Neurofibromatosis type 2 French cohort analysis using a comprehensive NF2 molecular diagnostic strategy.

OBJECTIVE: Neurofibromatosis type 2 (NF2) affects about one in 25,000 to 40,000 people. Most NF2 patients have private loss-of-function mutations scattered along the NF2 gene. Here, we present our NF2 investigation strategy. MATERIAL AND METHODS: We report a comprehensive NF2 mutation analysis of 221 NF2 French patients: 134 unrelated typical NF2 patients fulfilling the Manchester criteria and 87 unrelated patients presenting symptoms that partially fulfilled the Manchester criteria. RESULTS: A NF2 mutation was identified in 56 of the 221 patients, giving a global mutation detection rate of 25%. This rate reached 37% (49/134) for typical NF2 patients fulfilling the Manchester criteria and only 8% (7/87) for patients presenting symptoms suggestive of NF2. Six of these seven patients were under 25 of age. Our approach showed that 77% of NF2 identified variants were detected by coding exons sequencing. Multiplex ligation-dependent probe amplification allowed the identification of restricted rearrangements (23% of NF2 identified variants corresponding to complete deletion or partial deletion/duplication of NF2). CONCLUSION: High mutation detection rate can be achieved if well phenotyped NF2 patients are studied with multiple complementary and optimized techniques. NF2 somatic mosaicism detection was improved by frozen tumor samples molecular analysis.

Versatile and precise gene-targeting strategies for functional studies in mammalian cell lines.

The advent of programmable nucleases such as ZFNs, TALENs and CRISPR/Cas9 has brought the power of genetic manipulation to widely used model systems. In mammalian cells, nuclease-mediated DNA double strand break is mainly repaired through the error-prone non-homologous end-joining (NHEJ) repair pathway, eventually leading to accumulation of small deletions or insertions (indels) that can inactivate gene function. However, due to the variable size of the indels and the polyploid status of many cell lines (e.g., cancer-derived cells), obtaining a knockout usually requires lengthy screening and characterization procedures. Given the more precise type of modifications that can be introduced upon homology-directed repair (HDR), we have developed HDR-based gene-targeting strategies that greatly facilitate the process of knockout generation in cell lines. To generate reversible knockouts (R-KO), a selectable promoter-less STOP cassette is inserted in an intron, interrupting transcription. Loss-of-function can be validated by RT-qPCR and is removable, enabling subsequent restoration of gene function. A variant of the R-KO procedure can be used to introduce point mutations. To generate constitutive knockouts (C-KO), an exon is targeted, which makes use of HDR-based gene disruption together with NHEJ-induced indels on non-HDR targeted allele(s). Hence the C-KO procedure greatly facilitates simultaneous inactivation of multiple alleles. Overall these genome-editing tools offer superior precision and efficiency for functional genetic approaches. We provide detailed protocols guiding in the design of targeting vectors and in the analysis and validation of gene targeting experiments.

Uveal melanoma hepatic metastases mutation spectrum analysis using targeted next-generation sequencing of 400 cancer genes.

AIMS: Uveal melanoma (UM) is the most common malignant tumour of the eye. Diagnosis often occurs late in the course of disease, and prognosis is generally poor. Recently, recurrent somatic mutations were described, unravelling additional specific altered pathways in UM. Targeted next-generation sequencing (NGS) can now be applied to an accurate and fast identification of somatic mutations in cancer. The aim of the present study was to characterise the mutation pattern of five UM hepatic metastases with well-defined clinical and pathological features. METHODS: We analysed the UM mutation spectrum using targeted NGS on 409 cancer genes. RESULTS: Four previous reported genes were found to be recurrently mutated. All tumours presented mutually exclusive GNA11 or GNAQ missense mutations. BAP1 loss-of-function mutations were found in three UMs. SF3B1 missense mutations were found in the two UMs with no BAP1 mutations. We then searched for additional mutation targets. We identified the Arg505Cys mutation in the tumour suppressor FBXW7. The same mutation was previously described in different cancer types, and FBXW7 was recently reported to be mutated in UM exomes. CONCLUSIONS: Further studies are required to confirm FBXW7 implication in UM tumorigenesis. Elucidating the molecular mechanisms underlying UM tumorigenesis holds the promise for novel and effective targeted UM therapies.

[PSA testing, biopsy and cancer and benign prostate hyperplasia in France]. / Dosage du PSA, biopsie, cancer et hypertrophie bénigne de la prostate en France.

INTRODUCTION: Prostate-specific antigen (PSA) testing is high in France. The aim of this study was to estimate their frequency and those of biopsy and newly diagnosed cancer (PCa) according to the presence or absence of treated benign prostatic hyperplasia (BPH). PATIENTS AND METHODS: This study concerned men 40 years and older covered by the main French national health insurance scheme (73 % of all men of this age). Data were collected from the national health insurance information system (SNIIRAM). This database comprehensively records all of the outpatient prescriptions and healthcare services reimbursed. This information are linked to data collected during hospitalisations. RESULTS: The frequency of men without diagnosed PCa (10.9 millions) with at least one PSA test was very high in 2011 (men aged 40 years and older: 30 %, 70-74 years: 56 %, 85 years and older: 33 % and without HBP: 25 %, 41 % and 19 %). Men with treated BPH totalized 9 % of the study population, but 18 % of the men with at least one PSA test, 44 % of those with at least one prostate biopsy and 40 % of those with newly managed PCa. Over a 3-year period, excluding men with PCa, 88 % of men with BPH had at least one PSA test and 52 % had three or more PSA tests versus 52 % and 15 % for men without BPH. One year after PSA testing, men of 55-69 years with BPH more frequently underwent prostate biopsy than those without BPH (5.4 % vs 1.8 %) and presented PCa (1.9 % vs 0.9 %). CONCLUSIONS: PSA testing frequencies in France are very high even after exclusion of men with BPH, who can be a group with more frequent managed PCa. LEVEL OF EVIDENCE: 4.

IFNL3 (IL28B) polymorphism does not predict long-term response to interferon therapy in HBeAg-positive chronic hepatitis B patients.

UNLABELLED: The impact of IFNL3 (IL28B) polymorphism on response to interferon (IFN) treatment in patients infected with hepatitis B virus (HBV) is controversial. We aimed to investigate whether IFNL3 polymorphism (rs12979860) influences the long-term response of chronic hepatitis B (CHB) treatment to conventional IFN. DESIGN: Ninety-seven HBeAg-positive patients treated with IFN were evaluated in this study. Associations were investigated between IFNL3 genotypes and (i) HBeAg seroconversion at the end of treatment (EOT), (ii) sustained virological response (SVR) and (iii) HBsAg seroconversion through long-term follow-up (LTFU). Patients were followed for a median of 14 years. The majority of patients were infected with HBV genotype A (69.6%) and were Caucasian (77.9%). Ninety-five patients were genotyped at rs12979860. Similar IFNL3 distribution was observed among the different ethnicities (P = 0.62) or across HBV genotypes A through G (P = 0.70). Thirty-six patients experienced HBeAg seroconversion at EOT; HBeAg seroconversion rates were 37.0 and 35.5% in patients with CC and CT/TT genotypes, respectively (P = 0.82). Among the 44 patients (45%) who achieved a SVR, SVR rates were 48.9 and 39.6% in patients with CC and CT/TT IL28B genotypes, respectively (P = 0.80). HBsAg seroconversion occurred through LTFU in 28 patients. HBsAg seroconversion rates were 25.5 and 31.2% in patients with CC and CT/TT genotypes, respectively (P = 0.51). No significant relationship between IFNL3 rs12979860 and fibrosis stage was observed (P = 0.85). IFNL3 genotype was neither associated with SVR, nor with HBeAg seroconversion and long-term HBsAg seroconversion in HBeAg-positive CHB patients responding to IFN therapy.

Single agent and combination studies of pralatrexate and molecular correlates of sensitivity.

BACKGROUND: Pralatrexate is a dihydrofolate reductase (DHFR) inhibitor with high affinity for reduced folate carrier 1 (RFC-1) and folylpolyglutamate synthetase (FPGS), resulting in extensive internalization and accumulation in tumour cells. Pralatrexate is approved in the US for the treatment of relapsed or refractory peripheral T-cell lymphoma and is being investigated in various malignancies. Here, we evaluated molecular correlates of sensitivity to pralatrexate and explored combinations with a variety of anticancer agents. METHODS: Antiproliferative effects of pralatrexate were evaluated in 15 human-cancer cell lines using the MTT assay. Gene expression was evaluated using qRT-PCR. RESULTS: Pralatrexate and methotrexate had a similar pattern of cytotoxicity, pralatrexate being more potent. Pralatrexate potentiated the effects of platinum drugs, antimetabolites and EGFR inhibitors. Dose- and time-dependent cytotoxicity of pralatrexate correlated with high mRNA expression of FPGS. Acquired resistance to pralatrexate was associated with decreased RFC-1 expression, whereas methotrexate resistance correlated with increased DHFR expression, suggesting different mechanisms of acquired resistance. CONCLUSION: Pralatrexate was more potent than methotrexate in a panel of solid tumour lines. Our findings support the further clinical development of pralatrexate in combination with certain cytotoxics and targeted therapies, and suggest that RFC-1, FPGS and DHFR may be potential biomarkers of outcome.

Sequential therapy with adefovir dipivoxil and pegylated interferon alfa-2a for HBeAg-negative patients.

To assess the impact of sequential therapy with adefovir dipivoxil (ADV) and pegylated interferon alfa-2a (PEG-IFN) on virological (serum HBV-DNA) and serological (serum HBsAg) response in 20 consecutive HBeAg-negative patients. Patients received ADV for 20 weeks, then ADV and PEG-IFN for 4 weeks and lastly PEG-IFN for 44 weeks. Serum HBV-DNA and HBsAg were assessed at baseline, during therapy (weeks 20, 44 and 68) and follow-up (weeks 92 and 116). Sustained virological response (SVR) was defined as serum HBV-DNA <10 000 copies/mL (partial) or <70 copies/mL (complete) 24 weeks after stopping treatment. A serological response was defined as a serum HBsAg decrease ≥1 log(10) IU/mL at the end of treatment. Baseline median serum HBV-DNA and HBsAg levels were 7.6 log(10) copies/mL and 3.8 log(10) IU/mL, respectively. Ten patients (50%) achieved SVR, six of them had partial response and four complete response. Four patients (20%) achieved serological response. Complete SVRs showed a major and steep decline in HBsAg level with a median decrease of 0.5, 1.6 and 2.0 log(10) IU/mL at treatment week 20, 44 and 68, respectively. Partial SVRs showed a slight and slow decline in serum HBsAg level (0.1, 0.4, and 0.6 log IU/mL at weeks 20, 44 and 68, respectively). On-treatment serum HBsAg decrease had a high accuracy to predict SVR (AUROC = 0.88). Our results suggest that sequential therapy might be an interesting strategy for HBeAg-negative patients. Serum HBsAg kinetics seem to be an accurate tool to predict SVR. Large clinical trials are needed to explore this strategy with more potent analogues.

Expression and regulation by PPARgamma of hCG alpha- and beta-subunits: comparison between villous and invasive extravillous trophoblastic cells.

During human placental development trophoblast follows two differentiation pathways: the extravillous (EVCT) and the villous cytotrophoblasts (VCT) that display different phenotypes and functions. It is well established that human chorionic gonadotropin hormone (hCG) is mainly secreted by the endocrine VCT (syncytiotrophoblast) into the maternal compartment and stimulates the formation of the syncytiotrophoblast (ST) in an autocrine manner. We recently reported that the invasive EVCT also produces hCG that promotes trophoblast invasion in vitro. Herein, we compared hCG gene expression in primary culture of villous and extravillous trophoblasts obtained from the same first trimester human chorionic villi and differentiated in vitro into ST and invasive EVCT, respectively. Total hCG, free alpha and free beta subunits were quantified in cell supernatants by immunometric assays and normalized to DNA content. alpha and beta transcript levels were quantified by Q-PCR and normalized to cytokeratin 7. We show that free alpha-, free beta-subunits and total hCG are differently expressed and secreted by the two trophoblast subtypes during their differentiation in vitro. We found an alpha/beta ratio 100 times lower in invasive EVCT in comparison to the ST suggesting that beta subunit may not be step limiting for hCG production in EVCT. Finally we investigated the regulation of hCG gene expression by PPARgamma, a nuclear receptor that controls trophoblast differentiation and invasion. Interestingly, activation of PPARgamma by the agonist rosiglitazone gave opposite results in the endocrine VCT and invasive EVCT: alpha and beta subunit transcript levels and protein secretions were up regulated in VCT, whereas they were down regulated in EVCT. Our results demonstrated that hCG gene expression is differentially regulated in the two trophoblast lineages during their in vitro differentiation and modulated in an opposite way by PPARgamma.

Insulin resistance and geographical origin: major predictors of liver fibrosis and response to peginterferon and ribavirin in HCV-4.

BACKGROUND AND AIMS: Hepatitis C virus (HCV) genotype 4 (HCV-4) is increasing in prevalence in Western countries. However, little is known about the severity of the disease and response to treatment. The aim of this study was to assess the predictors (logistic regression) of severe fibrosis (METAVIR score F3-F4), and sustained virological response (SVR) to peginterferon and ribavirin in 226 consecutive HCV-4 patients (Egyptians 40%, Europeans 35% and Africans 24%). PATIENTS AND METHODS: Insulin resistance was assessed using the homeostasis model (HOMA-IR). Serum HCV-RNA level (bDNA) and subtypes of HCV (LiPA) were determined for all patients. RESULTS: Insulin resistance (HOMA-IR >3) was present in 105 patients (46%), and was associated with: age >45 years (OR, 2.614; 95% CI, 1.316 to 5.194), body mass index (BMI) >25 kg/m(2) (OR, 2.105; 95% CI, 1.048 to 4.229), serum HCV-RNA >800 000 IU/ml (OR, 3.143; 95% CI, 1.503 to 6.574), severe fibrosis (OR, 2.657; 95% CI, 1.214 to 5.818), and steatosis >30% (OR, 2.488; 95% CI, 1.105 to 5.602). Severe fibrosis was present in 67 patients (29%) and was associated with Egyptian origin (OR, 5.872; 95% CI, 2.747 to 12.553), excessive alcohol intake (OR, 5.311; 95% CI, 1.287 to 21.924), and HOMA-IR >3 (OR, 3.864; 95% CI, 1.859 to 8.034). 108 patients received a 48 week course of peginterferon plus ribavirin. SVR (undetectable serum HCV-RNA (TMA) 24 weeks after treatment stopping) was achieved in 59 patients (55%) and was associated with Egyptian origin (OR, 13.119; 95% CI, 3.089 to 55.706), HOMA-IR <2 (OR, 5.314; 95% CI, 1.953 to 14.459), and non-severe fibrosis (OR, 8.059; 95% CI, 2.512 to 25.855). CONCLUSION: Insulin resistance and geographical origin are major predictors of liver fibrosis and response to peginterferon and ribavirin in HCV-4 patients. Insulin resistance is frequently encountered in these patients, and correlated independently with serum HCV-RNA.

SOS1: a new player in the Noonan-like/multiple giant cell lesion syndrome.

Noonan-like/multiple giant cell lesion syndrome is a rare condition with phenotypic overlap with Noonan syndrome (NS) and cherubism. PTPN11 gene mutations were described in several individuals with this phenotype, and it is recently considered as a variant phenotype of NS. Gain-of-function mutations in the SOS1 gene were recently described as the second major cause of NS. Here, we report for the first time the involvement of SOS1 gene in a family with the Noonan-like/multiple giant cell lesion phenotype.

SPRED1 germline mutations caused a neurofibromatosis type 1 overlapping phenotype.

OBJECTIVE: Germline loss-of-function mutations in the SPRED1 gene have recently been identified in patients fulfilling the National Institutes of Health (NIH) diagnostic criteria for neurofibromatosis type 1 (NF1) but with no NF1 (neurofibromin 1) mutation found, suggesting a neurofibromatosis type 1-like syndrome. METHODS: 61 index cases with NF1 clinical diagnosis but no identifiable NF1 mutation were screened for SPRED1 mutation. RESULTS: We describe one known SPRED1 mutation (c.190C>T leading to p.Arg64Stop) and four novel mutations (c.637C>T leading to p.Gln213Stop, c.2T>C leading to p.Met1Thr, c.46C>T leading to p.Arg16Stop, and c.1048_1060del leading to p.Gly350fs) in five French families. Their NF1-like phenotype was characterised by a high prevalence of café-au-lait spots, freckling, learning disability, and an absence of neurofibromas and Lisch nodules in agreement with the original description. However, we did not observe Noonan-like dysmorphy. It is noteworthy that one patient with the p.Arg16Stop mutation developed a monoblastic acute leukaemia. CONCLUSIONS: In our series, SPRED1 mutations occurred with a prevalence of 0.5% in NF1 patients and in 5% of NF1 patients displaying an NF1-like phenotype. SPRED1 mutated patients did not display any specific dermatologic features that were not present in NF1 patients, except for the absence of neurofibromas that seem to be a specific clinical feature of NF1. The exact phenotypic spectrum and the putative complications of this NF1 overlapping syndrome, in particular haematological malignancies, remain to be further characterised. NIH diagnostic criteria for NF1 must be revised in view of this newly characterised Legius syndrome in order to establish a specific genetic counselling.

Gene expression and hepatitis C virus infection.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, with about 170 million people infected worldwide. Up to 70% of patients will have persistent infection after inoculation, making this disease a significant cause of morbidity and mortality. The severity of disease varies widely, from asymptomatic chronic infection to cirrhosis and hepatocellular carcinoma. Since the discovery of HCV, the treatment of hepatitis C has considerably improved. Recently, combination of pegylated interferons with ribavirin gives a response rate of about 55%. Treatment is indicated in patients with moderate or severe fibrosis. The tolerability of combination treatment is relatively poor, with a frequent flu-like syndrome and an impaired quality of life. In addition to viral and environmental behavioural factors, host genetic diversity is believed to contribute to the spectrum of clinical outcomes in HCV infection. The sequencing of the human genome, together with the development of high-throughput technologies that measure the function of the genome, have afforded unique opportunities to develop profiles that can distinguish, identify and classify discrete subsets of disease, predict the disease outcome or predict the response to treatment. This paper reviews the published literature on gene expression associated with HCV infection (HCV infection, fibrosis progression), and also according to response to treatment.

Tenascin-C expression relates to clinicopathological features in pilocytic and diffuse astrocytomas.

AIMS: Tenascin-C (TN-C) is an extracellular matrix brain glycoprotein for which conflicting in vitro and in vivo results are reported in the literature dealing with its involvement in astrocytoma aggressiveness, in particular astrocytoma invasion. In view of these conflicting results and the lack of data available on low-grade astrocytomas, the present study focuses on pilocytic World Health Organization (WHO) grade I, and diffuse WHO grade II astrocytomas, that is, two histological entities associated with very different invasive abilities. METHODS: Using real-time reverse transcription polymerase chain reaction and immunohistochemistry, we analysed the TN-C expression in normal brain tissue as well as in a series of 54 pilocytic and 53 grade II astrocytomas. CONCLUSIONS: Our data on normal brain showed that while TN-C is largely expressed in supratentorial white matter, it was largely absent in infratentorial white matter. Paralleling these observations, we showed that TN-C expression in low-grade astrocytomas similarly varies according to tumour site. Cox regression analysis evidenced that TN-C provided an independent prognostic value which is enhanced in the case of grade II astrocytomas for which positive TN-C expression is associated with a higher risk of recurrence. We also analysed TN-C expression specifically in vascular areas of low-grade astrocytomas without demonstrating any prognostic value for this additional feature. RESULTS: Similarly to normal brain, low-grade astrocytomas exhibit variations in TN-C expression with site, and this expression is associated with an independent prognostic value in terms of recurrence.

Liver gene expression signature to predict response to pegylated interferon plus ribavirin combination therapy in patients with chronic hepatitis C.

BACKGROUND AND AIMS: The gold standard treatment of chronic hepatitis C (CHC) is combined pegylated interferon and ribavirin. Considering side effects and treatment cost, prediction of treatment response before therapy is important. The aim of this study was to identify a liver gene signature to predict sustained virological response in patients with CHC. METHODS: Group A (training set) comprised 40 patients with CHC including 14 non-responders (NRs) and 26 sustained virological responders (SVRs). Group B (validation set) comprised 29 patients including 9 NRs and 20 SVRs. Eleven responder-relapsers were also included. A total of 58 genes associated with liver gene expression dysregulation during CHC were selected from the literature. Real-time quantitative RT-PCR assays were used to analyse the mRNA expression of these 58 selected genes in liver biopsy specimens taken from the patients before treatment. RESULTS: From the Group A data, three genes whose expression was significantly increased in NRs compared with SVRs were identified: IFI-6-16/G1P3, IFI27 and ISG15/G1P2. These three genes also showed significant differences in their expression profiles between NRs and SVRs in the independent sample (Group B). Supervised class prediction analysis identified a two-gene (IFI27 and CXCL9) signature, which accurately predicted treatment response in 79.3% (23/29) of patients from the validation set (Group B), with a predictive accuracy of 100% (9/9) and of 70% (14/20) in NRs and SVRs, respectively. The expression profiles of responder-relapsers did not differ significantly from those of NRs and SVRs, and 73% (8/11) of them were predicted as SVRs with the two-gene classifier. CONCLUSION: NRs and SVRs have different liver gene expression profiles before treatment. The most notable changes occurred mainly in interferon-stimulated genes. Treatment response could be predicted with a two-gene signature (IFI27 and CXCL9).

The angiopoietin pathway is modulated by PAR-1 activation on human endothelial progenitor cells.

OBJECTIVES: The importance of protease-activated receptor-1 (PAR-1) in blood vessel development has been shown in knock-out mice. As endothelial progenitor cells (EPCs) express functional PAR-1, we examined whether PAR-1 stimulation by the peptide SFLLRN interfered with the angiopoietin pathway, that is EPC commitment, proliferation and migration. METHODS AND RESULTS: Given the strong PAR-1 expression on CD34+ cells, we tested the effect of SFLLRN 75 micromol L(-1) on the emergence of EPCs from cord blood. PAR-1 activation did not modify the number of colonies or the day of emergence, in keeping with the lack of induction of angiopoietin 1 gene expression. Conversely, SFLLRN treatment of EPCs induced angiopoietin 2 gene expression and protein synthesis. Experiments with polyclonal blocking antibodies showed that angiopoietin 2 was involved in the proliferative effect of PAR-1 activation. PAR-1 activation also enhanced migration toward angiopoietin 1 in a Boyden chamber assay. CONCLUSIONS: Our study demonstrates that PAR-1-induced proliferation of EPCs involves angiopoietin 2. PAR-1 also enhances EPC migration toward angiopoietin 1. These findings might explain the role of thrombin in neovascularization via the angiopoietin pathway.

Quantitative gene expression in Budd-Chiari syndrome: a molecular approach to the pathogenesis of the disease.

BACKGROUND: Budd-Chiari syndrome (BCS) is associated with parenchymal changes leading to major architecture remodelling. In order to gain further insight into the pathogenesis of BCS, we investigated expression of a set of genes involved in the course of chronic liver diseases. METHODS: Quantitative expression of 35 selected genes involved in extracellular matrix regulation, growth factors, and angiogenesis was investigated in 13 cases of BCS and compared with 10 normal livers and 13 cirrhosis cases by real time reverse transcription-polymerase chain reaction. Differential gene expression was considered significant for genes showing at least a twofold variation, with p < 0.05. RESULTS: Expression of 14 genes was significantly increased in BCS versus normal liver, with the highest increase in superior cervical ganglion 10 (SCG10) gene. BCS cases were classified according to their evolution and morphological pattern as either acute or chronic in six and seven cases, respectively. Unsupervised hierarchical clustering of acute and chronic BCS cases on the basis of similarity in gene expression pattern led to distinction between the two groups. Expression of three genes was significantly different in acute versus chronic BCS (increase in matrix metalloproteinase 7 and SCG10, decrease in thrombospondin-1 for chronic BCS). Seventeen and 10 genes, mainly involved in extracellular matrix and vascular remodelling, were significantly deregulated in acute BCS versus normal liver and cirrhosis, respectively. CONCLUSION: These results show that BCS cases display a specific gene expression profile that is different from that of normal liver and cirrhosis; the molecular configuration of BCS can be readily distinguished by its evolution and morphological pattern.

Large variability of trophoblast gene expression within and between human normal term placentae.

Human placenta extracts are widely used in clinical and fundamental research, particularly to study the hormonal and exchange functions of the placenta. However, very little is known about the distribution of the main hormone mRNAs in the placenta as a whole. Total placenta extracts are heterogeneous in their cellular components, as they contain material of both fetal and maternal origin, and in their structure. Results vary greatly depending upon the location of the biopsy and the number of biopsies performed. We used real-time quantitative RT-PCR to determine whether transcripts corresponding to the main hormones secreted by the human placenta (e.g. hCG, HPL and PGH) are equally distributed within and between term placentae. We also measured cytokeratin 7 transcripts, which are specifically expressed in the trophoblast, and transcripts corresponding to nuclear receptors PPARgamma and RXRalpha. A comparison of the results obtained with 12 different samples from each of four normal term placentae revealed that the amounts of transcripts differ considerably within and between each placenta. This emphasizes the need to study large numbers of samples when looking for significant differences in gene expression.

Trophoblast production of a weakly bioactive human chorionic gonadotropin in trisomy 21-affected pregnancy.

Total human chorionic gonadotropin (hCG) is high in maternal serum at 14-18 wk of trisomy 21 (T21)-affected pregnancy, despite low placental hCG synthesis. We sought an explanation for this paradox. We first observed that, in T21-affected pregnancies, maternal serum hCG levels peaked at around 10 wk and then followed the same pattern throughout pregnancy as in controls, albeit at a higher (2.2-fold) level. After delivery, hCG clearance was not significantly different from that in controls. We isolated cytotrophoblasts from 29 T21-affected placentas (12-25 wk) and 13 gestational age-matched control placentas and cultured them for 3 d. In this large series, we confirmed that, in the culture medium of trophoblasts isolated from T21 placentas, hCG secretion was significantly lower (P < 0.003) than in controls, in contrast to the high hCG in maternal serum of the same patients. In T21 cultured trophoblasts, transcripts of sialyltransferase-1 and fucosyltransferase-1 were abnormally high. In corresponding culture medium, hCG was abnormally glycosylated; highly acidic [isoelectric points (pHi) = 4.5] as shown by isoelectric focusing, immunoblotting, and lectin binding; and weakly bioactive (46% of control) as determined using the Leydig cell model. In conclusion, T21 trophoblast cells produced hCG that was weakly bioactive and abnormally glycosylated but whose maternal clearance was unaltered.

Liver fibrosis is not associated with steatosis but with necroinflammation in French patients with chronic hepatitis C.

BACKGROUND: In chronic hepatitis C, it has been suggested that steatosis could accelerate progression of fibrosis. However, results of the few published studies are controversial. AIM: To determine the characteristics (epidemiological, biological, and histological) associated with steatosis and its relationship with liver lesions (grade of necroinflammation and stage of fibrosis) in patients with chronic hepatitis C. METHODS: From November 2000 to July 2001, untreated consecutive adults with chronic hepatitis C admitted for liver biopsy were included in this study. On the day of liver biopsy, a questionnaire for risk factors was completed prospectively, and a blood sample was obtained for laboratory analysis. RESULTS: Our study included 290 patients (143 men, 147 women). Mean body mass index (BMI) was 24 (3.8) kg/m(2). Proportions of patients with genotypes 1 and 3 were, respectively, 48% and 18%. A total of 135 patients (46.6%) had steatosis. Liver steatosis, in multivariate analysis, was associated with hepatitis C virus genotype 3, higher grade of necroinflammation, and higher BMI. There was no significant association between stage of fibrosis and liver steatosis. In multivariate analysis, high stage of fibrosis was associated with male sex, age over 50 years, high BMI, and high grade of necroinflammation. CONCLUSION: In our population of patients with chronic hepatitis C, steatosis does not seem to be an important determinant of liver fibrosis. High grade of necroinflammation is associated with a high stage of fibrosis.