Frequency of Participation in External Quality Assessment Programs Focused on Rare Diseases: Belgian Guidelines for Human Genetics Centers.

BACKGROUND: Participation in quality controls, also called external quality assessment (EQA) schemes, is required for the ISO15189 accreditation of the Medical Centers of Human Genetics. However, directives on the minimal frequency of participation in genetic quality control schemes are lacking or too heterogeneous, with a possible impact on health care quality. OBJECTIVE: The aim of this project is to develop Belgian guidelines on the frequency of participation in quality controls for genetic testing in the context of rare diseases. METHODS: A group of experts analyzed 90 EQA schemes offered by accredited providers and focused on analyses used for the diagnosis of rare diseases. On that basis, the experts developed practical recommendations about the minimal frequencies of participation of the Medical Centers of Human Genetics in quality controls and how to deal with poor performances and change management. These guidelines were submitted to the Belgian Accreditation Body and then reviewed and approved by the Belgian College of Human Genetics and Rare Diseases and by the National Institute for Health and Disability Insurance. RESULTS: The guidelines offer a decisional algorithm for the minimal frequency of participation in human genetics EQA schemes. This algorithm has been developed taking into account the scopes of the EQA schemes, the levels of experience, and the annual volumes of the Centers of Human Genetics in the performance of the tests considered. They include three key principles: (1) the recommended annual assessment of all genetic techniques and technological platforms, if possible through EQAs covering the technique, genotyping, and clinical interpretation; (2) the triennial assessment of the genotyping and interpretation of specific germline mutations and pharmacogenomics analyses; and (3) the documentation of actions undertaken in the case of poor performances and the participation to quality control the following year. The use of a Bayesian statistical model has been proposed to help the Centers of Human Genetics to determine the theoretical number of tests that should be annually performed to achieve a certain threshold of performance (eg, a maximal error rate of 1%). Besides, the guidelines insist on the role and responsibility of the national public health authorities in the follow-up of the quality of analyses performed by the Medical Centers of Human Genetics and in demonstrating the cost-effectiveness and rationalization of participation frequency in these quality controls. CONCLUSIONS: These guidelines have been developed based on the analysis of a large panel of EQA schemes and data collected from the Belgian Medical Centers of Human Genetics. They are applicable to other countries and will facilitate and improve the quality management and financing systems of the Medical Centers of Human Genetics.

Deorphanization and characterization of human olfactory receptors in heterologous cells.

Olfaction plays an indispensable role in human and animals in self and environmental recognition, as well as intra- and interspecific communication. Following the discovery of a family of olfactory receptors (ORs) by Buck and Axel in 1991, it has been established that the sense of smell begins with the molecular recognition of a chemical odorant by one or more ORs expressed in the olfactory sensory neurons. Therefore, characterization of the molecular interactions between odorant molecules and ORs is a key step in the elucidation of the general properties of the olfactory system and in the development of applications, i.e., design of new odorants, search for blockers, etc. The process putted in place at ChemCom to improve the expression of ORs at the cytoplasmic membrane of the HEK293 cell and assays enabling large-scale deorphanization, and to characterize the interaction between chemical odorants and ORs is described. The family of human ORs includes ca. 400 putatively functional ORs which are GPCRs (G protein-coupled receptors); to date over 100 human ORs have been deorphanized.

Profiling of olfactory receptor gene expression in whole human olfactory mucosa.

Olfactory perception is mediated by a large array of olfactory receptor genes. The human genome contains 851 olfactory receptor gene loci. More than 50% of the loci are annotated as nonfunctional due to frame-disrupting mutations. Furthermore haplotypic missense alleles can be nonfunctional resulting from substitution of key amino acids governing protein folding or interactions with signal transduction components. Beyond their role in odor recognition, functional olfactory receptors are also required for a proper targeting of olfactory neuron axons to their corresponding glomeruli in the olfactory bulb. Therefore, we anticipate that profiling of olfactory receptor gene expression in whole human olfactory mucosa and analysis in the human population of their expression should provide an opportunity to select the frequently expressed and potentially functional olfactory receptors in view of a systematic deorphanization. To address this issue, we designed a TaqMan Low Density Array (Applied Biosystems), containing probes for 356 predicted human olfactory receptor loci to investigate their expression in whole human olfactory mucosa tissues from 26 individuals (13 women, 13 men; aged from 39 to 81 years, with an average of 67±11 years for women and 63±12 years for men). Total RNA isolation, DNase treatment, RNA integrity evaluation and reverse transcription were performed for these 26 samples. Then 384 targeted genes (including endogenous control genes and reference genes specifically expressed in olfactory epithelium for normalization purpose) were analyzed using the same real-time reverse transcription PCR platform. On average, the expression of 273 human olfactory receptor genes was observed in the 26 selected whole human olfactory mucosa analyzed, of which 90 were expressed in all 26 individuals. Most of the olfactory receptors deorphanized to date on the basis of sensitivity to known odorant molecules, which are described in the literature, were found in the expressed olfactory receptors gene set.

Knockout mice reveal a role for P2Y6 receptor in macrophages, endothelial cells, and vascular smooth muscle cells.

P2Y receptors are G-protein-coupled receptors activated by extracellular nucleotides. The P2Y(6) receptor is selectively activated by UDP, and its transcript has been detected in numerous organs, including the spleen, thymus, intestine, blood leukocytes, and aorta. To investigate the biological functions of this receptor, we generated P2Y(6)-null mice by gene targeting. The P2Y(6) knockout (KO) mice are viable and are not distinguishable from the wild-type (WT) mice in terms of growth or fertility. In thioglycollate-elicited macrophages, the production of inositol phosphate in response to UDP stimulation was lost, indicating that P2Y(6) is the unique UDP-responsive receptor expressed by mouse macrophages. Furthermore, the amount of interleukin-6 and macrophage-inflammatory protein-2, but not tumor necrosis factor-alpha, released in response to lipopolysaccharide stimulation was significantly enhanced in the presence of UDP, and this effect was lost in the P2Y(6) KO macrophages. The endothelium-dependent relaxation of the aorta by UDP was abolished in KO P2Y(6) mice. The contractile effect of UDP on the aorta, observed when endothelial nitric-oxide synthase is blocked, was also abolished in P2Y(6)-null mice. In conclusion, we generated P2Y(6)-deficient mice and have shown that these mice have a defective response to UDP in macrophages, endothelial cells, and vascular smooth muscle cells. These observations might be relevant to several physiopathological conditions such as atherosclerosis or hypertension.

Loss of nucleotide regulation of epithelial chloride transport in the jejunum of P2Y4-null mice.

The P2Y(4) receptor is responsive to UTP in human and to ATP and UTP in rodents. With the aim of identifying its pharmacotherapeutic interest, we generated P2Y(4)-null mice by a classic gene targeting method. The proportion of genotypes was consistent with X-linked Mendelian transmission. Gene inactivation was checked by the complete disappearance of P2Y(4) receptor mRNA from liver, stomach, and intestine. The P2Y(4)-null mice had a grossly normal behavior, growth, and reproduction. Chloride secretion by the jejunal epithelium was assessed in Ussing chambers by the measurement of the short circuit current in the presence of phlorizin. We show here that the UTP- and ATP-induced chloride secretory responses observed in wild-type mice are abolished in P2Y(4)-null mice. This is the first clearcut demonstration of a biological role of the P2Y(4) receptor.

Extracellular adenine nucleotides modulate cytokine production by human monocyte-derived dendritic cells: dual effect on IL-12 and stimulation of IL-10.

To clarify the functional consequences of adenine nucleotides action on human monocyte-derived dendritic cells (DC), we have systematically compared the effects of adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), an ATP analog active on the P2Y(11) receptor, on the responses to three DC stimuli, TNF-alpha, LPS, sCD40L, tested at various concentrations, using two different IL-12 assays. We observed that ATPgammaS potentiated the IL-12p40 release induced by TNF-alpha, but also by lipopolysaccharides (LPS) and soluble CD40 ligand (sCD40L). This potentiation was observed as long as the IL-12p40 concentration under agonist stimulation remained below a threshold value close to 10 ng/ml; inhibition was observed above this value. The combinations ATPgammaS-TNF-alpha and ATPgammaS-sCD40L were unable to induce detectable bioactive IL-12p70 production and at concentrations of LPS that induced a significant stimulation of IL-12p70, the effect of ATPgammaS was purely inhibitory. Our results also show that ATPgammaS synergized with LPS and sCD40L, but not TNF-alpha, to stimulate IL-10 production. In conclusion, we have clarified the discrepancies in the literature concerning the action of adenine nucleotides on DC and our study supports the concept that, like prostaglandin E(2) and other agents increasing cyclic AMP, they favor either a Th2 response or tolerance.