Quality control of microbiota metagenomics by k-mer analysis.

BACKGROUND: The biological and clinical consequences of the tight interactions between host and microbiota are rapidly being unraveled by next generation sequencing technologies and sophisticated bioinformatics, also referred to as microbiota metagenomics. The recent success of metagenomics has created a demand to rapidly apply the technology to large case-control cohort studies and to studies of microbiota from various habitats, including habitats relatively poor in microbes. It is therefore of foremost importance to enable a robust and rapid quality assessment of metagenomic data from samples that challenge present technological limits (sample numbers and size). Here we demonstrate that the distribution of overlapping k-mers of metagenome sequence data predicts sequence quality as defined by gene distribution and efficiency of sequence mapping to a reference gene catalogue. RESULTS: We used serial dilutions of gut microbiota metagenomic datasets to generate well-defined high to low quality metagenomes. We also analyzed a collection of 52 microbiota-derived metagenomes. We demonstrate that k-mer distributions of metagenomic sequence data identify sequence contaminations, such as sequences derived from "empty" ligation products. Of note, k-mer distributions were also able to predict the frequency of sequences mapping to a reference gene catalogue not only for the well-defined serial dilution datasets, but also for 52 human gut microbiota derived metagenomic datasets. CONCLUSIONS: We propose that k-mer analysis of raw metagenome sequence reads should be implemented as a first quality assessment prior to more extensive bioinformatics analysis, such as sequence filtering and gene mapping. With the rising demand for metagenomic analysis of microbiota it is crucial to provide tools for rapid and efficient decision making. This will eventually lead to a faster turn-around time, improved analytical quality including sample quality metrics and a significant cost reduction. Finally, improved quality assessment will have a major impact on the robustness of biological and clinical conclusions drawn from metagenomic studies.

TP53 R249S mutation, genetic variations in HBX and risk of hepatocellular carcinoma in The Gambia.

In regions with high prevalence of chronic hepatitis B virus (HBV) infection and dietary aflatoxin B(1) (AFB(1)) exposure, hepatocellular carcinomas (HCCs) often contain TP53 mutation at codon 249 (R249S). Furthermore, a C-terminal truncated HBx protein expressed from hepatocyte integrated HBV is associated with HCC development. This study evaluates the association between R249S and HBX status in relation to HCC in West African population. HBX (complete or 3'-truncated) and HBS genes were assessed by PCR in cell-free DNA (CFDNA) from plasma of subjects recruited in a hospital-based case-control study (325 controls, 78 cirrhotic patients and 198 HCC cases) conducted in The Gambia. These samples had been previously analyzed for R249S and HBV serological status. Complete HBX sequence was frequently detected in CFDNA of HCC-R249S positive (77%, 43/56) compared with HCC-R249S-negative cases (44%, 22/50). Conversely, the proportion of 3'-truncated HBX gene was significantly higher in HCC-R249S negative than positive cases (34%, 17/50, compared with 12%, 7/56) (χ(2) = 12.12; P = 0.002; distribution of R249S negative and positive according to HBX status). Occult HBV infection (detected by PCR) was present in 24% of HCC previously considered as negative by HBV serology. Moreover, HBV mutation analysis revealed that double mutation at nucleotides 1762(T)/1764(A) was associated with diagnosis of cirrhosis or HCC {cirrhosis: odds ratio (OR): 9.50 [95% confidence interval (CI) 1.50-60.11]; HCC: OR: 11.29 [95% CI 2.07-61.47]}. These findings suggest that in HCC from The Gambia, complete HBX sequences are often associated with the presence of TP53 R249S mutation.

Interactions between hepatitis B virus and aflatoxin B(1): effects on p53 induction in HepaRG cells.

Infection by hepatitis B virus (HBV) and dietary exposure to aflatoxin B(1) (AFB(1)) are the main risk factors for the development of chronic liver disease and hepatocellular carcinoma (HCC). How these factors cooperate is still largely unknown. AFB(1) activation leads to DNA adduction and mutagenesis, with a specific mutation at codon 249 in TP53 (p.R249S). So far, only limited studies have addressed the effects of AFB(1) on HBV replication. We have analysed the effects of both risk factors on p53 induction during HBV infection in HepaRG, a cell line with hepatocyte-like morphology that metabolizes AFB(1) and supports HBV infection. Exposure to AFB(1) up to 5 µM induced a downregulation of HBV replication after 48 h, as measured by a decrease in viral antigens in the culture medium (HBsAg, HBeAg and large envelope protein) and in intracellular levels of HBV transcripts, DNA and HBsAg. Conversely, HBV infection did not significantly modify AFB(1)-DNA adduct formation or repair as assessed by immunodot-blot assay, and the induction of p53 in response to AFB(1) was similar in infected and non-infected HepaRG cells. Overall, our results suggest that AFB(1) exposure decreases HBV replication, whereas DNA damage by AFB(1) and subsequent p53 induction is not affected by the presence of the virus. Thus, in HepaRG cell line, AFB(1) and HBV do not cooperate to increase DNA damage by AFB(1). Further studies on the effects of both factors in a context of chronicity are needed to better understand synergistic effects.

Effects of the TP53 p.R249S mutant on proliferation and clonogenic properties in human hepatocellular carcinoma cell lines: interaction with hepatitis B virus X protein.

Aflatoxin B(1) (AFB(1)) is a risk factor for hepatocellular carcinoma (HCC) in many low-resource countries. Although its metabolites bind at several positions in TP53, a mutation at codon 249 (AGG to AGT, arginine to serine, p.R249S) accounts for 90% of TP53 mutations in AFB(1)-related HCC. This specificity suggests that p.R249S confers a selective advantage during hepatocarcinogenesis. Using HCC cell lines, we show that p.R249S has lost the capacity to bind to p53 response elements and to transactivate p53 target genes. In p53-null Hep3B cells, stable transfection of p.R249S or of another mutant, p.R248Q, did not induce significant changes in cell proliferation and survival after cytotoxic stress. In contrast, in a cell line that constitutively expresses both p.R249S and the hepatitis B virus antigen HBx (PLC/PRF/5), silencing of either p.R249S or HBx by RNA interference slowed down proliferation, with no additive effects when both factors were silenced. Furthermore, the two proteins appear to form a complex. In human HCC samples, mutation at codon 249 did not correlate with p.R249S protein accumulation or HBx truncation status. We suggest that p.R249S may contribute to hepatocarcinogenesis through interaction with HBx, conferring a subtle growth advantage at early steps of the transformation process, but that this interaction is not required for progression to advanced HCC.

Microbial ecology perturbation in human IgA deficiency.

Paradoxically, loss of immunoglobulin A (IgA), one of the most abundant antibodies, does not irrevocably lead to severe infections in humans but rather is associated with relatively mild respiratory infections, atopy, and autoimmunity. IgA might therefore also play covert roles, not uniquely associated with control of pathogens. We show that human IgA deficiency is not associated with massive quantitative perturbations of gut microbial ecology. Metagenomic analysis highlights an expected pathobiont expansion but a less expected depletion in some typically beneficial symbionts. Gut colonization by species usually present in the oropharynx is also reminiscent of spatial microbiota disorganization. IgM only partially rescues IgA deficiency because not all typical IgA targets are efficiently bound by IgM in the intestinal lumen. Together, IgA appears to play a nonredundant role at the forefront of the immune/microbial interface, away from the intestinal barrier, ranging from pathobiont control and regulation of systemic inflammation to preservation of commensal diversity and community networks.

Association between HBX status, aflatoxin-induced R249S TP53 mutation and risk of hepatocellular carcinoma in a case-control study from Thailand.

Hepatocellular carcinoma (HCC) is associated with hepatitis B virus (HBV) chronicity and dietary exposure to aflatoxin, a mutagen targeting codon 249 of tumor suppressor TP53 (R249S mutation). Based on a case-control in Thailand, we have measured R249S and the status of HBX gene in plasma DNA of 176 cases and 133 referents. Detection of HBX complete sequences was associated with R249S in HCC with no documented prior cirrhosis but not in HCC developing in a context of cirrhosis or in non-cancer chronic liver diseases. Thus, R249S may specifically cooperate with HBX in a pathway to HCC that bypasses cirrhosis.

Aflatoxin-induced TP53 R249S mutation in hepatocellular carcinoma in Thailand: association with tumors developing in the absence of liver cirrhosis.

Primary Liver Cancer (PLC) is the leading cause of death by cancer among males in Thailand and the 3(rd) among females. Most cases are hepatocellular carcinoma (HCC) but cholangiocarcinomas represent between 4 and 80% of liver cancers depending upon geographic area. Most HCC are associated with chronic infection by Hepatitis B Virus while a G → T mutation at codon 249 of the TP53 gene, R249S, specific for exposure to aflatoxin, is detected in tumors for up to 30% of cases. We have used Short Oligonucleotide Mass Analysis (SOMA) to quantify free circulating R249S-mutated DNA in plasma using blood specimens collected in a hospital case:control study. Plasma R249S-mutated DNA was detectable at low concentrations (≥ 67 copies/mL) in 53 to 64% of patients with primary liver cancer or chronic liver disease and in 19% of controls. 44% of patients with HCC and no evidence of cirrhosis had plasma concentrations of R249S-mutated DNA ≥ 150 copies/mL, compared to 21% in patients with both HCC and cirrhosis, 22% in patients with cholangiocarcinoma, 12% in patients with non-cancer chronic liver disease and 3% of subjects in the reference group. Thus, plasma concentrations of R249S-mutated DNA ≥ 150 copies/mL tended to be more common in patients with HCC developing without pre-existing cirrhosis (p = 0.027). Overall, these results support the preferential occurrence of R249S-mutated DNA in HCC developing in the absence of cirrhosis in a context of HBV chronic infection.

TP53 Mutations and HBX Status Analysis in Hepatocellular Carcinomas from Iran: Evidence for Lack of Association between HBV Genotype D and TP53 R249S Mutations.

High incidence of HCC is mostly due to the combination of two major risk factors, chronic infection with hepatitis B (HBV) and/or C (HCV) viruses and exposure to the mycotoxin aflatoxin B(1), which induces a particular mutation at codon 249 in TP53 (R249S). Eight genotypes of HBV are diversely found in high and low incidence areas. Regardless of documented strong associations between TP53 R249S mutation and HBV genotypes B, C, A or E, there is no report of such association for genotype D despite of the presence of aflatoxin in areas with high prevalence of HBV genotype D. In Iran, 3% of the population is chronically infected with HBV, predominantly genotype D. Twenty-one histologically confirmed HCC cases from Iran were analyzed for TP53 R249S and HBV double mutations 1762(T)/1764(A), hallmarks of more pathogenic forms of HBV. We did not detect any of these mutations. In addition, we report the only case identified so far carrying both R249S mutation and chronic HBV genotype D, a patient from The Gambia in West Africa. This paper suggests that association between HBV genotype D and aflatoxin-induced TP53 mutation is uncommon, explaining the relatively lower incidence of HCC in areas where genotype D is highly prevalent.

Mutations in TP53 and CTNNB1 in Relation to Hepatitis B and C Infections in Hepatocellular Carcinomas from Thailand.

Hepatocellular carcinoma (HCC) may develop according to two major pathways, one involving HBV infection and TP53 mutation and the other characterized by HCV infection and CTNNB1 mutation. We have investigated HBV/HCV infections and TP53/CTNNB1 mutations in 26 HCC patients from Thailand. HBV DNA (genotype B or C) was detected in 19 (73%) of the cases, including 5 occult infections and 3 coinfections with HCV. TP53 and CTNNB1 mutations were not mutually exclusive, and most of TP53 mutations were R249S, suggesting a significant impact of aflatoxin-induced mutagenesis in HCC development.

Seasonal variation in TP53 R249S-mutated serum DNA with aflatoxin exposure and hepatitis B virus infection.

BACKGROUND: Chronic hepatitis B virus (HBV) infection and dietary aflatoxin B1 (AFB1) exposure are etiological factors for hepatocellular carcinoma (HCC) in countries with hot, humid climates. HCC often harbors a TP53 (tumor protein p53) mutation at codon 249 (R249S). In chronic carriers, 1762T/1764A mutations in the HBV X gene are associated with increased HCC risk. Both mutations have been detected in circulating cell-free DNA (CFDNA) from asymptomatic HBV carriers. OBJECTIVE: We evaluated seasonal variation in R249S and HBV in relation to AFB1 exposure. METHODS: R249S was quantitated by mass spectrometry in CFDNA in a cross-sectional survey of 473 asymptomatic subjects (237 HBV carriers and 236 noncarriers) recruited in three villages in the Gambia over a 10-month period. 1762T/1764A HBV mutations were detected by quantitative polymerase chain reaction. In addition, the HBV S gene was sequenced in 99 subjects positive for HBV surface antigen (HBsAg). RESULTS: We observed a seasonal variation of serum R249S levels. Positivity for R249S and average concentration were significantly higher in HBsAg-positive subjects surveyed during April-July (61%; 5,690 ± 11,300 R249S copies/mL serum) than in those surveyed October-March [32% and 480 ± 1,030 copies/mL serum (odds ratio = 3.59; 95% confidence interval: 2.05, 6.30; p < 0.001)]. Positivity for HBV e antigen (HBeAg) (a marker of HBV replication) and viral DNA load also varied seasonally, with 15-30% of subjects surveyed between April and June HBeAg positive, compared with < 10% surveyed during other months. We detected 1762T/1764A mutations in 8% of carriers, half of whom were positive for R249S. We found HBV genotype E in 95 of 99 HBsAg-positive subjects. CONCLUSION: R249S is detectable in CFDNA of asymptomatic subjects. Evidence of temporal and quantitative variations suggests an interaction among AFB1 exposure, HBV positivity, and replication on TP53 mutation formation or persistence.

Hepatitis B and Hepatitis C Infection Biomarkers and TP53 Mutations in Hepatocellular Carcinomas from Colombia.

Hepatocellular Carcinoma (HCC) is a leading cause of cancer-related death worldwide. Globally, the most important HCC risk factors are Hepatitis B Virus (HBV) and/or Hepatitis C Virus (HCV), chronic alcoholism, and dietary exposure to aflatoxins. We have described the epidemiological pattern of 202 HCC samples obtained from Colombian patients. Additionally we investigated HBV/HCV infections and TP53 mutations in 49 of these HCC cases. HBV biomarkers were detected in 58.1% of the cases; HBV genotypes F and D were characterized in three of the samples. The HCV biomarker was detected in 37% of the samples while HBV/HCV coinfection was found in 19.2%. Among TP53 mutations, 10.5% occur at the common aflatoxin mutation hotspot, codon 249. No data regarding chronic alcoholism was available from the cases. In conclusion, in this first study of HCC and biomarkers in a Colombian population, the main HCC risk factor was HBV infection.

The aflatoxin-induced TP53 mutation at codon 249 (R249S): biomarker of exposure, early detection and target for therapy.

Hepatocellular Carcinoma (HCC) in high-incidence areas (sub-Saharan Africa, South-Eastern Asia) often contains a somatic mutation at codon 249 in TP53 (R249S). This mutation is rare in low-incidence areas of Europe and the United States. There is evidence that R249S occurs as the result of mutagenesis by aflatoxin in a context of HBV chronic infection. Here, we summarize the mechanisms of R249S formation and the possible role of p.R249S protein in HCC pathogenesis. Next, we discuss the significance of R249S as a biomarker to study the natural history of HCC and as a target for therapeutic approaches aimed at restoring wild-type p53 activity.