La Tronche, France
La Tronche, France

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Pugniere P.,Genomic Core Facility | Banzet S.,Operational Environments | Chaillou T.,Operational Environments | Mouret C.,Genomic Core Facility | Peinnequin A.,Genomic Core Facility
Analytical Biochemistry | Year: 2011

A large part of the reliability of reverse transcription quantitative polymerase chain reaction (RT-qPCR) data depends on technical variations. Such variations are mainly attributable to the reverse transcription step. Standardization is a key factor in decreasing the intersample variability. However, an ideal standardization is not always possible, and compromises must be found. Due to technical requirements, the current consensus is that a constant amount of total RNA should be used for the RT step (CA-RT). Because RNA isolation yields are variable, such a practice requires the use of variable volumes of nucleic acid extracts in RT reaction. We demonstrate that some RNA extracts contain both exogenous and endogenous inhibitors. These inhibitors induce a decrease in RT efficiency that significantly impairs the reliability of RT-qPCR data. Conversely, these inhibitors have a slight effect on the qPCR step. To overcome such drawbacks, we proposed to carry out the RT reaction with a constant volume of RNA extract by preserving a constant RNA amount through the supplementation of yeast transfer RNA (CV-RT). We show that CV-RT, compared with the usual CA-RT, allows us to decrease the RT-qPCR variability induced by intersample differences. Such a decrease is a prerequisite for the reliability of messenger RNA quantification. © 2011 Elsevier Inc. All rights reserved.

Magerus-Chatinet A.,University of Paris Descartes | Neven B.,University of Paris Descartes | Stolzenberg M.-C.,University of Paris Descartes | Daussy C.,University of Paris Descartes | And 16 more authors.
Journal of Clinical Investigation | Year: 2011

Autoimmune diseases develop in approximately 5% of humans. They can arise when self-tolerance checkpoints of the immune system are bypassed as a consequence of inherited mutations of key genes involved in lymphocyte activation, survival, or death. For example, autoimmune lymphoproliferative syndrome (ALPS) results from defects in self-tolerance checkpoints as a consequence of mutations in the death receptor-encoding gene TNF receptor superfamily, member 6 (TNFRSF6; also known as FAS). However, some mutation carriers remain asymptomatic throughout life. We have now demonstrated in 7 ALPS patients that the disease develops as a consequence of an inherited TNFRSF6 heterozygous mutation combined with a somatic genetic event in the second TNFRSF6 allele. Analysis of the patients' CD4-CD8- (double negative) T cells - accumulation of which is a hallmark of ALPS - revealed that in these cells, 3 patients had somatic mutations in their second TNFRSF6 allele, while 4 patients had loss of heterozygosity by telomeric uniparental disomy of chromosome 10. This observation provides the molecular bases of a nonmalignant autoimmune disease development in humans and may shed light on the mechanism underlying the occurrence of other autoimmune diseases.

Putoux A.,French Institute of Health and Medical Research | Putoux A.,University of Paris Descartes | Thomas S.,French Institute of Health and Medical Research | Thomas S.,University of Paris Descartes | And 46 more authors.
Nature Genetics | Year: 2011

KIF7, the human ortholog of Drosophila Costal2, is a key component of the Hedgehog signaling pathway. Here we report mutations in KIF7 in individuals with hydrolethalus and acrocallosal syndromes, two multiple malformation disorders with overlapping features that include polydactyly, brain abnormalities and cleft palate. Consistent with a role of KIF7 in Hedgehog signaling, we show deregulation of most GLI transcription factor targets and impaired GLI3 processing in tissues from individuals with KIF7 mutations. KIF7 is also a likely contributor of alleles across the ciliopathy spectrum, as sequencing of a diverse cohort identified several missense mutations detrimental to protein function. In addition, in vivo genetic interaction studies indicated that knockdown of KIF7 could exacerbate the phenotype induced by knockdown of other ciliopathy transcripts. Our data show the role of KIF7 in human primary cilia, especially in the Hedgehog pathway through the regulation of GLI targets, and expand the clinical spectrum of ciliopathies. © 2011 Nature America, Inc. All rights reserved.

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