Christophe Hirtz1, Jerome Vialaret1, Georges Nouadje2, Susanna Schraen3, Pascale Benlian4, Sandrine Mary5, Pascal Philibert6, Laurent Tiers1, Pauline Bros1, Constance Delaby1, Audrey Gabelle7, Sylvain Lehmann8. 1. CHRU de Montpellier, Hôpital St Eloi Université de Montpellier, INSERM U1183, IRMB, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France. 2. SEBIA, 91008 Evry, France. 3. Inserm U837, Lille, FRANCE Université Paris 7-Denis Diderot, France. 4. Université Lille 2 and UMR CNRS 8199 Integrative Genomics and Metabolic Disease Modeling, LILLE France; CHRU de Lille, Unité de Médecine Moléculaire des Maladies Métaboliques (U4M), Lille, FRANCE. 5. Université Lille 2 and UMR CNRS 8199 Integrative Genomics and Metabolic Disease Modeling, LILLE France. 6. CHRU de Montpellier, Service de Biochimie et d'Hormonologie, Montpellier, France. 7. CHRU de Montpellier, Hôpital St Eloi Université de Montpellier, INSERM U1183, IRMB, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France; Centre Mémoire de Ressources et de Recherche Languedoc-Roussillon, Département de Neurologie, CHRU de Montpellier, Université de Montpellier, Montpellier, France. 8. CHRU de Montpellier, Hôpital St Eloi Université de Montpellier, INSERM U1183, IRMB, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France. Electronic address: s-lehmann@chu-montpellier.fr.
Abstract
BACKGROUND: Apolipoprotein E (Apo E) is a 36 Kda glycoprotein involved in lipid transport. It exists in 3 major isoforms: E2, E3 and E4. ApoE status is known to be a major risk factor for late-onset Alzheimer's and cardiovascular diseases. Genotyping is commonly used to obtain ApoE status but can show technical issues with ambiguous determinations. Phenotyping can be an alternative, not requiring genetic material. We evaluated the ability to accurately type ApoE isoforms by 2 phenotyping tests in comparison with genotyping. METHODS: Two phenotyping techniques were used: (1) LC-MS/MS detection of 4 ApoE specific peptides (6490 Agilent triple quadripole): After its denaturation, serum was either reduced and alkylated, or only diluted, and then trypsin digested. Before analysis, desalting, evaporation and resuspension were performed. (2) Isoelectric focusing and immunoprecipitation: serum samples were neuraminidase digested, delipidated and electrophoresed on Hydragel ApoE (Sebia agarose gel) using Hydrasys 2 Scan instrument (Sebia, Lisses, France). ApoE isoforms bands were directly immunofixed in the gel using a polyclonal anti human ApoE antibody. Then, incubation of the gel with HRP secondary antibody followed by TTF1/TTF2 substrate allowed the visualization of ApoE bands. The results of the two techniques were compared to genotyping. RESULTS: Sera from 35 patients previously genotyped were analyzed with the 2 phenotyping techniques. 100% concordance between both phenotyping assays was obtained for the tested phenotypes (E2/E2, E2/E3, E2/E4, E3/E3, E3/E4, E4/E4). When compared to genotyping, 3 samples were discordant. After reanalyzing them by both phenotyping tests and DNA sequencing, 2/3 discrepancies were confirmed. Those can be explained by variants or rare ApoE alleles or by unidentified technical issues. 102 additional samples were then tested on LC-MS/MS only and compared to genotyping. The data showed 100% concordance. CONCLUSION: Our 2 phenotyping methods represent a valuable alternative to genotyping. LC-MS/MS has the advantage of being fully specific, with identification of the different isoforms and can be considered as a reference method. Sebia isofocusing technique was concordant with LC-MS/MS. Plus, it is a rapid, semi-automated assay that can be easily implemented in clinical laboratories.
BACKGROUND:Apolipoprotein E (Apo E) is a 36 Kda glycoprotein involved in lipid transport. It exists in 3 major isoforms: E2, E3 and E4. ApoE status is known to be a major risk factor for late-onset Alzheimer's and cardiovascular diseases. Genotyping is commonly used to obtain ApoE status but can show technical issues with ambiguous determinations. Phenotyping can be an alternative, not requiring genetic material. We evaluated the ability to accurately type ApoE isoforms by 2 phenotyping tests in comparison with genotyping. METHODS: Two phenotyping techniques were used: (1) LC-MS/MS detection of 4 ApoE specific peptides (6490 Agilent triple quadripole): After its denaturation, serum was either reduced and alkylated, or only diluted, and then trypsin digested. Before analysis, desalting, evaporation and resuspension were performed. (2) Isoelectric focusing and immunoprecipitation: serum samples were neuraminidase digested, delipidated and electrophoresed on Hydragel ApoE (Sebia agarose gel) using Hydrasys 2 Scan instrument (Sebia, Lisses, France). ApoE isoforms bands were directly immunofixed in the gel using a polyclonal anti humanApoE antibody. Then, incubation of the gel with HRP secondary antibody followed by TTF1/TTF2 substrate allowed the visualization of ApoE bands. The results of the two techniques were compared to genotyping. RESULTS: Sera from 35 patients previously genotyped were analyzed with the 2 phenotyping techniques. 100% concordance between both phenotyping assays was obtained for the tested phenotypes (E2/E2, E2/E3, E2/E4, E3/E3, E3/E4, E4/E4). When compared to genotyping, 3 samples were discordant. After reanalyzing them by both phenotyping tests and DNA sequencing, 2/3 discrepancies were confirmed. Those can be explained by variants or rare ApoE alleles or by unidentified technical issues. 102 additional samples were then tested on LC-MS/MS only and compared to genotyping. The data showed 100% concordance. CONCLUSION: Our 2 phenotyping methods represent a valuable alternative to genotyping. LC-MS/MS has the advantage of being fully specific, with identification of the different isoforms and can be considered as a reference method. Sebia isofocusing technique was concordant with LC-MS/MS. Plus, it is a rapid, semi-automated assay that can be easily implemented in clinical laboratories.
Authors: Wendy E Heywood; Anna Baud; Emily Bliss; Ernestas Sirka; Jonathan M Schott; Henrik Zetterberg; Daniela Galimberti; Neil J Sebire; Kevin Mills Journal: J Vis Exp Date: 2016-10-20 Impact factor: 1.355