Analyse de données d’expression transcriptomiques rythmées par des gènes-horloge : approche méthodologique et optimisation

Background

In microarray data, wide-scale correlations are numerous and increase the number of genes correlated to a test condition (phenotype, mutation status, etc.) either positively or negatively. Several methods have been developed to limit the effect of such correlations on the false discovery rate, but these may reject too many genes that have a mild or indirect impact on the studied condition. We propose here a simple methodology to correct this spurious effect without eliminating weak but true correlations.

Results

This methodology was applied to a microarray dataset designed to distinguish heterozygous BRCA1 mutation carriers from non-carriers. As our samples were collected at different times in the morning, we evaluated the effect of correlations due to circadian rhythm. The circadian system is a well-known correlation network, regulated by a small number of period genes whose expression varies throughout the day in predictable ways. The downstream effects of this variation on the expression of other genes, however, are incompletely characterized. We used two different strategies to correct this correlation bias, by either dividing or multiplying the expression of correlated genes by the expression of the considered period gene according to the sign of the correlation between the period gene and correlated gene (respectively positive or negative).

Conclusions

We observed a linear relationship between the number of false-positive/negative genes and the strength of the correlation of the candidate gene to the test condition. BRCA1 was highly correlated to the period gene Per1; our correction methodology enabled us to recover genes coding for BRCA1-interacting proteins which were not selected in the initial direct analysis. This methodology may be valuable for other studies and can be applied very easily in case of well-known correlation networks.     

Halorubrum pleomorphic virus-6 Membrane Fusion Is Triggered by an S-Layer Component of Its Haloarchaeal Host

(1) Background: Haloarchaea comprise extremely halophilic organisms of the Archaea domain. They are single-cell organisms with distinctive membrane lipids and a protein-based cell wall or surface layer (S-layer) formed by a glycoprotein array. Pleolipoviruses, which infect haloarchaeal cells, have an envelope analogous to eukaryotic enveloped viruses. One such member, Halorubrum pleomorphic virus 6 (HRPV-6), has been shown to enter host cells through virus-cell membrane fusion. The HRPV-6 fusion activity was attributed to its VP4-like spike protein, but the physiological trigger required to induce membrane fusion remains yet unknown. (2) Methods: We used SDS-PAGE mass spectroscopy to characterize the S-layer extract, established a proteoliposome system, and used R18-fluorescence dequenching to measure membrane fusion. (3) Results: We show that the S-layer extraction by Mg²⁺ chelating from the HRPV-6 host, Halorubrum sp. SS7-4, abrogates HRPV-6 membrane fusion. When we in turn reconstituted the S-layer extract from Hrr. sp. SS7-4 onto liposomes in the presence of Mg²⁺, HRPV-6 membrane fusion with the proteoliposomes could be readily observed. This was not the case with liposomes alone or with proteoliposomes carrying the S-layer extract from other haloarchaea, such as Haloferax volcanii. (4) Conclusions: The S-layer extract from the host, Hrr. sp. SS7-4, corresponds to the physiological fusion trigger of HRPV-6.     

Association between hereditary predisposition to common cancers and congenital multimalformations

In a previous article we reported that mutations favoring cancer at adulthood seemed to improve fertility and limit miscarriages. Because spontaneous abortion may result from anomalies in embryo, we questioned if an increased frequency of congenital malformation could be evidenced among cancer-prone families. Oncogenetics database (≈193 000 members) of the comprehensive cancer center Jean Perrin was crossed with regional registry of congenital malformations (≈10 000). Among children born between 1986 and 2011, 176 children with malformation matched in both databases. In breast/ovaries cancer-prone families, the risk for malformations was multiplied by 2.4 [1.2-4.5] in case of a BRCA1 mutation. Frequencies of malformation in BRCA2 and MMR mutated families were similar to families without a cancer syndrome. In comparison to malformations concerning a unique anatomical system, multimalformations were significantly more frequent in case of BRCA or MMR mutations: compared to families without cancer syndrome, the risk of multimalformations was multiplied by 4.1 [0.8-21.7] for cancer-prone families but with no known deleterious mutation, by 6.9 [1.2-38.6] in families with a known mutation but an unknown parental mutational status and by 10.4 [2.3-46.0] when one parent carried the familial mutation. No association with the type of anatomical system was found, nor with multiple births. These results suggest that BRCA and MMR genes play an important role in human embryogenesis and that if their function is lowered because of heterozygote mutations, congenital malformations are either more likely (BRCA1 mutations) and/or more susceptible to concern several anatomical systems.     

Estimation of the incidence of pleural mesothelioma according to death certificates in France

BACKGROUND: The number of cases of pleural mesothelioma in France has varied substantially according to methods of assessment. MATERIALS AND METHODS: We collected information from certifying physicians about 316 subjects who died between 1 July 1992 and 30 June 1993 in three regions of France with a cause of death coded as ICD-9 category 163. The ICD codes selected as the cause of death for 178 deaths between 1 January 1987 and 31 December 1992 histologically confirmed and diagnosed as pleural mesothelioma by an expert committee were examined. Finally, we used this information to estimate the number of deaths from pleural mesothelioma in France in 1992. RESULTS: In Part I, 45% (men: 54%; women: 28%) of the cases coded as ICD-9 section 163 were definitely or probably mesothelioma; 18% (men: 16%; women: 21%) possibly mesothelioma; and 37% (men: 30%; women: 51%) other tumors, primarily adenocarcinoma metastases. In Part II, 74% of the confirmed pleural mesotheliomas were coded in category 163 (men: 75%; women: 70%). Extrapolation nationwide indicated that 902 deaths were coded as ICD-9 163 in 1992: 521 cases involved definite or probable mesothelioma and 724 definite, probable, and possible cases. CONCLUSIONS: The analysis of this sample suggests that estimating the number of mesothelioma cases from the cause-of-death statistics may overestimate their incidence, but that death certificates appeared to report the diagnosis of histologically confirmed mesothelioma accurately.     

Nijmegen breakage syndrome gene (NBS1) is not the tumor suppressor gene at 8q21.3 involved in colorectal carcinoma

Genetic instability is characteristic of cancer cells, both at the chromosomal level (e.g., aneuploidy, aneusomy, translocations), and at the sequence level (e.g., microsatellite instability). Colorectal cancers (CRCs) can be divided into two groups: tumors of the proximal colon, where microsatellite instability is frequent and chromosomal aberrations are rare, and tumors of the distal colon, where microsatellite instability is less frequent and chromosomal aberrations are common. Constitutional chromosomal instability is a hallmark of the Nijmegen breakage syndrome (NBS), a disorder in which the NBS1 gene is mutated. In a previous study, we found an elevated frequency of allelic imbalance (AI) at the 8q21.3 NBS1 locus in proximal, though not distal CRCs. This result suggested that the loss of NBS1 might contribute to the genetic instability, and thus the malignant progression, of proximal CRC. We therefore sequenced the 16 exons of the NBS1 gene in all cases where we had observed AI. Of the 29 cases, none showed any sequence anomaly, although several polymorphisms were found. We also studied markers flanking the recently described Rad54B gene, which is a member of the Rad52 epistasis group to which NBS1 itself belongs. Mutations of this gene, located a few cM distal to NBS1, have been found in colorectal cancer and in primary lymphomas, and it may thus be the target of AI at 8q21. We found that AI at Rad54B was not frequent, and none was observed in cases showing AI at NBS1.     

in vitro effects of recombinant human interferon gamma on human mesothelioma cell lines

Malignant mesothelioma is a tumor arising from serous surfaces and often related to asbestos exposure. Malignant mesothelioma is resistant to various forms of therapy. Radiotherapy, surgery or chemotherapy only slightly improve prognosis. IFN-γ produces complete or partial responses in stage-l patients with malignant mesothelioma. The in vitro biological effect of IFN-γ on malignant mesothelioma cells remains poorly elucidated. In the present study, 32 well-characterized human mesothelioma cell lines (HMCL) were treated with r-hu IFN--γ at 4 doses and cell growth was determined by a colorimetric method (MTT assay). Among the 32 HMCLs tested, 11 exhibited significant cell-growth inhibition; 16 were insensitive to r-hu IFN-γ, and 5 were slightly inhibited. The sensitive cell lines were strongly inhibited by r-hu IFN-γ. Our results show that HMCL exhibit a large range of responses to r-hu IFN-γ, some of which can be compared with those obtained in vivo in humans.     

Allelic imbalance at NBS1 is frequent in both proximal and distal colorectal carcinoma

Nijmegen breakage syndrome (NBS) is a hereditary disorder involving chromosomal instability, cancer risk and radiosensitivity. NBS carriers have an increased risk of cancer, though the significance of mutations in the NBS1 gene in sporadic cancer has not yet been investigated. Because the loss of NBS1 is associated with increased chromosomal re-arrangements, and tumors of the colon are particularly prone to chromosomal anomalies, we have begun to study the NBS1 locus in colorectal cancer (CRC). DNA was isolated from 99 microdissected colorectal tumors, and microsatellite markers flanking the NBS1 locus at 8q21.3 as well as elsewhere on 8q were analyzed. Normal lymphocyte DNA from each patient served to normalize the amplification of each allele, and a reduction of at least 35% in the intensity of one allele was taken as evidence of allelic imbalance (AI). In proximal and distal CRCs we found 25.9 and 36.2% with AI at 8q21.3, respectively. AI in proximal CRC tended not to extend to marker D8S555 at 8q24.1, whereas in distal CRC the region of AI frequently included all the informative markers. AI of 8q21.3 was not associated with any clinical variable. These results suggest that 8q21.3 contains a tumor suppressor gene involved in proximal CRC, possibly NBS1. The large regions of AI make it difficult to determine the importance of AI at the NBS1 locus in distal CRC.     

In vitro biodegradation of chrysotile fibres by alveolar macrophages and mesothelial cells in culture: comparison with a pH effect

The modification of the chemistry of asbestos chrysotile fibres (Mg3(Si2O5)(OH)4) after their ingestion by cultured cells has been studied. Two types of cells involved in asbestos related pulmonary disease were used, rabbit alveolar macrophages (AM), recovered by bronchoalveolar lavage, and pleural mesothelial cells (PMC) obtained from the rat parietal pleura. Chemical characterisation of intracellular fibres was performed on unstained ultrathin sections by electron probe microanalysis. The results showed a progressive leaching of Mg, characterised by a time dependent decrease of Mg/Si. AM were more efficient than PMC at leaching intracellular chrysotile fibres since it took longer to obtain the same proportion of leached fibres with PMC than with AM. As in vitro Mg-leaching can be obtained by acid treatment, chrysotile fibres were incubated, either untreated or pretreated with cell membranes, at pH 4 or 7 for various times. The data show that the kinetic of leaching by AM was comparable with leaching at pH 4. The leaching by PMC was of the same order as leaching at pH 7. When membranes were adsorbed on to the fibres, a delayed leaching was observed. The results indicate that the solubilisation of chrysotile by AM could be an intraphagolysosomal event due to a pH effect. With PMC, however, it is not possible to draw this conclusion since nothing is known about the intracellular pH.     

HLA-AB and -DR types in patients with infectious mononucleosis (IM)

The frequency of HLA-A, -B and -DR antigens in 48 infectious mononucleosis (IM) patients was studied and the antigen frequencies were compared with those of a local panel. The resulting data do not confirm the association of IM and HLA-B35 as reported in a previous work. A high and significant frequency of blank HLA-DR phenotypes was noticed in IM population during the acute phase, but HLA-DR phenotypic expression was restored after recovery. This lack of expression could be the result of modified HLA-DR expression on circulating EBV infected B lymphocytes.     

Populations of circulating T lymphocytes in patients with alcoholic cirrhosis

The characteristics, the origin and the role in the determinism and/or in the course of hepatocellular necrosis of immune disorders in patients with alcoholic liver cirrhosis still remain unclear. In this study, the T-lymphocyte population was determined by sheep-rosette formation and labelling with OKT3-monoclonal antibodies. T-cell subsets were investigated using OKT4 and OKT8 monoclonal antibodies in the peripheral blood of 40 alcoholic patients with cirrhosis (CA) and of 23 non-cirrhotic alcoholic (ANC) patients. The results were compared to those in 34 healthy volunteers. A decrease in the percentage of T-lymphocytes in the two groups of alcoholic subjects was noted. However, this decrease was significant only in the CA group and only when OKT3 antibodies were used as markers. The lymphocyte-subset identified by OKT4 antibodies was decreased as well in the CA and ANC groups; however the OKT8+ subset was significantly decreased in the CA group only (p less than 0.001). No correlation was found between this decrease in OKT8+ subpopulation or between the increased OKT4/OKT8 ratio and all biochemical parameters of hepatic function measured in this study. The significance of this T-cell imbalance in CA has still to be elucidated, concerning both the functional activity of the reduced pool of OKT8+ cells (suppressor or cytotoxic?) and the mechanism of this decrease.