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http://dx.doi.org/10.1007/s11011-015-9745-2
2. AUTHOR'S PROOF
Metadata of the article that will be visualized in OnlineFirst
1 Article Title Acid glycosaminoglycan (aGAG) excretion is increased in children with
autism spectrum disorder, and it can be controlled by diet
2 Article Sub- Title
3 Article Copyright -
Year
Springer Science+Business Media New York 2015
(This will be the copyright line in the final PDF)
4 Journal Name Metabolic Brain Disease
5
Corresponding
Author
Family Name Bjørklund
6 Particle
7 Given Name Geir
8 Suffix
9 Organization Council for Nutritional and Environmental Medicine
10 Division
11 Address Toften 24, Mo i Rana 8610, Norway
12 e-mail bjorklund@conem.org
13
Author
Family Name Endreffy
14 Particle
15 Given Name Ildikó
16 Suffix
17 Organization Department of Pediatrics, Josa Andras County Hospital
18 Division
19 Address Nyiregyhaza, Hungary
20 e-mail
21
Author
Family Name Dicső
22 Particle
23 Given Name Ferenc
24 Suffix
25 Organization Department of Pediatrics, Josa Andras County Hospital
26 Division
27 Address Nyiregyhaza, Hungary
28 e-mail
29
Author
Family Name Urbina
30 Particle
31 Given Name Mauricio A.
3. AUTHOR'S PROOF
32 Suffix
33 Organization University of Exeter
34 Division Department of Biosciences, College of Life and
Environmental Sciences
35 Address Exeter, UK
36 Organization Universidad de Concepción
37 Division Departamento de Zoología, Facultad de Ciencias Naturales
y Oceanografía
38 Address Casilla 160-C, Concepción, Chile
39 e-mail
40
Author
Family Name Endreffy
41 Particle
42 Given Name Emoke
43 Suffix
44 Organization University of Szeged
45 Division Department of Pediatrics and Pediatric Health Care Center,
Faculty of Medicine
46 Address Szeged, Hungary
47 e-mail
48
Schedule
Received 15 June 2015
49 Revised
50 Accepted 4 October 2015
51 Abstract Autism research continues to receive considerable attention as the options for
successful management are limited. The understanding of the autism spectrum
disorder (ASD) etiology has now progressed to encompass genetic, epigenetic,
neurological, hormonal, and environmental factors that affect outcomes for
patients with ASD. Glycosaminoglycans (GAGs) are a family of linear,
sulfated polysaccharides that are associated with central nervous system (CNS)
development, maintenance, and disorders. Proteoglycans (PG) regulate diverse
functions in the central nervous system. Heparan sulfate (HS) and chondroitin
sulfate (CS) are two major GAGs present in the PGs of the CNS. As
neuroscience advances, biochemical treatments to correct brain chemistry
become better defined. Nutrient therapy can be very potent and has minimal to
no side effects, since no molecules foreign to the body are needed. Given
GAGs are involved in several neurological functions, and that its level can be
somewhat modulated by the diet, the present study aimed to evaluate the role
of GAGs levels in ASD symptoms. Both tGAG and its different fractions were
evaluated in the urine of ASD and healthy control childrens. As levels differed
between groups, a second trial was conduted evaluating if diet could reduce
tGAG levels and if this in turn decrease ASD symptoms. The present study
found that tGAG concentration was significantly higher in the urine of children
with ASD compared to healthy control children and this was also evident in
all GAG fractions. Within groups (controls and ASD), no gender differences in
GAG excretion were found. The use of a 90 days elimination diet (casein-free,
special carbohydrates, multivitamin/mineral supplement), had major effects in
reducing urinary tGAG excretion in children with ASD.
5. AUTHOR'S PROOF
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1
2
3 ORIGINAL ARTICLE
4 Acid glycosaminoglycan (aGAG) excretion is increased
5 in children with autism spectrum disorder,
6 and it can be controlled by diet
7 Ildikó Endreffy1
& Geir Bjørklund2
& Ferenc Dicső1
&
8 Mauricio A. Urbina3,4
& Emoke Endreffy5
9
10 Received: 15 June 2015 /Accepted: 4 October 2015
11 # Springer Science+Business Media New York 2015
12 Abstract Autism research continues to receive considerable
13 attention as the options for successful management are limit-
14 ed. The understanding of the autism spectrum disorder (ASD)
15 etiology has now progressed to encompass genetic, epigenet-
16 ic, neurological, hormonal, and environmental factors that af-
17 fect outcomes for patients with ASD. Glycosaminoglycans
18 (GAGs) are a family of linear, sulfated polysaccharides that
19 are associated with central nervous system (CNS) develop-
20 ment, maintenance, and disorders. Proteoglycans (PG) regu-
21 late diverse functions in the central nervous system. Heparan
22 sulfate (HS) and chondroitin sulfate (CS) are two major GAGs
23 present in the PGs of the CNS. As neuroscience advances,
24 biochemical treatments to correct brain chemistry become bet-
25 ter defined. Nutrient therapy can be very potent and has min-
26 imal to no side effects, since no molecules foreign to the body
27 are needed. Given GAGs are involved in several neurological
28 functions, and that its level can be somewhat modulated by the
29 diet, the present study aimed to evaluate the role of GAGs
30 levels in ASD symptoms. Both tGAG and its different
31fractions were evaluated in the urine of ASD and healthy
32control childrens. As levels differed between groups, a second
33trial was conduted evaluating if diet could reduce tGAG levels
34and if this in turn decrease ASD symptoms. The present study
35found that tGAG concentration was significantly higher in the
36urine of children with ASD compared to healthy control chil-
37dren and this was also evident in all GAG fractions. Within
38groups (controls and ASD), no gender differences in GAG
39excretion were found. The use of a 90 days elimination diet
40(casein-free, special carbohydrates, multivitamin/mineral sup-
41plement), had major effects in reducing urinary tGAG excre-
42tion in children with ASD.
43Keywords Autism . Glycosaminoglycans . Proteoglycans .
44Neurodevelopment disorders . Epigenetics . Nutrition
45Introduction
46Autism spectrum disorder (ASD) is a heterogeneous group of
47behaviorally defined neurodevelopmental disorders. This
48group of disorders is presented from birth or very early during
49development and affect essential human behaviours such as
50imagination, the ability to communicate ideas and feelings,
51social interaction, and the establishment of relationships with
52others (Bjørklund 2013). Although there is strong evidence
53for genetic factors in ASD, it appears to be a polygenic con-
54dition (Betancur 2011), and also responsive to a range of en-
55vironmental events. Although, its precise etiology is un-
56known, ASD is clearly a complex human genetic disorder that
57involves interactions between genes and environment (Freitag
58et al. 2010; Li et al. 2014). Future integrative epigenomic
59analyses of genetic risk factors for environmental exposures
60and methylome analyses are expected to be important for un-
61derstanding the complex etiology of ASD (LaSalle 2014).
* Geir Bjørklund
bjorklund@conem.org
1
Department of Pediatrics, Josa Andras County Hospital,
Nyiregyhaza, Hungary
2
Council for Nutritional and Environmental Medicine, Toften 24,
8610 Mo i Rana, Norway
3
Department of Biosciences, College of Life and Environmental
Sciences, University of Exeter, Exeter, UK
4
Departamento de Zoología, Facultad de Ciencias Naturales y
Oceanografía, Universidad de Concepción, Casilla 160-C,
Concepción, Chile
5
Department of Pediatrics and Pediatric Health Care Center, Faculty
of Medicine, University of Szeged, Szeged, HungaryQ1
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62 Autism was once considered as a rare disorder, however,
63 the reported incidence and prevalence have drastically in-
64 creased during the last decades. Although, this estimate could
65 be affected by a change in diagnosis, increased awareness,
66 new therapeutic options and specific approaches to the educa-
67 tion of children with ASD (Macedoni-Lukšič et al. 2015),
68 today the prevalence of ASD in the US is estimated to be at
69 least 1 in 88 births (Q2 CDC 2008; Blumberg et al. 2013).
70 Nutrition affects multiple aspects of neuroscience including
71 neurodevelopment, neurogenesis and functions of neurons,
72 synapses and neural networks in specific brain regions.
73 Nutrition-gene interactions play a critical role in these re-
74 sponses, leading in turn to major effects on brain health, dys-
75 function, and disease. Individual differences in multiple gene
76 variants, including mutations, single nucleotide polymor-
77 phism (SNPs) and copy number variants (CNVs), significant-
78 ly modify the effects of nutrition on gene expression. A further
79 layer of regulation is added by differences in the epigenome,
80 and nutrition is one of many epigenetic regulators that can
81 modify gene expression without changes in DNA sequence
82 (Dauncey 2013).
83 Epigenetic marks are modifications of DNA and histones
84 (Jalili et al. 2013). They are considered to be permanent within
85 a single cell during development and are hereditable across
86 cell division. Programming of neurons through epigenetic
87 mechanisms is believed to be critical in neural development.
88 Mutation of genes encoding proteins that translate epigenetic
89 markings (MBDs) may lead to ASD. Copy number variations,
90 which are identified in a significant percentage of patients
91 with developmental disorders, may also lead to alterations in
92 the fine structure of the brain through epigenetic effects (as
93 illustrated by maternal 15q duplication in the regulation of
94 specific candidate genes that are critical for brain develop-
95 ment). Better understanding of epigenetic mechanisms in
96 ASD and neurodevelopmental disorders will ultimately help
97 in the development of novel treatment options (Rangasamy
98 et al. 2013).
99 Heparan sulfate (HS) regulates diverse cell-surface sig-
100 naling events, and its roles in the development of the ner-
101 vous system recently have been increasingly uncovered by
102 studies using genetic models carrying mutations of genes
103 encoding enzymes for its synthesis. Roles of HS in neural
104 development have been studied in animal models carrying
105 mutations in Ext1 and other genes encoding enzymes in-
106 volved in HS synthesis. These genetic studies revealed that
107 HS is necessary for the specific functioning of certain brain
108 structures, such as the cerebellum and the olfactory bulbs,
109 cortical neurogenesis, and a variety of axon path-finding
110 processes (Kantor et al. 2004). Pearson et al. (2013) suggest
111 that aberrant function extracellular matrix glycosaminogly-
112 can (GAG) localized to the subventricular zone of the lat-
113 eral ventricles may be a biomarker for ASD, and potentially
114 involved in the etiology of the disorder.
115Considering that several GAG play an active role in some
116areas of the brain and that the extracellular matrix function of
117GAG in the lateral ventricles has been proposed as an ASD
118biomarker, the present study aimed to: 1) evaluate potential
119differences in GAG levels in ASD patients compared to con-
120trol subjects, and 2) explore the potential role of diet on GAG
121modulation, and its effects on ASD symptoms. We hypothe-
122sized that GAG levels will differ between ASD and control
123patients and that diet play a major role in regulating GAG
124levels. These findings may have important implications for
125ASD patients and their treatment.
126Materials and methods
127Patients and sampling
128The present study was approved by the local ethics committee
129(Josa Andras County Hospital, Nyiregyhaza, Hungary), and
130informed consistent was obtained from the parents of each
131child.
132A total of 106 children (26 females and 80 males; range 2–
13314 years) with a diagnosis of ASD were recruited for this
134study from the Pediatric Department at Josa Andras County
135Hospital. Each child was evaluated to ensure that they met the
136criteria for autistic disorder based on the American Psychiatric
137Association (APA 1994). Their diagnosis was also confirmed
138using the Autism Diagnostic Interview-Revised (ADI-R; Lord
139et al. 1994). None of the children with ASD had any additional
140medical conditions such as epilepsy, cerebral palsy or motor
141deficiencies.
142Thirty one (31) age-matched controls (seven females and
14327 males; range 2–14 years) from kindergarten and elementa-
144ry schools in Eastern Hungary were also included in the pres-
145ent study. These children were recruited to the study by phy-
146sicians and other healthcare professionals, and were examined
147to ensure that they didn’t had any neurological or
148neurodevelopmental issues.
149GAG (total and fractions) analytical methods
150Urine samples were collected to quantify total glycosamino-
151glycan (tGAG). Since some preservatives may interfere with
152analyses (Di Ferrante 1967), samples were quickly frozen af-
153ter collection and kept frozen until analysis (including trans-
154port to the laboratory).
155Total glycosaminoglycan was measured as the amount of
156Alcian Blue precipitated from 50 μl of urine at pH 5.8 in the
157presence of 50 mmol l−1
MgCl2 using the technique described
158by Whiteman (1973). In order to determine the percentage
159ratio of the different aGAG (acid glycosaminoglycan) frac-
160tions, the method of Di Ferrante (1967) was used with minor
161modifications (Endreffy and Dicsö 1988). Urine samples were
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162 precipitated with CPC (N-cetylpyridinium chloride) at pH 4.0,
163 stabilized (so that timing of the reaction is less critical) and
164 stored at 4 °C until analyzed (Endreffy and Dicsö 1988). The
165 aGAG reference standard solutions (HA=hyaluronic acid,
166 HS=heparan sulfate, Co-4/6-S=chondroitin-4/6-sulfate,
167 DS=dermatan sulfate, KS=keratan sulfate, and UA=uronic
168 acid) were obtained from the Pediatric Clinics of Pécs and
169 Szeged Medical Universities, Hungary.
170 Twenty milliliter first-morning urine sample was mixed
171 with 0.5 ml 5 % (v/w) aqueous CPC, and acetic acid 10 %
172 (v/w) was then added at pH 4. Samples were then incubated at
173 25 °C for 4 h and then at 4 °C for 12 h. At the end of the
174 incubation, samples were centrifuged at 8000xg for 10 min.
175 All supernatant was removed, and the pellet was washed with
176 alcohol (96 % v/w) twice. Samples were centrifuged at 8,
177 000xg for 10 min and the excess alcohol removed. This sec-
178 ond precipitate was dissolved in 2 ml of 0.6 M NaCl, follow-
179 ing centrifugation at 8,000xg for 10 min. 2 ml of this super-
180 natant was treated with 8 ml of absolute alcohol and stored at
181 4 °C for 12 h. The sample was subsequently centrifuged at 8,
182 000xg for 10 min, and the resulting precipitate dissolved in
183 0.1 ml 0.6 M NaCl. GAG fractions without hyaluronidase
184 digestion were then quantified in 30 μl. Samples were placed
185 on a thin layer of silica gel, and the plates were developed at
186 23 °C for 90 min in a n-propanol, ammonium, water solvent,
187 at 40: 60: 5 (v:v:v).
188 GAG fractions were also quantified following hyaluroni-
189 dase digestion. Briefly, 10 μg hyaluronidase (1000 USP/U/
190 mg) was added to the aGAG test (Kieselgel 60, Merck), incu-
191 bated at 37 °C for 6 h and centrifuged at 8,000xg for 10 min.
192 30 μl of the supernatant was placed on a thin layer of silica gel
193 and was developed at 25 °C as above.
194 The dried plate was sprayed with a 2 % (v/w) orcinol ethyl
195 acetate solution, dried, and sprayed with 20 % (v/v) sulfuric
196 acid. The plate was kept at 100 °C until the appearance of the
197 HS blue, DS and KS violet spots. Hyaluronidase digests
198 hyaluronic acid and chondroitin-4/6-sulfate, the spots were
199 then quantitated by densitometry (Clinisan-2, Philips).
200 Elimination diet
201 A second experiment was conducted to test if GAG (total or
202 fractions) excretion could be modulated by the diet. Twenty
203 ASD patients aging between 3 and 11 years were selected to
204 follow a special diet for 3 months. The diet was adjusted to
205 suit the taste and habits of the family, and to avoid any foods
206 suspected of causing detrimental consequences for patient’s
207 appetite, palatability or dislike. A control group of 6 ASD
208 patients with similar characteristics was maintained with a free
209 diet during the same 3 month period. No patient was receiving
210 stimulant medication or antipsychotic drugs. The study began
211 with an initial physical evaluation conducted by a physician to
212 determine that the children were in reasonable physical health.
213Twenty children took the elimination diet and supplement for
2143 months (lactose-free and specific carbohydrate diet, vita-
215mins, minerals, and omega-3 oils). On the last day of the
2163 month period on the elimination and free diet, the mothers
217of the patients evaluated qualitative changes in sleep patterns,
218gastrointestinal symptoms, receptive language, general behav-
219iour, eye contact, expressive language, and sociability after the
220study (Table 2). Urine samples for tGAG analysis were taken,
221handled, and analyzed as described before, at the beginning
222and at the end of the 3 month period for each single patient.
223Statistical analysis
224Values are presented as mean±SEM. Potential differences in
225the tGAG excretion rates and their different fractions between
226ASD patients and controls were tested by a t-test. The poten-
227tial effect of the 3 month restriction and free diets, in ASD
228patients, on tGAG excretion rates was tested by paired t-tests
229within each group (restriction and free diets). Normal distri-
230bution and homogeneity of variances were checked before
231any further analysis, and all meet these assumptions. Differ-
232ences were considered significant with a p-value less than
2330.05).
234Results
235The present study found a significantly elevated tGAG excre-
236tion in 106 ASD affected children compared to 31 controls
237(Fig. 1). The mean value of the excretion in the patients was:
238183.3±5.9 mg tGAG g creatinine−1
, 95 % confidence interval
239132.7 to 176.5. On the other hand 28.71±3.6 mg tGAG g
240creatinine−1
excretion was found in the controls. The main
241GAG fractions were: heparan sulfate and chondroitin-4/6-sul-
242fate. But the excretions of all GAG fractions were significantly
243increased in the patients with ASD (Fig. 2). There was no
Fig. 1 Excretion of total glycosaminoglycan (tGAG) in 106 ASD
children and 31 age-matched controls. Asterisks denote differences
between the groups as determined by t-test
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244 significant difference between ASD affected boys and girls in
245 excretion of tGAG and GAG fractions (Table 1).
246 Nutrients and ASD: dietary factors
247 Patients that followed the elimination diet (lactose-free, spe-
248 cial carbohydrates, multivitamin/mineral supplement), occu-
249 pational therapy and adaptive sports, showed significant re-
250 duction in urinary tGAG excretion compared to the beginning
251 of the experiment (p<0.0001; Fig. 3). Before the elimination
252 diet, ASD patients showed an average tGAG excretion of
253 203.7±12.7, a value that decreased to 106.8±12.8 after
254 3 months on an elimination diet. tGAG excretion rate
255 remained unchanged in the control group (free diet) after the
256 3 months period, with an average value of 205.0±22.3 (p=
257 0.095, Fig. 3).
258Results from the parent’s evaluation of several behavioral
259and physical changes after the elimination diet showed minor
260improvements after the 3 month period (Table 2).
261Discussion
262Epigenetic mechanisms such as DNA methylation may be
263able to explain some of the variability observed with both
264environmental and genetic risk factors in ASD (Rangasamy
265et al. 2013). Epigenetic modifications to nucleotides or chro-
266matin provide long-lived effects on gene expression and phe-
267notype without modifying the DNA sequence. Epigenetic
268mechanisms such as DNA methylation can be altered by en-
269vironmental changes (Dolinoy et al. 2007; Jirtle and Skinner
2702007). Several environmental exposures have been correlated
271with reduced global DNA methylation in humans (James et al.
2722004; Dolinoy et al. 2007; Rusiecki et al. 2008). Deficiencies
Fig. 2 Excretion of different glycosaminoglycan (GAG) fractions in 106
ASD children and 31 age-matched controls. Asterisks denote differences
between the groups as determined by t-test
t1:1 Table 1 Excretion of total glycosaminoglycan (tGAG) and GAG
fractions (mg g creatine−1
; values in mean±SEM) in 74 ASD children
(17 females and 57 males)
t1:2 GAG/fractions ASD boys ASD girls p-value
t1:3 tGAG 196.7±6.892 185.6±13.85 0.4536
t1:4 hyaluronic acid (HA) 9.404±0.8332 11.18±1.324 0.2976
t1:5 heparan sulfate (HS) 59.75±4.622 57.06±8.565 0.7812
t1:6 chondroitin 4/6 sulfate (Co-4/6) 87.54±4.228 72.35±5.849 0.0744
t1:7 dermatan sulfate (DS) 11.05±0.9427 12.35±2.149 0.5345
t1:8 keratan sulfate (KS) 20.26±2.918 20.29±4.319 0.9958
t1:9 uronic acid (UA) 9.228±0.8115 10.88±1.437 0.3283
Fig. 3 Excretion of total glycosaminoglycan (tGAG) in 20 ASD children
and 31 age-matched controls before and after a 3 month elimination diet.
Asterisks signal statistical differences between the groups at the start and
the end of the 3 month period
t2:1Table 2 Impressions by mothers of 20 ASD children at the end of the
study, either after 3 months of elimination or normal diets
t2:2Category No diet (score) Elimination diet (score)
t2:3Sleep 3.9 6.0
t2:4Gastrointestinal symptoms 2.8 5.1
t2:5Receptive language 4.9 5.8
t2:6General behaviour 4.3 5.2
t2:7Eye contact 4.9 5.4
t2:8Expressive language 4.8 5.7
t2:9Sociability 5.1 5.9
t2:10Overall 4.4 5.6
1=much worse, 2=worse, 3=slightly better, 4=no change, 5=slightly
better, 6=better, 7=much better
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273 in methylation and oxidative stress pathways have been im-
274 plicated in ASD (James et al. 2004).
275 Sokol et al. (2006) reported a high level of total plasma
276 amyloid-β precursor protein (sAPP), including sAPPα, in a
277 small sample of young children with severe ASD and aggres-
278 sion. These children presented sAPP levels≥2 fold higher than
279 those of children without ASD.
280 APP is a large (695–770 amino acid) glycoprotein pro-
281 duced in brain microglia, astrocytes, oligodendrocytes, and
282 neurons. Proliferation, migration, differentiation, myelination,
283 and synaptogenesis are all steps involved in neuronal devel-
284 opment and maturation (Ray et al. 2011). Ray et al. (2011)
285 showed that levels of sAPPα, sAPPβ (proteolytic cleavage
286 products of APP by α- and β-secretase, respectively) are un-
287 balanced in ASD and may contribute to an anabolic environ-
288 ment leading to brain overgrowth.
289 The Aβ-amyloid fibrils are composed predominantly of
290 fragments of a larger transmembrane glycoprotein, the β-
291 amyloid precursor protein (βAPP). The molecular modifica-
292 tion in βAPP and tau that give rise to the isoforms found in the
293 Aβ-amyloid fibrils and paired helical filaments remain un-
294 known (Pérez et al. 1998). The demonstration that GAGs
295 induced the polymerization of full-length tau under physiolog-
296 ical conditions follows on from earlier studies reporting on the
297 effects of these sulfated polysaccharides on the polymeriza-
298 tion of βAPP fragments and suggests that GAGs may play a
299 specific role in the amyloidosis seen in Alzheimer’s disease
300 (AD) (Pérez et al. 1998).
301 Analysis of this neuroprotective effect of the sulfated gly-
302 cans revealed different efficiencies, with heparan sulfate (HS)
303 being the most effective, followed by keratan sulfate (KS) and
304 then the chondroitin sulfates (Co-4/6-S and DS). The role of
305 GAGs has also been suggested in neural development or re-
306 generation. It has also been indicated that aberrant extracellu-
307 lar matrix GAG function localized to the subventricular zone
308 of the lateral ventricles could be used as a biomarker for ASD,
309 and potentially involved in the etiology of the disorder (Pear-
310 son et al. 2013).
311 There have been several studies of the nutritional and met-
312 abolic status of children with ASD, but each focused on the
313 study of only a few biomarkers. The studies have demonstrat-
314 ed that children with ASD have impaired methylation, de-
315 creased glutathione and increased oxidative stress, and those
316 studies demonstrated that nutritional supplementation with
317 methylcobalamin, folinic acid, and trimethylglycine is benefi-
318 cial (James et al. 2004, 2006, 2009). Many significant differ-
319 ences were observed in the ASD group compared to the
320 neurotypical group, including low levels of biotin, plasma
321 glutathione, plasma uridine, plasma ATP, red blood cell
322 NADH, plasma sulfate (free and total), and plasma tryptophan
323 (Chauhan et al. 2004; Adams et al. 2011).
324 Alterations in GAGs have been reported in a variety of
325 neurological disorders, including autism, epilepsy,
326Parkinson’s disease and schizophrenia. The present results
327suggest that GAG excretion could be relevant to interpreting
328and directing future investigations into the elusive etiology of
329ASD. The sulfate-dependent developmental process affects
330neurons in brain regions whose function appears to be intrinsic
331to the disabilities found in autism. Sulfate is also necessary for
332the integrity of the gastrointestinal lining.
333Levels between 30 and 60 mg GAG g creatinine−1
of Acid
334glycosaminoglycan-uria are considered normal, while levels
335of 100–200 mg GAG g creatinine−1
have been classified as
336mildly elevated. Our results confirm and agree with previous
337findings, as our control group presented levels of 28.71±
3383.6 mg tGAG g creatinine−1
, while the ASD grouppresented
3396.4 fold higher levels of tGAG than the control group (183.3±
3405.9 mg tGAG g creatinine-1). This is in agreement with what
341is considered mildly elevated.
342Research using knockout of genes involved in HS biosyn-
343thesis have provided more physiologically relevant informa-
344tion about the role of HS in mammalian neural development
345(Yamaguchi et al. 2010). To define the role of HS in brain
346physiology, were generated conditional Ext1-knockout mice
347targeted to postnatal neurons. These conditional Ext1 mutant
348mice normally develop without any detectable morphological
349changes in the brain. Remarkably, these mice recapitulate nu-
350merous autism-like behavioral phenotypes encompassing the
351three core deficits of autism. Results from electrophysiologi-
352cal analyses indicate that removal of HS compromises gluta-
353matergic synaptic transmission by affecting the synaptic local-
354ization of AMPA receptors. These results demonstrate that HS
355is required for normal functioning of glutamatergic synapses.
356Moreover, the development of several autism-like deficits in
357these mice suggests that the cellular and molecular conditions
358resulting from the elimination of neuronal HS recapitulate
359critical parts of the pathogenic mechanisms of autism (Irie
360et al. 2012). These studies implicate that a decrease in N-
361sulfated HS in specific regions of the brain could be contrib-
362uting to the etiology of ASD and may serve as a biomarker for
363the disorder.
364The excretion of GAG, as detected by thin layer chroma-
365tography, before and after special diet in ASD children is
366evidence for the role of GAGs in ASD (Yamaguchi et al.
3672010; Irie et al. 2012). In the present study, the excretion of
368GAG in children with ASD was ≥3 folds higher than that of
369the healthy control children.
370Acknowledgments The authors thank the children with ASD and their
371parents for their consensus, collaboration, and hard work as participants
372in this research study. Further, the physicians Denes Kovendi and Eva
373Satorhegyi are thanked for help with diagnosis.
374Compliance with ethical standards
375Conflict of interest The authors declare no potential conflicts of inter-
376est with respect to the authorship, and/or publication of this article.
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377 Ethical approval All procedures performed in studies involving hu-
378 man participants were in accordance with the ethical standards of the
379 institutional and/or national research committee and with the 1964 Hel-
380 sinki declaration and its later amendments or comparable ethical
381 standards.
382
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AUTHOR QUERIES
AUTHOR PLEASE ANSWER ALL QUERIES.
Q1. Please check if the affiliation/s are captured and presented correctly.
Q2. CDC 2012 has been changed to CDC 2008 as per the reference list. Please check if okay.
