IgA antibodies in Rett syndrome

http://aut.sagepub.comAutism

DOI: 10.1177/1362361306062024 2006; 10; 189 Autism

K. L. Reichelt and O. Skjeldal IgA antibodies in Rett syndrome

http://aut.sagepub.com/cgi/content/abstract/10/2/189 The online version of this article can be found at:

Published by:

http://www.sagepublications.com

On behalf of:

The National Autistic Society

can be found at:Autism Additional services and information for

http://aut.sagepub.com/cgi/alerts Email Alerts:

http://aut.sagepub.com/subscriptions Subscriptions:

http://www.sagepub.com/journalsReprints.navReprints:

http://www.sagepub.com/journalsPermissions.navPermissions:

http://aut.sagepub.com/cgi/content/refs/10/2/189SAGE Journals Online and HighWire Press platforms):

(this article cites 21 articles hosted on the Citations

© 2006 The National Autistic Society, SAGE Publications. All rights reserved. Not for commercial use or unauthorized distribution. at WELCH MEDICAL LIBRARY on August 1, 2007 http://aut.sagepub.comDownloaded from

http://www.nas.org.uk

http://aut.sagepub.com/cgi/alerts

http://aut.sagepub.com/subscriptions

http://www.sagepub.com/journalsReprints.nav

http://www.sagepub.com/journalsPermissions.nav

http://aut.sagepub.com/cgi/content/refs/10/2/189

http://aut.sagepub.com

189

IgA antibodies in Rettsyndrome

K . L . R E I C H E L T The National Hospital, Oslo, Norway

O. S K J E L D A L The National Hospital, Oslo, Norway

A B S T R A C T The level of IgA antibodies to gluten and gliadin proteinsfound in grains and to casein found in milk, as well as the level of IgGto gluten and gliadin, have been examined in 23 girls with Rettsyndrome and 53 controls. Highly statistically significant increaseswere found for the Rett population compared to the controls. Thereason for this remains unknown, but because IgA antibodies reflectthe uptake of proteins and/or epitopes of proteins from the gut, thismay be indicative of increased protein uptake.

A D D R E S S Correspondence should be addressed to: K . L . R E I C H E LT , Institute ofPediatric Research, University of Oslo, Rikshospitalet, N-0027 Oslo, Norway. e-mail:[email protected]

Introduction

Rett syndrome is a neuro-developmental disorder and one of the leadingcauses of mental retardation in girls, affecting 1 per 10,000. Although thereis recent evidence of abnormal early development in Rett syndrome(Burford et al., 2003), most children with this disorder appear to theparents to develop normally until 6–18 months of age, after which timethey undergo a period of neurological regression. Although special neuro-logical symptoms may be detected in some from early infancy (Wright etal., 2003), these are usually not noticed by the parents until later. Typicalchanges are loss of purposeful hand use (dyspraxia), deceleration of headcircumference, peculiar stereotyped hand movements, transient autisticbehaviour, respiratory abnormalities, bruxism and seizures. The period ofrapid deterioration is often followed by a stagnation phase which lasts intoadulthood. The phenotype can vary, with some girls having a serious formof the disease and some having a milder clinical picture.

In males the disorder is apparently mostly fatal, but a few cases havebeen described. Approximately 84 percent of the Rett patients have agenetic lesion in the X-linked MECP-2 encoding methyl-CpG-bindingprotein 2 that binds specifically to methylated DNA and acts as a general

autism © 2006SAGE Publicationsand The National

Autistic SocietyVol 10(2) 189–197; 062024

1362-3613(200603)10:2

www.sagepublications.comDOI: 10.1177/1362361306062024

K E Y W O R D S

casein;gliadin;gluten;

IgAantibodies;

Rett syndrome



transcription repressor, which affects a series of genes in an epigenetic way(Amir et al., 1999; Chen et al., 2001).

IgA antibodies are formed in the mucosa of the gut and secreted to thegut, protecting against foreign proteins, and are a measure of proteinuptake from the gut. These antibodies are distributed to all mucusmembranes in the body and are for instance increased in celiac disease. IgGantibodies are the circulating antibodies against foreign proteins.

Increased levels of IgA antibodies to gluten, gliadin and casein havebeen found in pervasive developmental disorders (PDD: Cade et al., 2000;Lucarelli et al., 1995; Reichelt et al., 1990). Rett syndrome is also classi-fied as one of the PDD syndromes and some of these patients initially showtransient autistic symptoms. The purpose of this article is to examinewhether girls with Rett show increased uptake of proteins from the gut(measured as IgA antibody levels in serum) against dietary proteins.

Participants and methods

The clinical group consisted of 23 females all diagnosed with Rettsyndrome, based on well established criteria (Hagberg, 1995; Hagberg andSkjeldal, 1994). Their age range was from 1 to 36 years with a median of11 years (mean = 13). Controls were all healthy females of age range 1 to55 years with median of 12 years (mean 16) and N = 53. The Rett partici-pants were volunteered by their parents, to whom the purpose of the exam-ination had been explained.

Blood was drawn by vene-puncture and centrifuged and serum wassent to the immunological laboratory (Nycomed Pharma) and assayed forIgA antibodies against gliadin, gluten, lactalbumin, lactoglobulin, caseinand ovalbumin. In those cases where IgA antibodies were above the uppercutoff for celiac disease, the celiac typical endomycium (later anti-transglutaminase antibodies) were also measured. IgG antibodies to glutenand gliadin and total IgA were also measured by using enzyme-linkedimmunosorbent assay (ELISA) as described by Scott et al. (1985a; 1985b).Costar EIA Microplates (no. 3490) were coated with antigens at the follow-ing concentrations: casein and lactalbumin 0.001 g/l; gliadin and gluten0.01 g/l; lactoglobulin 0.05 g/l; and ovalbumin 0.1 g/l. Serum sampleswere tested at 1:400 dilution for IgA activity by the ELISA method (Scottet al., 1984). Optical density (OD) was read at 405 nm. Every microplateincluded three serum samples tested with each antigen. On the basis ofreproducibility, tests showed a day-to-day variation of less than 0.1 ODunits (Scott et al., 1985a; 1985b). Recently Fürsts Laboratory in Oslo havebought this laboratory but they use the same techniques, equipment, staffand consultants.

A U T I S M 10(2)

190



Urine peptides were measured as described by Solaas et al. (2002).Briefly, urine equivalent to 250 nanomoles creatinine was separated byHigh Pressure Liquid Chromatography (HPLC) and the peptides that areretained on a C-18 reverse phase column at pH 2, and eluted by acetoni-tril gradient after hippuric acid, are measured at 215 nm as the area underthe curve. As a control for purity, the ratio 215 to 280 was routinelymeasured (Reichelt and Knivsberg, 2003). That the peaks are indeedpeptides has been extensively discussed (Reichelt et al., 1998).

StatisticsBecause the distribution of IgA antibodies in the normal population is veryskewed towards the lower levels, the non-paired and non-parametricMann–Whitney U-test was used. The statistical characteristics U and U� aregiven as well as the p-value.

Results

Table 1 shows the overall results of the two groups for IgA. The Rett popu-lation is different from the normal controls for gluten, gliadin and casein,as seen in Figure 1 for gliadin and in Figure 2 for gluten. The statistics givea p-value of 0.001 for gluten and gliadin and 0.005 for casein.

Lactalbumin and lactoglobulin were not different from the controls.Thus for lactoglobulin the Mann–Whitney statistic had values U = 310 andU� = 345 and p = 0.785 (not significant). For lactalbumin U = 249 and U�= 411 and p = 0.124 (not significant).

Two of the girls with Rett but none of the controls had no demon-strable specific IgA levels without a general IgA deficiency, for which wehave no explanation. None had endomycium or anti-transglutaminaseincreases, which are special markers for celiac disease; therefore the Rett

R E I C H E L T & S K J E L D A L : I g A A N T I B O D I E S I N R E T T

191

Table 1 IgA antibodies in girls with Rett and controls

Gliadin Gluten Casein

(N = 23) (N = 54) (N = 23) (N = 53) (N = 23) (N = 54)

Mean 0.229 0.080 0.252 0.061 0.343 0.115Median 0.190 0.080 0.200 0.060 0.165 0.08095% CI:

min. 0.139 0.056 0.143 0.049 0.168 0.077max. 0.319 0.104 0.360 0.073 0.519 0.153

The values are expressed as optical density units from ELISA as described in Scott et al. (1985a; 1985b).Two-tailed p for gliadin in Rett against controls was < 0.0001, as is the value for gluten. Two-tailed p-value for casein in Rett against controls was = 0.0005.



AU

TIS

M10(2)

192 nidailg AgI

0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

1

3515947454341493735333139272523212917151311197531

o.n elpmaS

OD

un

its

1seireS2seireS

Figure 1 The level of IgA antibodies against gliadin in serum in Rett syndrome (series 1) and 53 controls (series 2). The Mann–WhitneyU-statistic = 249 and U�� = 969.5, and the two-tailed p-value was < 0.0001(highly significant)

©

2006 Th

e Natio

nal A

utistic S

ociety, S

AG

E P

ub

lication

s. All rig

hts reserved

. No

t for co

mm

ercial use o

r un

auth

orized

distrib

utio

n.

at WE

LCH

ME

DIC

AL LIB

RA

RY

on August 1, 2007

http://aut.sagepub.comD

ownloaded from


girls did not have celiac disease. Total IgA antibody level in the serum ofthe Rett group had a range of 0.25 to 3.91 and the given control valuesare 0.7 to 3.7. The total IgA levels were not statistically different for girlswith Rett compared to controls.

Total IgG levels were also measured for gluten and gliadin (see Table 2).Here we found IgG levels for gliadin with U = 165 and U� = 1077 and atwo-tailed p-value < 0.0001. For gluten we found U = 158 and U� = 1138and again a two-tailed p-value < 0.0001 (highly significant).

Eight girls had their antibody levels tested again after 2 years. Nostatistical differences were found. For example, IgA to gluten in one partici-pant was 0.42, and at retest was 0.39. Another participant was measuredat 0.94 and at 0.97 on the retest. A third participant came out with 0.15and a retest value of 0.20.

IgE (or typically allergy related antibodies) levels in 11 of the 23 girlswith Rett were measured by ELISA immune assay at the same laboratory,and none were above the upper limit (average level expressed as percent-age of upper IgE limit of the controls = 10.7 ± 8.9 percent, N = 11).

No correlation of age and antibody levels was shown.Plotting peptide levels in urine, obtained as published by Solaas et al.


193

Correlation between peptide levels andgliadine IgA levels in Rett syndrome

0

–0.10 500 1000 1500 2000 2500

Peptide level

3000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Gliadine IgAlevel

Figure 2 The levels of IgA antibodies against gluten in OD units measured byenzyme-linked immunosorbent assay (ELISA: Scott et al., 1985b) plotted againstpeptide levels measured as area under the UV 215 nm curve (Solaas et al., 2002). Theslope of the linear regression line is different from zero with p = 0.02 (two-tailed).Spearman r = –0.362 (corrected for ties) and 95 percent confidence interval is –0.612to –0.048. In the patients with the highest peptide levels we did not find the MECP2mutation



(2002), against IgA antibody levels towards gluten, a negative correlationof peptide levels to IgA antibodies against gliadin (Figure 1) was found.The same was found for gliadin IgA and IgG against peptide levels (notshown). However, the few excessively high peptide containing sampleswould dominate the correlation.

Discussion

Our data indicate that as a group the girls with Rett syndrome show higherIgA antibody levels in serum against gluten, gliadin and casein proteinscompared to controls. They do not show this for lactalbumin, lactoglobu-lin and ovalbumin. Because endomycium antibodies or trans-glutaminaseantibodies were not increased, this is not a celiac condition. It is, however,indicative of increased uptake of some proteins from the gut in Rettsyndrome. The increased IgG antibody levels may also be seen as a possiblebyproduct of increased uptake with concomitant tissue damage. Increasedpterin levels in Rett syndrome (Messahel et al., 2000) have been proposedas a marker of immune system activation in Rett. This could also explainthe antibody increases but not why the antibody increase is selective.

Healthy persons also absorb intact proteins from the gastro-intestinaltract (Husby et al., 1985; Kilshaw and Cant, 1984). Furthermore the uptakeof active enzymes has been demonstrated (Gardner, 1995; Ross et al.,1973; Schoutsen and De Jung, 1984) as has the uptake of theproteinase/peptidase known as botulinum toxin. This fact is reinforced byfinding intact antigens from ingested proteins in mothers’ milk (Axelssonet al., 1986; Stuart et al., 1984; Troncone et al., 1987). It is normal to haveIgG antibodies against most food proteins and usually also IgA antibodies,

A U T I S M 10(2)

194

Table 2 IgG antibody levels in serum of girls with Rett and controls

Gliadin IgG Gluten IgG

Rett Controls Rett Controls(N = 23) (N = 54) (N = 24) (N = 54)

Mean 1.11 0.46 1.17 0.43Median 1.06 0.39 1.12 0.3995% CI:

min. 0.93 0.27 0.93 0.33max. 1.29 0.57 1.40 0.54

All numbers are in OD units from ELISA.The two-tailed p < 0.0001 is found for gluten in Rett girls compared to controls. Two-tailed p < 0.0001is also found for gliadin in Rett girls compared to controls.



but with a different profile and a lower average. Furthermore peptides aretaken up post-prandially (Chabance et al., 1998), and protein epitopes thatinduce antibodies are usually peptidic in nature. Ingested proteins mayinduce antibodies, for instance against myelin oligodendrocyte glycopro-tein in experimental autoimmune encephalomyelitis (Stefferl et al., 2000).However, because lactoglobulin and lactalbumin and ovalbumin did notshow this increase, the increased uptake must be somewhat selective.

What increased uptake means is more difficult to explain. It could bepart of a general membrane leakiness, because we have earlier foundincreased protein levels in urine from girls with Rett syndrome (Reicheltet al., 2001) and in more than 80 percent of examined cases hyperpep-tiduria is often quite severe (Solaas et al., 2002). The severe stress of thedisorder may also increase gut permeability for casein, gliadin and glutenprecursor proteins; this may possibly be relevant as an explanation also forRett girls, but is so far speculative.

Recently, ataxia with polyneuritis and cerebellar volume decrease andespecially Purkinje cell loss has been characterized (Hadjivassiliou et al.,1998; 2002) with anti-gliadin and anti-gluten antibodies but withoutceliac disease. Only further research can elucidate these intricacies.However, an increased gut uptake of some proteins may perhaps be relatedto the developmental inhibition which is found in Rett syndrome.

ReferencesA M I R , R . E . , VA N D E N V E Y V E R , I . B. , WA N, M. , T R A N, C.Q. , F R A N C K E , U. &

Z O G H B I , H .Y. (1999) ‘Rett Syndrome Is Caused by Mutations in X-Linked MECP2,Encoding Methyl-CpG-Binding Protein 2’, Nature Genetics 23: 185–8.

A X E L S S O N, I . , JAC O B S S O N, I . , L I N D B E R G, T. & B E N E D I K T S S O N, B. (1986) ‘BovineLacto-Globulin in Human Milk’, Acta Paediatrica Scandinavica 75: 702–7.

B U R F O R D, B. , K E R R , A .M. & M AC L E O D, H.A . (2003) ‘Nurse Recognition of EarlyDeviation in Development in Home Videos of Infants with Rett Disorder’, Journal ofIntellectual Disability Research 47: 588–96.

C A D E , R . J. , P R I V E T T E , R . , F R E G LY, M. , ROW L A N D, N. , S U N, Z . , WAG E M A K E R , H .& E D E L S T E I N, C. (2000) ‘Autism and Schizophrenia: Nutritional Disorders’,Nutrition & Neuroscience 2: 57–72.

C H A B A N C E , B. , M A RT E AU, P. , R A M B AU D, J.C. , M I G L I O R E- S A M O U R, D. , B OY NA R D,M. , P E R ROT I N, P. , G U I L L E T, R . , J O L L E S , P. & F I AT, A .M. (1998) ‘Casein PeptideRelease and Passage to the Blood in Humans during Digestion of Milk and Yogurt’,Biochimie 80: 155–65.

C H E N, R .Z . , A K B A R I A N, S . , T U D O R, M. & JA N I S C H, R . (2001) ‘Deficiency ofMethyl-CpG Binding Protein-2 in CNS Neurons Results in a Rett-Like Phenotype inMice’, Nature Genetics 27: 327–31.

G A R D N E R, M.L .G. (1995) ‘A Review of Current Knowledge of GastrointestinalAbsorption of Intact Proteins Including Medicinal Preparations of ProteolyticEnzymes’, in M.L .G. G A R D N E R & K . J. S T E F F E N S (eds) Absorption of Orally AdministeredEnzymes, pp. 1–8. Berlin: Springer.


195



H A D J I VA S S I L I O U, M. , G R Ü N E WA L D, R .A . , C H AT TO PA D H YAY, A .K . , DAV I E S - J O N E S ,G.A .B. , G I B S O N, A . & JA R R E T T, J.A . (1998) ‘Clinical, Radiological,Neuro-Physiological and Neuro-Pathological Characteristics of Gluten Ataxia’,Lancet 352: 1582–5.

H A D J I VA S S I L I O U, M. , B O S C O L O, S . , DAV I S - J O N E S , G.A .B. , G R Ü N E WA L D, R .A . ,N OT, T. , S A N D E R S , D. S . , S I M P S O N, J. E . , TO N G I O R G I , E . , W I L L I A M S O N, C.A . &WO O D RO O F E , N.M. (2002) ‘The Humoral Response in the Pathogenesis of GlutenAtaxia’, Neurology 58: 1221–6.

H AG B E R G, B. (1995) ‘Rett Syndrome: Clinical Peculiarities and Biological Mysteries’,Acta Paediatrica 84: 971–6.

H AG B E R G, B. & S K J E L DA L , O.H . (1994) ‘Rett Variants: A Suggested Model forInclusion Criteria’, Pediatric Neurology 11: 5–11.

H U S B Y, S . , J E N S E N I U S , J. C. & S V E H AG, S . E . (1985) ‘Passage of Un-DegradedDietary Antigen into the Blood of Healthy Adults’, Scandinavian Journal of Immunology22: 83–92.

K I L S H AW, P. J. & C A N T, A . J. (1984) ‘The Passage of Maternal Dietary Proteins intoHuman Breast Milk’, International Archives of Allergy & Applied Immunology 75: 8–15.

L U C A R E L L I , S . , F R E D I A N I , T. , Z I N G O N I , A .M. , F E R RU Z Z I , F. , G I A R D I N I , O. ,QU I N T I E R I , F. , B A R B ATO, M. , D ’ E U F E M I A , P. & C A R D I , E . (1995) ‘Food Allergyand Infantile Autism’, Panminerva Medica 37: 137–41.

M E S S A H E L , S . , P H E A S E N T, A . E . , PA L L , H . & K E R R , A .M. (2000) ‘Abnormalities inUrinary Pterin Levels in Rett Syndrome’, European Journal of Paediatric Neurology 4:211–17.

R E I C H E LT, K . L . & K N I V S B E R G, A .M. (2003) ‘Can the Pathophysiology of Autism BeExplained by the Nature of the Discovered Urine Peptides?’, Nutrition & Neuroscience6: 19–28.

R E I C H E LT, K . L . , E D R E M, J. & S C OT T, H . (1990) ‘Gluten, Milk Proteins and Autism:Results of Dietary Intervention on Behavior and Urinary Peptide Excretion’, Journalof Applied Nutrition 42: 1–11.

R E I C H E LT, W.H. , E K , J. , S T E N S RU D, M. & R E I C H E LT, K . L . (1998) ‘PeptideExcretion in Coeliac Disease’, Journal of Pediatric Gastroenterology & Nutrition 26: 305–9.

R E I C H E LT, K . L . , G A R D N E R, M.L .G. , WA R I N G, R .H . , NA I D U, S . , K N I V S B E R G, A .M.& N Ø D L A N D, M. (2001) ‘Rett Syndrome: Urinary Peptide, Sulphate andImmunoglobulin Abnormalities’, in T H E AU T I S M R E S E A R C H U N I T, S U N D E R L A N D

U N I V E R S I T Y (ed.) 2001: An Autism Odyssey, pp. 245–8. Sunderland: ARU.RO S S , D. J. , P TA S Z Y N S K I , M. & O S T E R , K .A . (1973) ‘The Presence of Ectopic

Xanthine Oxidase in Atherosclerotic Plaques and Myocardial Tissues’, PSEBM 144:523–6.

S C OT T, H . , FAU S A , O. , E K , J. & B A N D T Z A E G, P. (1984) ‘Immune Response Patternin Celiac Disease: Serum Antibodies to Dietary Antigens Measured by an EnzymeLinked Immunosorbent Assay (ELISA)’, Clinical & Experimental Immunology 57: 25–32.

S C OT T, H . , RO G N U M, T.O. , M I D T V E D T, T. J. & B R A N D T Z A E G, P. (1985a) ‘AgeRelated Changes in Human Serum Antibodies to Dietary and Colonic BacteriaAntigens Measured by Enzyme-Linked Immunoabsorbent Assay’, Acta Pathologica,Microbiologica et Immunologica Scandinavica 93: 65–70.

S C OT T, H . , RO G N U M, T.O. & B R A N D T Z A E G, P. (1985b) ‘Performance Testing ofAntigen Coated Polystyrene Microplates for ELISA Measurements of SerumAntibodies to Bacterial and Dietary Antigens’, Acta Pathologica, Microbiologica etImmunologica Scandinavica 93: 117–23.

A U T I S M 10(2)

196



S C H O U T S E N, N. & D E J O N G, J.W. (1984) ‘Xanthine Oxidase in Rabbit Plasma AfterApplication of a Bovine Milk Preparation to Small Intestine’, Arch. Int. de Physiol.Biochim. 92: 379–84.

S O L A A S , K .M. , S K J E L DA L , O. , G A R D N E R, M.L .G. , K A S E , B. F. & R E I C H E LT, K . L .(2002) ‘Urinary Peptides in Rett Syndrome’, Autism 6 (3): 315–29.

S T E F F E R L , A . , S C H U B A RT, A . , S TO R C H, M. , A M I N I , A . , M AT H E R, I . , L A S S M A N N,H. & L I N I N G TO N, C. (2000) ‘Butyrophilin, a Milk Poprotein, Modulates theEncephalitogenic T Cell Response to Myelin Oligodendrocyte Glycoprotein inExperimental Autoimmune Encephalomyelitis’, Journal of Immunology 165: 2859–65.

S T UA RT, C.A . , T W I S L E TO N, R . , N I C H O L A S , M. & H I D E , D.W. (1984) ‘Passage ofCow’s Milk Protein in Breast Milk’, Clinical Allergy 14: 533–5.

T RO N C O N E, R . , S C A R C E L L O, A . , D O NAT I E L L O, A . , C A N NATA RO, P. , TA R A B U S C O,A . & AU R I C C H I O, S . (1987) ‘Passage of Gliadin into Human Breast Milk’, ActaPaediatrica Scandinavica 76: 453–6.

W R I G H T, M. , VA N D E R L I N D E N, M.L . , K E R R , A .M. , B U R F O R D, B. , A R ROW S M I T H,G. & M I D D L E TO N, I . (2003) ‘Motion Analysis of Stereotyped Hand Movements inRett Syndrome’, Journal of Intellectual Disability Research 47: 85–9.


197



IgA antibodies in Rett syndrome

Documents

Transcript of IgA antibodies in Rett syndrome