Human Plasma Glycome in Attention-Deficit Hyperactivity Disorder and Autism Spectrum Disorders

Abstract

Over a half of all proteins are glycosylated, and their proper glycosylation is essential for normal function. Unfortunately, because of structural complexity of nonlinear branched glycans and the absence of genetic template for their synthesis, the knowledge about glycans is lagging significantly behind the knowledge about proteins or DNA. Using a recently developed quantitative high throughput glycan analysis method we quantified components of the plasma N-glycome in 99 children with attention-deficit hyperactivity disorder (ADHD), 81 child and 5 adults with autism spectrum disorder, and a total of 340 matching healthy controls. No changes in plasma glycome were found to associate with autism spectrum disorder, but several highly significant associations were observed with ADHD. Further structural analysis of plasma glycans revealed that ADHD is associated with increased antennary fucosylation of biantennary glycans and decreased levels of some complex glycans with three or four antennas. The design of this study prevented any functional conclusions about the observed associations, but specific differences in glycosylation appears to be strongly associated with ADHD and warrants further studies in this direction. Over a half of all proteins are glycosylated, and their proper glycosylation is essential for normal function. Unfortunately, because of structural complexity of nonlinear branched glycans and the absence of genetic template for their synthesis, the knowledge about glycans is lagging significantly behind the knowledge about proteins or DNA. Using a recently developed quantitative high throughput glycan analysis method we quantified components of the plasma N-glycome in 99 children with attention-deficit hyperactivity disorder (ADHD), 81 child and 5 adults with autism spectrum disorder, and a total of 340 matching healthy controls. No changes in plasma glycome were found to associate with autism spectrum disorder, but several highly significant associations were observed with ADHD. Further structural analysis of plasma glycans revealed that ADHD is associated with increased antennary fucosylation of biantennary glycans and decreased levels of some complex glycans with three or four antennas. The design of this study prevented any functional conclusions about the observed associations, but specific differences in glycosylation appears to be strongly associated with ADHD and warrants further studies in this direction. Attention-deficit hyperactivity disorder (ADHD) 1The abbreviations used are:ADHDattention-deficit hyperactivity disorderASDautism spectrum disorderHILIChigh performance liquid chromatographyHPLChigh pressure liquid chromatography. and autism spectrum disorders (ASDs) are both highly heritable multifactorial and clinically heterogeneous childhood-onset neuro-developmental disorders (1.Faraone S.V. Khan S.A. Candidate gene studies of attention-deficit/hyperactivity disorder.J. Clin. Psychiatry 67 Suppl. 2006; 8: 13-20Google Scholar, 2.Levy S.E. Mandell D.S. Schultz R.T. Autism. Lancet. 2009; 374: 1627-1638Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). The hallmarks of ADHD are severe inattention, hyperactivity, and impulsivity whereas the characteristic symptoms of ASD include impaired communication and social interaction skills as well as repetitive and restricted behavior and interests (3.American Psychiatric Association Diagnostic and statistical manual of mental disorders. 4th edn, Text Revision. American Psychiatric Association, Washington, DC2000Google Scholar). Several pieces of evidence point toward some shared heritability and thus genetic factors contributing to the occurrence of both ADHD an ASD (4.Rommelse N.N. Franke B. Geurts H.M. Hartman C.A. Buitelaar J.K. Shared heritability of attention-deficit/hyperactivity disorder and autism spectrum disorder.Eur. Child Adolesc. Psychiatry. 2010; 19: 281-295Crossref PubMed Scopus (391) Google Scholar). Children with ADHD and ASD frequently share symptoms of impaired communication skills, impaired social interactions (4.Rommelse N.N. Franke B. Geurts H.M. Hartman C.A. Buitelaar J.K. Shared heritability of attention-deficit/hyperactivity disorder and autism spectrum disorder.Eur. Child Adolesc. Psychiatry. 2010; 19: 281-295Crossref PubMed Scopus (391) Google Scholar), and develop difficulties in familial, educational, and academic functioning (5.Biederman J. Attention-deficit/hyperactivity disorder: a selective overview.Biol. Psychiatry. 2005; 57: 1215-1220Abstract Full Text Full Text PDF PubMed Scopus (959) Google Scholar, 6.Wall D.P. Esteban F.J. Deluca T.F. Huyck M. Monaghan T. Velez de Mendizabal N. Goñi J. Kohane I.S. Comparative analysis of neurological disorders focuses genome-wide search for autism genes.Genomics. 2009; 93: 120-129Crossref PubMed Scopus (39) Google Scholar). The abnormal functioning of the dopaminergic and serotonergic systems (5.Biederman J. Attention-deficit/hyperactivity disorder: a selective overview.Biol. Psychiatry. 2005; 57: 1215-1220Abstract Full Text Full Text PDF PubMed Scopus (959) Google Scholar, 7.Nedic G. Pivac N. Hercigonja D.K. Jovancevic M. Curkovic K.D. Muck-Seler D. Platelet monoamine oxidase activity in children with attention-deficit/hyperactivity disorder.Psychiatry Res. 2010; 175: 252-255Crossref PubMed Scopus (24) Google Scholar, 8.Hercigonja Novkovic V. Rudan V. Pivac N. Nedic G. Muck-Seler D. Platelet serotonin concentration in children with attention-deficit/hyperactivity disorder.Neuropsychobiology. 2009; 59: 17-22Crossref PubMed Scopus (22) Google Scholar, 9.Sharp S.I. McQuillin A. Gurling H.M. Genetics of attention-deficit hyperactivity disorder (ADHD).Neuropharmacology. 2009; 57: 590-600Crossref PubMed Scopus (103) Google Scholar), among other neurotransmitters, were repeatedly found in ADHD and ASD, and therefore the candidate genes common for both disorders are the dopaminergic transporter gene, dopaminergic receptor type 3 and type 4 genes, serotonin transporter gene, and genes for catechol-o-methyl-transferase and monoamine oxidase type A (4.Rommelse N.N. Franke B. Geurts H.M. Hartman C.A. Buitelaar J.K. Shared heritability of attention-deficit/hyperactivity disorder and autism spectrum disorder.Eur. Child Adolesc. Psychiatry. 2010; 19: 281-295Crossref PubMed Scopus (391) Google Scholar), although genome-wide association studies did not confirm with certainty all these candidates (10.Franke B. Neale B.M. Faraone S.V. Genome-wide association studies in ADHD.Hum. Genet. 2009; 126: 13-50Crossref PubMed Scopus (339) Google Scholar). A shared genetic basis is further suggested by the higher frequency of ADHD and ASD traits in children with a defined genetic diagnosis such as 22q11 deletion syndrome, XXYY syndrome, and microdeletions or duplications in the chromosome 15q13.2-q13.3 region (11.Tartaglia N. Davis S. Hench A. Nimishakavi S. Beauregard R. Reynolds A. Fenton L. Albrecht L. Ross J. Visootsak J. Hansen R. Hagerman R. A new look at XXYY syndrome: medical and psychological features.Am. J. Med. Genet. A. 2008; 146A: 1509-1522Crossref PubMed Scopus (118) Google Scholar, 12.Miller D.T. Shen Y. Weiss L.A. Korn J. Anselm I. Bridgemohan C. Cox G.F. Dickinson H. Gentile J. Harris D.J. Hegde V. Hundley R. Khwaja O. Kothare S. Luedke C. Nasir R. Poduri A. Prasad K. Raffalli P. Reinhard A. Smith S.E. Sobeih M.M. Soul J.S. Stoler J. Takeoka M. Tan W.H. Thakuria J. Wolff R. Yusupov R. Gusella J.F. Daly M.J. Wu B.L. Microdeletion/duplication at 15q13.2q13.3 among individuals with features of autism and other neuropsychiatric disorders.J. Med. Genet. 2009; 46: 242-248Crossref PubMed Scopus (264) Google Scholar). However, the molecular agents related to various biological processes and interactions between different risk factors responsible for development of ADHD and ASD remain elusive (6.Wall D.P. Esteban F.J. Deluca T.F. Huyck M. Monaghan T. Velez de Mendizabal N. Goñi J. Kohane I.S. Comparative analysis of neurological disorders focuses genome-wide search for autism genes.Genomics. 2009; 93: 120-129Crossref PubMed Scopus (39) Google Scholar). attention-deficit hyperactivity disorder autism spectrum disorder high performance liquid chromatography high pressure liquid chromatography. Protein N-glycosylation is an ancient metabolic pathway invented before the diversification of Archea, Bacteria, and Eukarya, which still exists in all three domains of life (13.Weerapana E. Imperiali B. Asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems.Glycobiology. 2006; 16: 91R-101RCrossref PubMed Scopus (274) Google Scholar, 14.Calo D. Kaminski L. Eichler J. Protein glycosylation in Archaea: Sweet and Extreme.Glycobiology. 2010; 20: 1065-1076Crossref PubMed Scopus (109) Google Scholar). At least 2000 different glycan determinants have been added to the backbone of mammalian polypeptides (15.Cummings R.D. The repertoire of glycan determinants in the human glycome.Mol. Biosyst. 2009; 5: 1087-1104Crossref PubMed Scopus (356) Google Scholar) and the complexity of the glycoproteome is estimated to be several orders of magnitude greater than for the proteome itself (16.Lee R.T. Lauc G. Lee Y.C. Glycoproteomics: protein modifications for versatile functions.EMBO Rep. 2005; 6: 1018-1022Crossref PubMed Scopus (27) Google Scholar). N-glycosylation is essential for multicellular life and its complete absence is embryonically lethal (17.Marek K.W. Vijay I.K. Marth J.D. A recessive deletion in the GlcNAc-1-phosphotransferase gene results in peri-implantation embryonic lethality.Glycobiology. 1999; 9: 1263-1271Crossref PubMed Scopus (118) Google Scholar). Disregulation of glycosylation is associated with a wide range of diseases, including cancer, diabetes, and cardiovascular, congenital, immunological, and infectious disorders (18.Crocker P.R. Paulson J.C. Varki A. Siglecs and their roles in the immune system.Nat. Rev. Immunol. 2007; 7: 255-266Crossref PubMed Scopus (1416) Google Scholar, 19.Marth J.D. Grewal P.K. Mammalian glycosylation in immunity.Nat. Rev. Immunol. 2008; 8: 874-887Crossref PubMed Scopus (529) Google Scholar, 20.Ohtsubo K. Marth J.D. Glycosylation in cellular mechanisms of health and disease.Cell. 2006; 126: 855-867Abstract Full Text Full Text PDF PubMed Scopus (2092) Google Scholar), but because of experimental difficulties in analyzing complex glycan structures, the knowledge about glycan structure and function is still lagging behind the knowledge about other macromolecules (21.Lauc G. Sweet secret of the multicellular life.Biochim. Biophys. Acta. 2006; 1760: 525-526Crossref PubMed Scopus (16) Google Scholar). However, recent technological advances have allowed reliable, high-throughput quantification of N-glycans (22.Royle L. Campbell M.P. Radcliffe C.M. White D.M. Harvey D.J. Abrahams J.L. Kim Y.G. Henry G.W. Shadick N.A. Weinblatt M.E. Lee D.M. Rudd P.M. Dwek R.A. HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software.Anal. Biochem. 2008; 376: 1-12Crossref PubMed Scopus (386) Google Scholar, 23.Huhn C. Selman M.H. Ruhaak L.R. Deelder A.M. Wuhrer M. IgG glycosylation analysis.Proteomics. 2009; 9: 882-913Crossref PubMed Scopus (255) Google Scholar, 24.Selman M.H. McDonnell L.A. Palmblad M. Ruhaak L.R. Deelder A.M. Wuhrer M. Immunoglobulin G glycopeptide profiling by matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry.Anal. Chem. 2010; 82: 1073-1081Crossref PubMed Scopus (100) Google Scholar), which now permit large scale studies aimed at understanding of the role of protein glycosylation in complex diseases. Variations in glycosylation are of great physiological significance because changes in glycans significantly change the structure and function of polypeptide parts of glycoproteins (25.Skropeta D. The effect of individual N-glycans on enzyme activity.Bioorg. Med. Chem. 2009; 17: 2645-2653Crossref PubMed Scopus (142) Google Scholar). Proper glycosylation of membrane receptors is particularly important as it modulates adaptive properties of the cell and affects communication between cells (26.Dennis J.W. Lau K.S. Demetriou M. Nabi I.R. Adaptive Regulation at the Cell Surface by N-Glycosylation.Traffic. 2009; 10: 1569-1578Crossref PubMed Scopus (163) Google Scholar). Altered glycosylation and ensuing differences in function of various receptors could be a predisposing factor in different psychiatric disorders, and indeed some of them were reported to be associated with altered protein glycosylation (27.Barisic K. Lauc G. Dumic J. Pavlovic M. Flogel M. Changes of glycoprotein patterns in sera of humans under stress.Eur. J. Clin. Chem. Clin. Biochem. 1996; 34: 97-101PubMed Google Scholar, 28.Bauer D. Haroutunian V. Meador-Woodruff J.H. McCullumsmith R.E. Abnormal glycosylation of EAAT1 and EAAT2 in prefrontal cortex of elderly patients with schizophrenia.Schizophr. Res. 2010; 117: 92-98Crossref PubMed Scopus (90) Google Scholar, 29.Baysal B.E. Willett-Brozick J.E. Badner J.A. Corona W. Ferrell R.E. Nimgaonkar V.L. Detera-Wadleigh S.D. A mannosyltransferase gene at 11q23 is disrupted by a translocation breakpoint that co-segregates with bipolar affective disorder in a small family.Neurogenetics. 2002; 4: 43-53Crossref PubMed Scopus (27) Google Scholar, 30.Maguire T.M. Thakore J. Dinan T.G. Hopwood S. Breen K.C. Plasma sialyltransferase levels in psychiatric disorders as a possible indicator of HPA axis function.Biol. Psychiatry. 1997; 41: 1131-1136Abstract Full Text PDF PubMed Scopus (32) Google Scholar, J. G. C.A. Glycosylation of as for 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). we the analysis of plasma N-glycans in patients with ADHD and The study 99 children with ADHD from the psychiatric to the with the children and their psychological and psychological and the for in attention-deficit/hyperactivity disorder: in and Clin. Psychiatry. 59: Google Scholar). were for all were of or and were from the with were disorder, disorder, disorder and disorders, all by psychiatric on or with or selective serotonin healthy children their as controls. or other psychiatric were to and the Child with or children with ASD were were from the by the patients have a diagnosis of ASD to were by a child and a in the for in and have been by a and patients with genetic were The for in this study normal of all have a normal and and were total the individuals children and 5 adults with ASD and individuals were from a a with of at all The study in with the and the were in any about the and as reported A. O. O. Rudan I. Campbell H. C. A. I. N. J. Rudd P.M. Lauc G. and of Plasma Res. 2009; 8: PubMed Scopus Google Scholar). Plasma proteins were in a of and N-glycans were by with in a 96-well plate to the throughput of glycans were with glycans were to interaction high performance liquid chromatography on a at with to with as A and as B. were with with and of and The an of and from which the for the individual glycans were to The were all in the for and for the and of glycan as The of glycan in were as the of total of total glycan and total serum were to the of by The analysis a at with in as A and to with in as B. were on the to their the higher the A used for of the glycan were in 96-well the of of and to to 5 at and Washington, before to the differences in levels of in between ADHD and of the glycans were to an ADHD glycan and a glycan were and were on were from both glycan and in before the of of and to to 5 at and Washington, before to the of glycans the structural features were by the the from or glycan glycan in glycan that were reported are in were quantified from to the of and features were defined glycans glycans glycans biantennary glycans biantennary glycans glycans glycans glycans glycans glycans glycans glycans glycan between ADHD and N-glycans were a with N-glycans as the and ADHD as the and were used as The analysis the and of the statistical analysis A and for Scholar). between ASD and N-glycans were a with N-glycans as the and ASD as the and were used as The structure as a used a because a is not for this structure and to The is in and therefore individual of related individuals than a of The analysis the statistical analysis L. J. analysis in J. Genet. Full Text Full Text PDF PubMed Scopus Google Scholar). on associations between mental disorders and plasma N-glycans were for results because of we a we used an of we are of the that this method is to traits such as plasma as the method is on the of we have this in the Using a recently developed method for high throughput quantitative analysis of glycans we the plasma glycome in 99 children with ADHD and healthy children were for in quantified of The of the glycan is in Further glycosylation features were from the as in the and in the total of glycan for these glycan features are in of plasma glycome in children with ADHD and controls. in glycan are in and the other glycans traits were is in the and between ADHD and N-glycans were a with N-glycans as the and ADHD as the and were used as with statistical significance the of are in with ADHD as a in a new glycans significantly change with A. O. O. M. I. M. Rudd P.M. Campbell H. Rudan I. Lauc G. of mass plasma and on human plasma 2010; 20: PubMed Scopus Google Scholar), thus associations between ADHD and N-glycans were a with N-glycans as the ADHD as the and and as for four associations of glycan levels were found to be whereas further were to statistical significance The changes in plasma glycans in ADHD were the in glycan and and a in of and ADHD are in the with and could not have been the from other which to it for structural glycan in are glycan of and and biantennary glycan with three antennary antennary and antennary which of the is associated with we glycans from ADHD and glycans by chromatography and to and were with an enzyme which antennary ADHD and glycan were by to the of from in that although is the of in is the of in ADHD and that it is the in that with ADHD. for this by the of with the enzyme S.V. Khan S.A. Candidate gene studies of attention-deficit/hyperactivity disorder.J. Clin. Psychiatry 67 Suppl. 2006; 8: 13-20Google Scholar, 2.Levy S.E. Mandell D.S. Schultz R.T. Autism. Lancet. 2009; 374: 1627-1638Abstract Full Text Full Text PDF PubMed Scopus (538) Google Psychiatric Association Diagnostic and statistical manual of mental disorders. 4th edn, Text Revision. American Psychiatric Association, Washington, DC2000Google D.P. Esteban F.J. Deluca T.F. Huyck M. Monaghan T. Velez de Mendizabal N. Goñi J. Kohane I.S. Comparative analysis of neurological disorders focuses genome-wide search for autism genes.Genomics. 2009; 93: 120-129Crossref PubMed Scopus (39) Google Scholar) that not that whereas in the glycan structure in is in ADHD is increased and the glycan structure in further by of the complete plasma glycome with S.V. Khan S.A. Candidate gene studies of attention-deficit/hyperactivity disorder.J. Clin. Psychiatry 67 Suppl. 2006; 8: 13-20Google Scholar, 2.Levy S.E. Mandell D.S. Schultz R.T. Autism. Lancet. 2009; 374: 1627-1638Abstract Full Text Full Text PDF PubMed Scopus (538) Google Psychiatric Association Diagnostic and statistical manual of mental disorders. 4th edn, Text Revision. American Psychiatric Association, Washington, DC2000Google D.P. Esteban F.J. Deluca T.F. Huyck M. Monaghan T. Velez de Mendizabal N. Goñi J. Kohane I.S. Comparative analysis of neurological disorders focuses genome-wide search for autism genes.Genomics. 2009; 93: 120-129Crossref PubMed Scopus (39) Google Scholar) which that are changes in the of in ADHD analysis of glycans associated with ADHD. by from the glycan of and which both at of from glycan with S.V. Khan S.A. Candidate gene studies of attention-deficit/hyperactivity disorder.J. Clin. Psychiatry 67 Suppl. 2006; 8: 13-20Google Scholar, 2.Levy S.E. Mandell D.S. Schultz R.T. Autism. Lancet. 2009; 374: 1627-1638Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, Psychiatric Association Diagnostic and statistical manual of mental disorders. 4th edn, Text Revision. American Psychiatric Association, Washington, DC2000Google N.N. Franke B. Geurts H.M. Hartman C.A. Buitelaar J.K. Shared heritability of attention-deficit/hyperactivity disorder and autism spectrum disorder.Eur. Child Adolesc. Psychiatry. 2010; 19: 281-295Crossref PubMed Scopus (391) Google Scholar) that to The new at to which on the of of from ADHD glycan with S.V. Khan S.A. Candidate gene studies of attention-deficit/hyperactivity disorder.J. Clin. Psychiatry 67 Suppl. 2006; 8: 13-20Google Scholar, 2.Levy S.E. Mandell D.S. Schultz R.T. Autism. Lancet. 2009; 374: 1627-1638Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, Psychiatric Association Diagnostic and statistical manual of mental disorders. 4th edn, Text Revision. American Psychiatric Association, Washington, DC2000Google N.N. Franke B. Geurts H.M. Hartman C.A. Buitelaar J.K. Shared heritability of attention-deficit/hyperactivity disorder and autism spectrum disorder.Eur. Child Adolesc. Psychiatry. 2010; 19: 281-295Crossref PubMed Scopus (391) Google Scholar) of and that in ADHD is an of the of with The type of glycan were on 81 children and their healthy and for the glycan features are in this study in a used to statistical significance of the observed No significant associations between glycan levels and ASD were of plasma glycome in children with ASD and controls. in glycan are in and the other glycans traits were is in the and between ASD and N-glycans were a with N-glycans as the and ASD as the and were used as with ASD as in a new have the analysis of human plasma N-glycome in patients with ADHD and we have found differences between children with ASD and their healthy several significant associations between plasma glycans and ADHD were we have that the observed association between ADHD and increased increased which is from from the in levels of biantennary glycans with antennary ADHD associated with a of changes in and of these changes and were highly whereas the four were the significance of between and the is highly these results that the levels of and glycans are altered in ADHD. these changes were with the association between ADHD and the in of plasma The of glycans that found to be increased in ADHD is of biantennary whereas all glycan that were decreased in ADHD of and is important for the of adaptive properties of the cell membrane (26.Dennis J.W. Lau K.S. Demetriou M. Nabi I.R. Adaptive Regulation at the Cell Surface by N-Glycosylation.Traffic. 2009; 10: 1569-1578Crossref PubMed Scopus (163) Google Scholar) and it reported that the increased in the cell membrane and membrane receptor C. J. P. M. Nabi I.R. J.L. J.W. Regulation of receptors by and PubMed Scopus Google Scholar). the structure of glycans to individual membrane receptors significantly affects their functional properties (25.Skropeta D. The effect of individual N-glycans on enzyme activity.Bioorg. Med. Chem. 2009; 17: 2645-2653Crossref PubMed Scopus (142) Google Scholar), thus it is that differences in glycosylation could in altered and a for the development of ADHD. is important to that we have levels of individual glycans in glycan from several different and because glycosylation is and small in plasma levels could have important functional we the study of human plasma which revealed that by the of proteins and A. O. O. Rudan I. Campbell H. C. A. I. N. J. Rudd P.M. Lauc G. and of Plasma Res. 2009; 8: PubMed Scopus Google Scholar). However, a individual the of plasma glycome is O. J. M. A. I. Lauc G. of in human 2009; 19: PubMed Scopus Google Scholar) and factors have on the of glycans A. O. O. M. I. M. Rudd P.M. Campbell H. Rudan I. Lauc G. of mass plasma and on human plasma 2010; 20: PubMed Scopus Google Scholar). altered that be associated with specific were to in a M. S. M. A. O. O. K. W. Rudd P.M. Campbell H. Rudan I. Lauc G. from normal human plasma 2010; 20: PubMed Scopus Google Scholar). we have a new of increased and decreased and glycans that with whereas plasma N-glycome not found to be significantly altered in that the in glycan could be of the predisposing factors for ADHD because it reported to be associated with the activity of membrane receptors (26.Dennis J.W. Lau K.S. Demetriou M. Nabi I.R. Adaptive Regulation at the Cell Surface by N-Glycosylation.Traffic. 2009; 10: 1569-1578Crossref PubMed Scopus (163) Google Scholar). this to be this a new for the development of for ADHD. with