-889 C>T, IL1B-511 C>T, IL6-174 G>C and TNFA-308 G>A, according to LVSD (left ventricular ejection fraction, <40%). Saliva IL-1ß, serum tumour necrosis factor-α and C-reactive protein were assayed in basal (T0 and T2, before and after coronary angiography) and stress (T1) conditions. MAIN RESULTS: The 42.1% of patients with LVSD had a 1.5-fold higher frequency of IL1B T allele (P<0.001). IL1B-511TT was associated with LVSD (P=0.008) and with a decrease in IL-1ß level in saliva at T1 (P=0.013). IL-1ß was the highest at T1 (P<0.001) and was associated with left ventricular ejection fraction (P=0.002). The IL1B TT genotype and the C-reactive protein were the two independent predictors of LVSD in multivariate analysis, with an odds ratio of 2.7 (95% confidence interval: 1.3-5.5; P=0.008) and 1.1 (95% confidence interval: 1.1-1.2; P<0.001), respectively. CONCLUSION: IL1B was a predictor of LVSD and of the decreased IL-1ß response to stress. This suggests that IL1B exerts an influence on LVSD through its effect on IL-1ß secretion.

Environmental influence on the worldwide prevalence of a 776C->G variant in the transcobalamin gene (TCN2).

BACKGROUND: A 776C-->G variant (dbSNP ID: rs1801198) in the transcobalamin gene (TCN2; MIM# 275350) decreases the cellular and plasma concentration of transcobalamin and thereby influences the cellular availability of vitamin B(12). OBJECTIVE: To evaluate the worldwide prevalence of this variant and its association with homocysteine plasma level. METHODS: The study was performed in 1433 apparently healthy subjects, including Afro-Americans and Afro-Africans and in 251 Afro-Africans participants with severe malaria. RESULTS: The frequencies of the 776G allele were the highest in China (0.607; 95% CI 0.554 to 0.659), low in West Africa (Bénin and Togo, 0.178; 0.154 to 0.206), and intermediate in France (0.445; 0.408 to 0.481), Italy (0.352; 0.299 to 0.409), Morocco (0.370; 0.300 to 0.447) and Mexico (0.374; 0.392 to 0.419). The 776G genotype was more frequent in Afro-Americans from New York (16.7; 8.4 to 30.7) and in Afro-African patients with severe malaria (6.0%; 95% CI 3.7 to 9.6) than in healthy Afro-African volunteers (p = 0.0004 and p = 0.033, respectively), while no difference was observed for MTHFR 677TT and 677T alleles. A disequilibrium of TCN2 genotype distribution was recorded in patients with severe malaria, with a twofold higher GG genotype than expected (p = 0.010). An association between the TCN2 polymorphism and homocysteine was observed only in Mexico and France, the two countries with the highest rate of low plasma concentration of vitamin B(12) (<100 pmol/l). CONCLUSION: Given the dramatic heterogeneity of the 776G allele frequency worldwide, this polymorphism may be prone to a selective pressure or confers an evolutionary advantage in confronting environmental factors, one of which is malaria.

Prevalence of methylenetetrahydrofolate reductase 677T and 1298C alleles and folate status: a comparative study in Mexican, West African, and European populations.

BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR) 677C-->T polymorphism is heterogeneously distributed worldwide, with the highest and lowest frequencies of the T allele in Mexico and Africa, respectively, and a south-to-north gradient in Europe. Distribution of MTHFR 1298A-->C is less well known. It has been hypothesized that 677T frequency could result in part from gene-nutrient interactions. OBJECTIVE: The objective was to compare the association of 677T and 1298C alleles with plasma concentrations of homocysteine, folate, and vitamin B-12 in geographical areas with contrasting 677T allele frequencies. DESIGN: Healthy young adults (n = 1277) were recruited in Mexico City, the West African countries of Bénin and Togo, France, and Sicily (Italy). Homocysteine, folate, and vitamin B-12 were measured in plasma, and MTHFR polymorphisms were measured in genomic DNA. RESULTS: Mexico City and Sicily reported the highest and Bénin and Togo reported the lowest plasma concentrations of folate. Mexico City had the highest 677T allele prevalence and the lowest influence of 677TT genotype on homocysteine, whereas the opposite was observed in Africa. The prevalence of the 1298C allele was lowest in the Mexicans and Africans and highest in the French. The percentage of the 677T genotype was significantly associated with the folate concentrations in 677CC carriers in a univariate analysis (R = 0.976; 95% CI: 0.797, 0.996; P < 0.0002) and in a multiple regression model that included homocysteine, vitamin B-12, and age (P = 0.0002). CONCLUSION: Our data agree with the hypothesis of a gene-nutrient interaction between MTHFR 677C-->T polymorphism and folate status that may confer a selective advantage of TT-homozygous genotype when dietary intake of folate is adequate, at least in the areas studied.