Genetic investigation of the ubiquitin-protein ligase E3A gene as putative target in Angelman syndrome

BACKGROUND Angelman syndrome (AS) is caused by maternal chromosomal deletions, imprinting defects, paternal uniparental disomy involving chromosome 15 and the ubiquitin-protein ligase UBE3A gene mutations. However the genetic basis remains unclear for several patients. AIM To investigate the involvement of UBE3A gene in AS and identifying new potential genes using exome sequencing. METHODS We established a cohort study in 50 patients referred to Farhat Hached University Hospital between 2006 and 2021, with a strong suspicion of AS and absence of chromosomal aberrations. The UBE3A gene was screened for mutation detection. Two unrelated patients issued from consanguineous families were subjected to exome analysis. RESULTS We describe seven UBE3A variants among them 3 none previously described including intronic variants c.2220+14T>C (intron14), c.2507+43T>A (Exon15) and insertion in Exon7: c.30-47_30-46. The exome sequencing revealed 22 potential genes that could be involved in AS-like syndromes that should be investigated further. CONCLUSION Screening for UBE3A mutations in AS patients has been proven to be useful to confirm the diagnosis. Our exome findings could rise to new potential alternative target genes for genetic counseling

Search for alternative conditions for the use of chlorinated solvents in Non-Aqueous Reversed Phases liquid chromatography. Application to the analysis of lipids present in complex media

Déterminer la composition en triacylglycérols des huiles végétales est un défi important à relever aussi bien en biologie végétale que dans le domaine médical ou celui de l’industrie agro-alimentaire. La chromatographie en phase liquide à polarité inversée de phases en milieu non aqueux (NARP-LC) est la méthode la plus utilisée, avec cependant une utilisation récurrente de solvants chlorés. Le premier objectif de ce travail a été de proposer des conditions analytiques alternatives n’utilisant pas de solvants chlorés. Pour ce faire, nous avons établi le diagramme de force éluante sur phases stationnaires en C18 des phases mobiles binaires constituées d’acétonitrile comme solvant faible et de divers solvants forts (acétone, iso-propanol, acétate d’éthyle, butanol) à quatre températures différentes (25, 43, 63 et 85°C). La comparaison dans des conditions iso-éluantes de l’analyse de 9 huiles de graines contenant une large gamme de TAG nous a permis de montrer que le mélange MeCN/BuOH 74/26 à 25°C est le meilleur choix, en terme de sélectivité pour l’analyse des TAG. Ce qui répond à notre premier objectif. Dans un second temps, nous avons comparé le potentiel séparatif des phases stationnaires de nouvelle génération à petit diamètre de particules, partiellement ou totalement poreuses. L’optimisation des conditions chromatographiques nous a permis de décrire deux systèmes chromatographiques, très performants, en termes d’efficacité et de rapidité. Le premier permet la séparation de TAG contenant des acides gras polyinsaturés isomères de position en C18 : 3 et C18 : 2. L’identification de ces isomères particuliers a été réalisée grâce à la synthèse d’informations complémentaires, obtenues en GC/MS, LC/MS ainsi que l’utilisation de lois de rétention chromatographiques. En outre, ce travail nous a permis de proposer un tableau récapitulatif regroupant un très grand nombre de TAG, qui n’ont jamais été décrits à notre connaissance. Le second permet une analyse rapide de l’huile d’olive, en moins de 5 min, tout en respectant les consignes qualitatives imposées par l’organisme «Conseil Oléique International (COI)». Comparée aux méthodes officielles couramment utilisées, elle mérite d’être proposée comme méthode de référence pour le contrôle de qualité de l’appellation de ces huiles.The determination of triacylglycerols in vegetable oils is an important challenge in plant biology, in the medical field, and in food industry. Nowadays, non-Aqueous Reversed Phase Liquid Chromatography (NARP-LC) using chlorinated solvents is commonly used for this purpose. The first objective of this work was to develop alternative analytical conditions that can avoid using chlorinated solvents. In a first step, by using C18 stationary phases, we have established the eluotropic solvent strength scale as a function of temperature of several binary mobile phases consisting of acetonitrile as weak solvent and various strong solvents including acetone, isopropanol, ethyl acetate, and butanol. The comparison of the results obtained under iso-eluotropic conditions for nine seed oils containing a wide range of TAG enabled us to show that the MeCN/BuOH (74/26, v/V) mixture operating at 25 °C are the best mobile phase conditions for TAG analysis, in terms of selectivity, thus avoiding the use of chlorinated solvents. In a second step, we compared the separation of TAGs on new generation of fully or partially porous stationary phase particles of small diameter. After optimizing the separating conditions, the obtained data allowed us to propose two highly efficient chromatographic systems. The first system enables the efficient separation of C18:3 and C18:2 positional isomers of C18 polyunsaturated fatty acids containing TAG. For the identification of these TAG isomers, it was necessary to combine the data obtained by GC-MS and LC -MS as well as the data obtained by the application of some chromatographic retention laws. Taken together, these results allowed us to provide a list containing a large number of TAG unknown to date. The second system allows rapid analysis of olive oil in less than 5 min. This system obeying the guidelines of the International Olive Council can be proposed as a candidate reference method for rapid quality control of olive oils

Recherche de conditions alternatives à l’utilisation de solvants chlorés en Chromatographie Liquide Non-Aqueuse à Polarité Inversée de Phases. Application à l’analyse des lipides présents dans les milieux complexes

The determination of triacylglycerols in vegetable oils is an important challenge in plant biology, in the medical field, and in food industry. Nowadays, non-Aqueous Reversed Phase Liquid Chromatography (NARP-LC) using chlorinated solvents is commonly used for this purpose. The first objective of this work was to develop alternative analytical conditions that can avoid using chlorinated solvents. In a first step, by using C18 stationary phases, we have established the eluotropic solvent strength scale as a function of temperature of several binary mobile phases consisting of acetonitrile as weak solvent and various strong solvents including acetone, isopropanol, ethyl acetate, and butanol. The comparison of the results obtained under iso-eluotropic conditions for nine seed oils containing a wide range of TAG enabled us to show that the MeCN/BuOH (74/26, v/V) mixture operating at 25 °C are the best mobile phase conditions for TAG analysis, in terms of selectivity, thus avoiding the use of chlorinated solvents. In a second step, we compared the separation of TAGs on new generation of fully or partially porous stationary phase particles of small diameter. After optimizing the separating conditions, the obtained data allowed us to propose two highly efficient chromatographic systems. The first system enables the efficient separation of C18:3 and C18:2 positional isomers of C18 polyunsaturated fatty acids containing TAG. For the identification of these TAG isomers, it was necessary to combine the data obtained by GC-MS and LC -MS as well as the data obtained by the application of some chromatographic retention laws. Taken together, these results allowed us to provide a list containing a large number of TAG unknown to date. The second system allows rapid analysis of olive oil in less than 5 min. This system obeying the guidelines of the International Olive Council can be proposed as a candidate reference method for rapid quality control of olive oils.Déterminer la composition en triacylglycérols des huiles végétales est un défi important à relever aussi bien en biologie végétale que dans le domaine médical ou celui de l’industrie agro-alimentaire. La chromatographie en phase liquide à polarité inversée de phases en milieu non aqueux (NARP-LC) est la méthode la plus utilisée, avec cependant une utilisation récurrente de solvants chlorés. Le premier objectif de ce travail a été de proposer des conditions analytiques alternatives n’utilisant pas de solvants chlorés. Pour ce faire, nous avons établi le diagramme de force éluante sur phases stationnaires en C18 des phases mobiles binaires constituées d’acétonitrile comme solvant faible et de divers solvants forts (acétone, iso-propanol, acétate d’éthyle, butanol) à quatre températures différentes (25, 43, 63 et 85°C). La comparaison dans des conditions iso-éluantes de l’analyse de 9 huiles de graines contenant une large gamme de TAG nous a permis de montrer que le mélange MeCN/BuOH 74/26 à 25°C est le meilleur choix, en terme de sélectivité pour l’analyse des TAG. Ce qui répond à notre premier objectif. Dans un second temps, nous avons comparé le potentiel séparatif des phases stationnaires de nouvelle génération à petit diamètre de particules, partiellement ou totalement poreuses. L’optimisation des conditions chromatographiques nous a permis de décrire deux systèmes chromatographiques, très performants, en termes d’efficacité et de rapidité. Le premier permet la séparation de TAG contenant des acides gras polyinsaturés isomères de position en C18 : 3 et C18 : 2. L’identification de ces isomères particuliers a été réalisée grâce à la synthèse d’informations complémentaires, obtenues en GC/MS, LC/MS ainsi que l’utilisation de lois de rétention chromatographiques. En outre, ce travail nous a permis de proposer un tableau récapitulatif regroupant un très grand nombre de TAG, qui n’ont jamais été décrits à notre connaissance. Le second permet une analyse rapide de l’huile d’olive, en moins de 5 min, tout en respectant les consignes qualitatives imposées par l’organisme «Conseil Oléique International (COI)». Comparée aux méthodes officielles couramment utilisées, elle mérite d’être proposée comme méthode de référence pour le contrôle de qualité de l’appellation de ces huiles

Homozygous mutation in murine retrovirus integration site 1 gene associated with a non‐syndromic form of isolated familial achalasia

Background Achalasia is a condition characterized by impaired function of esophageal motility and incomplete relaxation of the lower esophagus sphincter, causing dysphagia and regurgitation. Rare cases of early-onset achalasia appear often in combination with further symptoms in a syndromic form as an inherited disease. Methods Whole genome sequencing was used to investigate the genetic basis of isolated achalasia in a family of Tunisian origin. We analyzed the function of the affected protein with immunofluorescence and affinity chromatography study. Key Results A homozygous nonsense mutation was detected in murine retrovirus integration site 1 (MRVI1) gene (Human Genome Organisation Gene Nomenclature Committee (HGNC) approved gene symbol:IRAG1) encoding the inositol 1,4,5-trisphosphate receptor 1 (IP(3)R1)-associated cyclic guanosine monophosphate (cGMP) kinase substrate (IRAG). Sanger sequencing confirmed co-segregation of the mutation with the disease. Sequencing of the entireMRVI1gene in 35 additional patients with a syndromic form of achalasia did not uncover further cases withMRVI1mutations. Immunofluorescence analysis of transfected COS7 cells revealed GFP-IRAG with the truncating mutation p.Arg112* (transcript variant 1) or p.Arg121* (transcript variant 2) to be mislocalized in the cytoplasm and the nucleus. Co-transfection with cGMP-dependent protein kinase 1 isoform beta (cGK1 beta) depicted a partial mislocalization of cGK1 beta due to mislocalized truncated IRAG. Isolation of protein complexes revealed that the truncation of this protein causes the loss of the interaction domain of IRAG with cGK1 beta. Conclusions & Inferences In individuals with an early onset of achalasia without further accompanying symptoms,MRVI1mutations should be considered as the disease-causing defect

Genetic investigation of the ubiquitin-protein ligase E3A gene as putative target in Angelman syndrome

BACKGROUND Angelman syndrome (AS) is caused by maternal chromosomal deletions, imprinting defects, paternal uniparental disomy involving chromosome 15 and the ubiquitin-protein ligase UBE3A gene mutations. However the genetic basis remains unclear for several patients. AIM To investigate the involvement of UBE3A gene in AS and identifying new potential genes using exome sequencing. METHODS We established a cohort study in 50 patients referred to Farhat Hached University Hospital between 2006 and 2021, with a strong suspicion of AS and absence of chromosomal aberrations. The UBE3A gene was screened for mutation detection. Two unrelated patients issued from consanguineous families were subjected to exome analysis. RESULTS We describe seven UBE3A variants among them 3 none previously described including intronic variants c.2220+14T>C (intron14), c.2507+43T>A (Exon15) and insertion in Exon7: c.30-47_30-46. The exome sequencing revealed 22 potential genes that could be involved in AS-like syndromes that should be investigated further. CONCLUSION Screening for UBE3A mutations in AS patients has been proven to be useful to confirm the diagnosis. Our exome findings could rise to new potential alternative target genes for genetic counseling