Kazuhiro Aoki1, Adam D Heaps2, Kevin A Strauss2,3, Michael Tiemeyer1,4. 1. Complex Carbohydrate Research Center, University of Georgia, Athens, GA, Greece. 2. Clinic for Special Children, Strasburg, PA, United States. 3. Lancaster General Hospital, Lancaster, PA, United States. 4. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, Greece.
Abstract
BACKGROUND: Among Amish communities of North America, biallelic mutations of ST3GAL5 (c.694C > T) eliminate synthesis of GM3 and its derivative downstream a- and b-series gangliosides. Systemic ganglioside deficiency is associated with infantile onset psychomotor retardation, slow brain growth, intractable epilepsy, deafness, and cortical visual impairment. We developed a robust quantitative assay to simultaneously characterize glycan and ceramide moieties of plasma glycosphingolipids (GSLs) among ST3GAL5 c.694C > T homozygotes (n = 8), their heterozygous siblings (n = 24), and wild type control (n = 19) individuals. METHODS: Following extraction and saponification of total plasma lipids, GSLs were purified on a tC18 cartridge column, permethylated, and subjected to nanospray ionization mass spectrometry utilizing neutral loss scanning and data-dependent acquisition. Plasma GSLs were quantified against appropriate synthetic standards. RESULTS: Our method demonstrated linearity from 5 to 250 μl of plasma. Recovery of synthetic GSLs spiked into plasma was 99-104% with no matrix interference. Quantitative plasma GSL profiles discriminated among ST3GAL5 genotypes: GM3 and GD3 were undetectable in ST3GAL5 c.694C > T homozygotes, who had markedly elevated lactosylceramide (19.17 ± 4.20 nmol/ml) relative to heterozygous siblings (9.62 ± 2.46 nmol/ml) and wild type controls (6.55 ± 2.16 nmol/ml). Children with systemic ganglioside deficiency had a distinctive shift in ceramide composition toward higher mass species. CONCLUSIONS: Our quantitative glycolipidomics method discriminates among ST3GAL5 c.694C > T genotypes, can reveal subtle structural heterogeneity, and represents a useful new strategy to diagnose and monitor GSL disorders in humans.
BACKGROUND: Among Amish communities of North America, biallelic mutations of ST3GAL5 (c.694C > T) eliminate synthesis of GM3 and its derivative downstream a- and b-series gangliosides. Systemic ganglioside deficiency is associated with infantile onset psychomotor retardation, slow brain growth, intractable epilepsy, deafness, and cortical visual impairment. We developed a robust quantitative assay to simultaneously characterize glycan and ceramide moieties of plasma glycosphingolipids (GSLs) among ST3GAL5 c.694C > T homozygotes (n = 8), their heterozygous siblings (n = 24), and wild type control (n = 19) individuals. METHODS: Following extraction and saponification of total plasma lipids, GSLs were purified on a tC18 cartridge column, permethylated, and subjected to nanospray ionization mass spectrometry utilizing neutral loss scanning and data-dependent acquisition. Plasma GSLs were quantified against appropriate synthetic standards. RESULTS: Our method demonstrated linearity from 5 to 250 μl of plasma. Recovery of synthetic GSLs spiked into plasma was 99-104% with no matrix interference. Quantitative plasma GSL profiles discriminated among ST3GAL5 genotypes: GM3 and GD3 were undetectable in ST3GAL5 c.694C > T homozygotes, who had markedly elevated lactosylceramide (19.17 ± 4.20 nmol/ml) relative to heterozygous siblings (9.62 ± 2.46 nmol/ml) and wild type controls (6.55 ± 2.16 nmol/ml). Children with systemic ganglioside deficiency had a distinctive shift in ceramide composition toward higher mass species. CONCLUSIONS: Our quantitative glycolipidomics method discriminates among ST3GAL5 c.694C > T genotypes, can reveal subtle structural heterogeneity, and represents a useful new strategy to diagnose and monitor GSL disorders in humans.
Authors: Luigi Boccuto; Kazuhiro Aoki; Heather Flanagan-Steet; Chin-Fu Chen; Xiang Fan; Frank Bartel; Marharyta Petukh; Ayla Pittman; Robert Saul; Alka Chaubey; Emil Alexov; Michael Tiemeyer; Richard Steet; Charles E Schwartz Journal: Hum Mol Genet Date: 2013-09-10 Impact factor: 6.150
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Authors: Amir Boukhris; Rebecca Schule; José L Loureiro; Charles Marques Lourenço; Emeline Mundwiller; Michael A Gonzalez; Perrine Charles; Julie Gauthier; Imen Rekik; Rafael F Acosta Lebrigio; Marion Gaussen; Fiorella Speziani; Andreas Ferbert; Imed Feki; Andrés Caballero-Oteyza; Alexandre Dionne-Laporte; Mohamed Amri; Anne Noreau; Sylvie Forlani; Vitor T Cruz; Fanny Mochel; Paula Coutinho; Patrick Dion; Chokri Mhiri; Ludger Schols; Jean Pouget; Frédéric Darios; Guy A Rouleau; Wilson Marques; Alexis Brice; Alexandra Durr; Stephan Zuchner; Giovanni Stevanin Journal: Am J Hum Genet Date: 2013-06-06 Impact factor: 11.025