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 Autism is a serious neurodevelopmentaldisorder characterized by impairments in socialinteraction, abnormalities in verbal and nonver-bal communication, and restricted, stereotypedinterests and behaviors (American Psychiatric Association 1994). Although a large proportionof individuals with autism manifest abnormaldevelopment from birth, a subset of at least20–30% experience a regression with onsetbetween 18 and 24 months of age after aperiod of apparently normal development(Lainhart etal. 2002). Autistic disorder is themost severe form of autism spectrum disorders(ASDs), which include Asperger’s syndromeand pervasive developmental disorders (PDDs)not otherwise specified. Approximately 70% of individuals with autistic disorder have somedegree of mental retardation, and about half arenonverbal or have very impaired speech.Seizures are present by adolescence in about30% of children with ASD, and between 5 and10% of autism cases occur in association withother serious medical conditions such as fragile X, tuberous sclerosis, and Angelman’s syn-drome (Fombonne 2003). Gastrointestinalproblems and sleep disturbances are alsothought to be common comorbidities;however, population-based prevalence esti-mates for these conditions are currently lack-ing. Males are four times as likely as females tohave autism, but this ratio approaches oneamong individuals with severe cognitiveimpairment (Gillberg and Wing 1999). Mostindividuals with autism cannot live indepen-dently as adults (Rapin 1999). Over the past20 years, the prevalence of autism has report-edly risen, with much public debate surround-ing the reasons for this increase. Early reportsestimated prevalence at 4–5 per 10,000 births(Fombonne 1999). Data published in the lastfew years suggest that autistic disorder occursin at least 1–2 per 1,000 births, and the preva-lence of the broader autism spectrum may beas high as 4–6 per 1,000 (Chakrabarti andFombonne 2005; Yeargin-Allsopp etal. 2003).The causes and contributing factors forautism are poorly understood. The number of children with a diagnosis of autism as deter-mined by the California Department of Developmental Services (DDS) has been ris-ing continuously for over a decade (CaliforniaDDS 2003). Although diagnostic changesand improvements in detection probably contribute to this increase (Chakrabarti andFombonne 2005; Croen etal. 2002), a truerise in incidence may also be occurring(Blaxill etal. 2003). Evidence for geneticcauses is strong, yet concordance in monozy-gotic twins suggests that a minimum of 40%of autism cases are likely to have an environ-mental cause. No single gene has yet beenspecifically linked to autism with replicability,but the disorder is believed to be polygenic. A few specific environmental factors are associ-ated with autistic behaviors—prenatal expo-sures to thalidomide (Rodier and Hyman1998), valproic acid (Christianson etal.1994), or rubella (Chess etal. 1978)—butthese are likely to play a negligible role, if any,in incident cases in Western countries overthe last decade or so.Mechanisms of pathogenesis have yet tobe delineated. Contrary to early beliefs thatautism resulted from bad parent–child inter-actions (Bettelheim 1967), it is now widely accepted that aberrant brain developmentunderlies autism pathogenesis (Bauman andKemper 2003; Courchesne etal. 1988; Pivenetal. 1990; Rodier etal. 1997). Autopsy stud-ies demonstrate structural changes in thebrain, and imaging and electrophysiology investigations reveal neurophysiologic differ-ences in information processing between chil-dren with autism and those with typicaldevelopment (Dawson etal. 2002; Maziadeetal. 2000; McPartland etal. 2004; Rapinand Dunn 2003; Rosenhall etal. 2003).Neuroimmunomodulatory factors may also
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 Address correspondence to I. Hertz-Picciotto,Department of Public Health Sciences, TB #168,University of California, Davis, CA 95616 USA.Telephone: (530) 752-3025. Fax: (530) 752-3239.E-mail: ihp@ucdavis.edu We thank K. Jose for the data and subject trackingsystems, M. Rose for her excellent project manage-ment, and L. Delwiche and P. Krakowiak for datamanagement and programming.This work was supported by the NationalInstitutes of Health (1 P01 ES11269) and by theU.S. Environmental Protection Agency through theScience to Achieve Results (STAR) program(R829388).The authors declare they have no competingfinancial interests.Received 8 July 2005; accepted 6 April 2006.
The CHARGE Study: An Epidemiologic Investigation of Genetic andEnvironmental Factors Contributing to Autism
Irva Hertz-Picciotto,
1,2 
Lisa A. Croen,
3 
Robin Hansen,
2,4 
Carrie R. Jones,
1,2 
Judy van de Water,
2,5 
and Isaac N. Pessah 
2,6 
1
Division of Epidemiology, Department of Public Health Sciences, School of Medicine, and
2
Medical Investigations ofNeurodevelopmental Disorders (MIND) Institute, University of California–Davis, Davis, California, USA;
3
Division of Research,Kaiser Foundation Research Institute, Kaiser Permanente, Oakland, California, USA;
4
Department of Pediatrics, and
5
Department ofRheumatology/Allergy and Clinical Immunology, School of Medicine, and
6
Department of Molecular Biosciences, Schoolof Veterinary Medicine, University of California–Davis, Davis, California, USA
Causes and contributing factors for autism are poorly understood. Evidence suggests thatprevalence is rising, but the extent to which diagnostic changes and improvements in ascertain-ment contribute to this increase is unclear. Both genetic and environmental factors are likely tocontribute etiologically. Evidence from twin, family, and genetic studies supports a role for aninherited predisposition to the development of autism. Nonetheless, clinical, neuroanatomic,neurophysiologic, and epidemiologic studies suggest that gene penetrance and expression may beinfluenced, in some cases strongly, by the prenatal and early postnatal environmental milieu.Sporadic studies link autism to xenobiotic chemicals and/or viruses, but few methodologically rig-orous investigations have been undertaken. In light of major gaps in understanding of autism, a large case–control investigation of underlying environmental and genetic causes for autism andtriggers of regression has been launched. The CHARGE (Childhood Autism Risks from Geneticsand Environment) study will address a wide spectrum of chemical and biologic exposures, suscep-tibility factors, and their interactions. Phenotypic variation among children with autism will beexplored, as will similarities and differences with developmental delay. The CHARGE study infra-structure includes detailed developmental assessments, medical information, questionnaire data,and biologic specimens. The CHARGE study is linked to University of California–Davis Centerfor Children’s Environmental Health laboratories in immunology, xenobiotic measurement, cellsignaling, genomics, and proteomics. The goals, study design, and data collection protocols aredescribed, as well as preliminary demographic data on study participants and on diagnoses of thoserecruited through the California Department of Developmental Services Regional Center System.
Key words: 
autism, autistic spectrum disorder, developmental delay, environment, genetics, mentalretardation, pervasive developmental disorder.
Environ Health Perspect 
114:1119–1125 (2006).doi:10.1289/ehp.8483 available via 
http://dx.doi.org/ 
[Online 6 April 2006]
 
play a role (Silva etal. 2004; Vargas etal.2005). Cytokine profiles, lymphocyte activa-tion, and other immunologic parameters differbetween individuals with and without autism(Ashwood and Van de Water 2004a, 2004b;Croonenberghs etal. 2002). Distributions of neuropeptides and neurotrophins at birthappeared to be altered among children wholater developed autism (Nelson etal. 2001).Results from twin and family studies sug-gest a strong genetic contribution to the etiol-ogy of autism. Beginning with the classic work by Folstein and Rutter (1977), data from threepopulation-based twin studies have demon-strated a higher concordance rate amongmonozygotic compared with dizygotic twins(Cook 1998). Strong familial aggregation of autism has also been demonstrated. The sib-ling recurrence risk (i.e., the probability of developing autism given a person’s sibling isautistic) has been estimated at 2–14% (Jordeetal. 1990; Ritvo etal. 1989; Smalley etal.1988), a 10- to 20-fold increase over the gen-eral population prevalence. A family history of social deficits, language abnormalities, andpsychiatric disorders has also been observed incase–control and clinic-based studies (Folsteinand Rutter 1988; Piven and Palmer 1999). Autism co-occurs with several knowngenetic disorders, such as tuberous sclerosis(Smalley 1998), Angelman syndrome(Steffenburg etal. 1996), phenylketonuria, Joubert syndrome (Ozonoff etal. 1999), andMöbius syndrome (Johansson etal. 2001),and chromosomal abnormalities such as frag-ile X syndrome (Reiss and Freund 1990).More than 90% of autism cases, however,have none of the above syndromes.Linkage, association, and cytogenetic stud-ies have been conducted. Numerous candidategenes for autism have been suggested based ontheir functional role, location within candidatechromosome regions, and positive associations with the disease (Korvatska etal. 2002).Replication of findings has been elusive(Wassink etal. 2004), probably because of thepolygenic etiology, heterogeneity of thephenotype, and, assuming a role for gene–environment interaction, variation in exposuredistributions across populations. An epigeneticmechanism related to Rett syndrome is alsoplausible (Samaco etal. 2005). Genomewidescans to identify regions marked by differinggene expression are considered key at thisstage. One such scan hints at the possiblegenetic basis for the well-established sex ratioof four males to one female (Stone etal.2004). A comparison of tuberous sclerosispatients with and without autism demon-strated 31 genes for which expression differed(Tang etal. 2004); because both groupsshared the tuberous sclerosis diagnosis, the dif-ferentially expressed genes may be related toautism, although they are not necessarily causal. It is plausible that a substantial propor-tion of autism cases could be due to multiplegenes interacting with one or more environ-mental factors (Cederlund and Gillberg 2004;Glasson etal. 2004).Neuroanatomic and epidemiologic investi-gations support a prenatal or early postnatalorigin. Courchesne etal. (1988) observed cere-bellar abnormalities consistent with abnormali-ties in cell migration between the third andfifth month of gestation. Magnetic resonanceimaging studies point to migrational errors thatresult in disorganized columns of the cerebralcortex (Casanova etal. 2002). Anthropometricindicators, such as brain size and growth trajec-tory (Herbert 2005), suggest overall cerebralvolume to be larger in mid-childhood, withgrowth that accelerates early and then deceler-ates, although this phenotype may apply toonly a subset of cases. Neuroimaging studiesindicate involvement of specific brain regions,including the amygdala, hippocampus, andcorpus callosum (Brambilla etal. 2003;Schumann etal. 2004).Studies of environmental factors alsorelate to the prenatal origin of autism. Chessetal. (1978) reported that, within a cohort of about 250 children with congenital rubella,7% were later diagnosed with autism. A case–control study using both maternalreports and medical records of illnesses duringpregnancy showed relative risks of 4.1 forinfluenza and 3.3 for rubella (Deykin andMacMahon 1979). Daily maternal smokingduring early pregnancy was reported to belinked to autism in a large case–control epi-demiology study (odds ratio = 1.4; 95% con-fidence interval, 1.1–1.8) (Hultman etal.2002), although in our estimation, theseanalyses may have inappropriately adjustedfor potentially intermediate variables. Thelink between autism and early 
in utero 
expo-sure to thalidomide places the timing of theinsult coincident with neural tube closure inthe fourth to fifth week of gestation (Rodierand Hyman 1998). Case reports of autism inchildren gestationally exposed to valproic acid(Christianson etal. 1994; Rodier etal. 1997; Williams etal. 2001) are concordant withexperimental animal studies (Ingram etal.2000). A small number of cases of autismafter maternal infection with cytomegalovirus(Markowitz 1983; Stubbs etal. 1984),measles or mumps (Deykin and MacMahon1979), or herpes (Ritvo etal. 1990) as well asone case each of syphilis and toxoplasmosis(Rutter and Bartak 1971) have been reported.Taken together, the literature suggests aprominent genetic component involving mul-tiple gene loci, but also a likely contribution
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Figure 1.
Environmental exposures and sources of information in the CHARGE study. The left-hand boxindicates five classes of exposures that are candidates as environmental factors contributing to autism.The right-hand box lists sources of data available on CHARGE study participants. Arrows show a fewexamples of how specific exposures can be assessed. For example, pesticide exposures and/or theirmetabolites can be assessed in several ways (black arrows): laboratory assays can be conducted onblood (serum) and urine specimens; the interview collects information on applications in the home andalso obtains residential histories that can be linked to exposure databases on commercial pesticideapplications in California. Metals (blue arrows) can be measured in hair and in newborn blood spotsobtained from the State Genetics Diseases Branch biospecimen bank or assessed by interview questionson fish consumption or use of household products. Exposures to infectious agents (dashed arrows) canbe determined from medical records, self-reports, and assays on serum samples to test for seropositivityfor antibodies to specific viruses.
Persistentpollutants (flameretardants, etc.)PesticidesMetalsViruses, bacteria, andother infectionsMedical proceduresand pharmaceuticalsBiospecimensBloodHair (past exposures)Baby lock (first year of life)Mother’s hair (if long enough, prenatal)UrineBuccal cellsNewborn blood spotsInterviewsDietConsumer productsLifestyleResidential informationMedical historyLinkage to exposure databasesAir, water, pesticides, hazardous wasteMedical recordsObstetricLabor and deliveryNeonatalPediatric
 
from both chemical and microbial agents. It islikely that further understanding will requireconsideration of critical windows duringgestation and possibly early infancy, as well asinteractions between genetic or epigeneticpredisposition and environmental factors.
CHARGE Study Aims
In light of the enormous gap in our under-standing of the causes of both autism anddevelopmental delay (DD), a large epidemio-logic study was initiated in 2002. TheChildhood Autism Risk from Genetics and theEnvironment (CHARGE) study is addressinga wide spectrum of environmental exposures,endogenous susceptibility factors, and theinterplay between these two (CHARGE2006).To structure the search for etiologic factors, we are beginning with known neuro-developmental toxicants and hints from theimmunologic evidence. Additionally, physio-logic differences that might provide clues aboutsusceptibility and mechanisms are being exam-ined through characterization of metabolic,immunologic, and gene expression profiles, as well as genetic polymorphisms. Figure 1 showsfive broad classes of exposures of interest: pesti-cides, metals, persistent pollutants with knownor suspected neurodevelopmental or immuno-logic toxicity, medications and other treat-ments, and infections. Exposures from both theprenatal and early childhood periods are beinginvestigated, with data primarily from threesources:
a 
) extensive interviews with parents;
b 
)laboratory analysis of xenobiotics in blood,urine, and hair specimens; and
c 
) prenatal,labor and delivery, neonatal, and pediatricmedical records.CHARGE study specimens are analyzed forimmunologic, cell activation, xenobiotic,lipomic, and genomic markers in laboratories of the University of California–Davis (UC Davis)Center for Children’s Environmental Health(CCEH) (Table 1). Metals have been assayedin blood samples from > 300 index children, with a focus on mercury, lead, arsenic, cad-mium, and manganese. Immunologic profilesare being characterized, including cellularresponses to bacterial antigenic stimulation,general immunoglobulins, and production of chemokines and cytokines. Already, prelimi-nary results have demonstrated significant dif-ferences between children with autism andchildren from the general population in leptinconcentrations (Ashwood P, Kwong C, HansenR, Hertz-Picciotto I, Croen L, Krakowiak P,etal., unpublished observations). A detailed lipomics screen is being appliedto the plasma from the first few hundred chil-dren. Affymetrix GeneChip microarrays(Affymetrix, Santa Clara, CA) have been gen-erated from an initial sample of children andanalyzed to determine whether a genomic fin-gerprint for autism can be identified; results will be replicated on a further set. Brominatedflame retardants are being measured in 80–100children, and metabolites of pyrethroid pesti-cides will be evaluated in urine specimens.The CHARGE study also benefits fromCCEH hypothesis-driven experimental researchon animal models for autism in mice and non-human primates and
in vitro 
investigations of immune and neurogenic cells aimed at uncov-ering molecular mechanisms. A common data-base coordinates the archival, retrieval, andanalysis of samples, and the combination of population-based epidemiology with state-of-the-art molecular and cellular methods providesa powerful basis for interdisciplinary collabora-tive research. With future funding, theCHARGE study will undertake targeted evalu-ation of candidate genes, such as those responsi-ble for regulation of xenobiotic metabolizingenzymes, cell signaling in both neurons andimmune cells, and immune cell activation.Currently, the study is also characterizingphenotypic variation within the autism casegroup and relating these phenotypes to theexposures and physiologic profiles of interest.For example, we have begun to compareimmune function in regressive autism (children who have lost previously acquired social or lan-guage skills) with those with early onset (chil-dren who never acquired those skills). Otherphenotypic subtypes include, for example, highversus low cognitive function and presence ver-sus absence of gastrointestinal symptoms,macrocephaly, and sleep disturbances.
Design and Subject Recruitment
The CHARGE study appears to be the firstlarge-scale, population-based epidemiologicinvestigation focusing primarily on environ-mental exposures, as well as their interactions with genes, as underlying causes for autism. Ituses the case–control design, which providesthe most efficient sampling for studies of con-ditions that are rare or of multifactorial etiol-ogy. A further advantage is the focus on aspecific outcome, which translates into closescrutiny of diagnoses and rigorous measure-ment for the most highly suspect risk factors.The CHARGE study population is sam-pled from three strata: children with autism(full-syndrome autism, not those with a “spec-trum disorder”), children with DD but notautism, and children selected from the generalpopulation without regard for developmentalcharacteristics. All participating children (cur-rently >500, with an ultimate goal of between1,000 and 2,000) meet the following criteria:
a 
) between the ages of 24 and 60 months,
b 
)living with at least one biologic parent,
c 
)having a parent who speaks English orSpanish,
d 
) born in California, and
e 
) residingin the catchment areas of a specified list of regional centers (RCs) in California. No fur-ther exclusions are made based on genetics orfamily phenotype.Children with autism and children withmental retardation or DD are identifiedthrough RCs that contract with the CaliforniaDDS to determine eligibility and coordinateservices for persons with developmental disabili-ties. Eligibility in the DDS/RC system does notdepend on citizenship or financial status. Thus,the system is widely used across socioeconomiclevels and racial/ethnic groups. Referrals arefrom pediatricians, other clinical providers,schools, friends, and family members.The DDS/RC system is mandated to pro-vide services for individuals with autism, as well as for those with other PDDs who havemental retardation (IQ < 70) or are substan-tially handicapped. One investigation esti-mated that 75–80% of the total population of children with an autism diagnosis in the state were enrolled in the DDS system (Croen etal.2002). Among preschoolers, the figure may belower, with fewer mild cases. Additionally, thisproportion may decline with recent changes toeligibility requirements that emphasize theextent of disability. Children with Asperger’sor PDDs not otherwise specified withoutmental retardation are not generally eligiblefor DDS/RC services and therefore are notactively recruited into the CHARGE study.Potential cases of autism for the CHARGEstudy are defined as those who are eligible forservices based on a DDS/RC diagnosis of autism. Families with a child who has receiveda diagnosis but is not in the RC system are alsoinvited. The second study group, children with
Childhood autism: environment and genetics
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Table 1.
Biospecimen use for susceptibility and exposure markers.
a
Child’s bloodChild’s urineNewborn blood spotHairImmune markersCytokinesXXImmunoglobulins (general)XXAntigen-specific Ig responsesXXCell activationXLipid profilesXBrominated flame retardantsXPesticide metabolitesXMetalsXXXGenomicsXGeneticsX
a 
Not an exhaustive list of assays.
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