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

An Crepel,Fran Borovečki,Mario Ćurković,Vesna Novković Hercigonja,Mislav Novokmet,Matea Nikolac,Ana Knežević,Jean Steyaert,Gordana Nedić,Olga Gornik,Maja Pučić,Hilde Peeters,Nela Pivac,Gordan Lauc,Wilmar Igl,Katarina Dodig Ćurković,Igor Rudan,Dorotea Muck-Seler,Irma Redžić

doi:10.1074/mcp.m110.004200

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.

Highlights

Attention-deficit hyperactivity disorder (ADHD)1 and autism spectrum disorders (ASDs) are both highly heritable multifactorial and clinically heterogeneous childhood-onset neurodevelopmental disorders [1, 2]
We have found no differences between children with ASD and their healthy siblings, several significant associations between plasma glycans and ADHD were identified (Table I)
We have convincingly demonstrated that the observed association between ADHD and increased DG7 derived from the increase in relative levels of biantennary glycans with antennary fucose (A2FG2)

Summary

EXPERIMENTAL PROCEDURES

Sample Population and Study Design—The study included 99 medication-free children with ADHD (9.00 Ϯ 2.78 years old), recruited sequentially from the Polyclinic Kocijan/Hercigonja, Zagreb, diagnosed using psychiatric interview (APA, 1994), according to the DSM-IV criteria, separate interviews with the children and their parents, psychological interview and psychological tests, physical examination, and the Conners’ Rating Scale for Parents-short version [32]. The following was added: 1 ␮l of 250 mM sodium phosphate incubation buffer (pH 6.0), 0.5 ␮l (0.0025 U) of ABS, Arthrobacter ureafaciens sialidase (releases ␣2–3,6,8 sialic acid, Prozyme) and H2O to make up to 5 ␮l This was incubated overnight (16 –18 h) at 37 °C and passed through AcroPrepTM 96 Filter Plates, 350 ␮l well, 10K (Pall Corporation, Port Washington, NY) before applying to the HPLC. Fucosidase Digestion—To examine differences in levels of structures present in peak DG7 between ADHD samples and controls, aliquots of the 2AB-labeled glycans were mixed together to make an ADHD glycan pool and a control glycan pool. These pooled samples were dried down and concentrated pool samples were applied on HILIC column. The following was added: 1 ␮l of 250 mM sodium acetate incubation buffer (pH 5.0), 1 ␮l (0.003 mU) of AMF, Almond meal fucosidase

TABLE I

Glycan feature

RESULTS

TABLE II

Linear regression with ASD as predictor

DISCUSSION