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June 01, 2009

HLA-DR4 as a Risk Allele for Autism Acting in Mothers of Probands Possibly During Pregnancy

Author Affiliations

Author Affiliations: Departments of Neurology (Drs Johnson and Williams, Ms Sreenath, and Mr Stenroos) and Pediatrics (Drs Mars, Williams, and Lambert) and the Center for Childhood Neurotoxicology and Exposure Assessment (Drs Johnson, Mars, and Lambert and Mr Stenroos), University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School, Piscataway, New Jersey; Departments of Statistics and Genetics (Dr Buyske), Rutgers University, New Brunswick, New Jersey; BioSciences Research Associates, Cambridge, Massachusetts (Ms Stein); and Center for Blood Research, Harvard University, Boston, Massachusetts (Ms Stein).


Copyright 2009 American Medical Association. All Rights Reserved. Applicable FARS/DFARS Restrictions Apply to Government Use.2009

Arch Pediatr Adolesc Med. 2009;163(6):542-546. doi:10.1001/archpediatrics.2009.74

Objectives  To test whether HLA-DR4 acts in the mother, possibly during pregnancy, to contribute to the phenotype of autistic disorder in her fetus.

Design  Transmission disequilibrium testing in case mothers and maternal grandparents.

Setting  Previous studies have consistently shown increased frequency of HLA-DR4 in probands with autism and their mothers, but not their fathers. However, this has been documented only in case-control studies and not by a more direct study design to determine whether HLA-DR4 acts in mothers during pregnancy to contribute to autism in their affected offspring.

Participants  We genotyped for HLA-DR alleles in members of 31 families with parents and maternal grandparents. Probands with autism were tested using the Autism Diagnostic Observation Schedule–Western Psychological Services and Autism Diagnostic Interview, Revised. There was 80% power to detect an odds ratio of 3.6. Participants were all families from New Jersey and were similar in number to earlier studies of autism and HLA-DR4.

Outcome Measures  Analysis was by standard transmission disequilibrium testing. As a secondary test we examined the possibility of maternal imprinting.

Results  Significant transmission disequilibrium for HLA-DR4 was seen (odds ratio, 4.67; 95% confidence interval, 1.34-16.24; P = .008) for transmissions from maternal grandparents to mothers of probands, supporting a role for HLA-DR4 as an autism risk factor acting in mothers during pregnancy. Transmission disequilibrium was not seen for HLA-DR4 transmissions from parents to probands or from mothers to probands.

Conclusions  The HLA-DR4 gene may act in mothers of children with autism during pregnancy to contribute to autism in their offspring. Further studies are required to confirm these findings.

In most cases in which a gene has been associated with a disorder, the disease allele acts in the affected individual. Alternatively, a disease allele may act in the mother to contribute to the phenotype of her affected child. A reasonable hypothesis is that such maternal genes act during pregnancy (postpartum action through nursing seems unlikely); however, action before conception in the ovum and the cells that influence it is also possible. So far, there are more than 30 descriptions of such maternally acting alleles, so-called teratogenic alleles.1,2

Children with autism show deviation from the normal developmental pattern with impaired social interactions and communication, restricted interests, and repetitive stereotyped patterns of behavior that are evident prior to 36 months of age.3,4 Clinical genetic studies and modeling studies suggest that autism may be caused by multiple interacting gene loci5,6 and that environmental and epigenetic factors may contribute.6,7

Neuropathological,8,9 cytoarchitectonic,10 and minicolumn studies11,12 all support the prenatal origin of certain brain abnormalities in autism. Consequently, it is possible that maternal genes acting during pregnancy could contribute to the autism phenotype in the fetus.

A number of studies have associated HLA-DR4 with autism, some of them suggesting its action in mothers of children with autism, possibly during pregnancy. Warren and colleagues13 studied the frequency of a major histocompatibility complex extended haplotype that contains HLA-DR4 in a case-control design with 21 individuals with autism, their parents, and 62 controls. Compared with controls, the major histocompatibility complex extended haplotype, B44-SC30-DR4, was significantly increased in both cases and mothers, but not in fathers, of individuals with autism. Daniels et al14 confirmed this finding in a case-control study, adding 23 new individuals with autism and the parents of 18 of them and comparing them with 64 controls. All of the families in both studies were of northern European ancestry and lived in northern Utah. Subsequently, Warren et al15 studied 50 children with autism and 79 control subjects, all of northern European ancestry and all but 2 living in northern Utah, and found that certain alleles of the third hypervariable region of HLA-DRβ1 had a very strong association with children with autism, especially alleles within HLA-DR4. As an explanation of their striking maternal findings, Warren et al raised the question of whether a gene acting in the mother during pregnancy might contribute to autism in her fetus.

More recently, Torres et al16 carried out a case-control study of individuals with autism spectrum disorder from 31 families from Oregon, 34 families from Utah, and 38 families in the Autism Genetic Resource Exchange (AGRE), all white, compared with allele frequencies in white control subjects from the National Marrow Donor Program, and found that HLA-DR4 occurred more frequently in children with autism spectrum disorder than controls.

Recently, Lee et al17 carried out a case-control study of HLA-DR4 in autism in 16 families from eastern Tennessee and 33 families from AGRE who were selected from all parts of the United States and in which multiple male participants had autism and compared them with a control group of 475 healthy unrelated adults who were evaluated at the Johns Hopkins Hospital as potential bone marrow donors. All families with individuals with autism and control families were white. Compared with controls, children with autism and their mothers, but not their fathers, for the east Tennessee group had a significantly higher frequency of HLA-DR4 alleles than control subjects; specifically, the mothers were 5.54 times (95% confidence interval [CI], 1.74-18.67) and their children with autism were 4.20 times (95% CI, 1.37-13.27) more likely to have HLA-DR4 than control individuals.17 The HLA-DR4 frequencies in children with autism in the AGRE group, their mothers, and their fathers were not significantly different from controls.17 The authors interpreted their findings in the eastern Tennessee group as consistent with a hypothesis that maternal-fetal immune interaction in utero could affect fetal brain development; such an immune interaction could conceivably involve both HLA and related genes in both genetic and epigenetic mechanisms.

Although these studies suggested a maternal effect of HLA-DR4 for autism, all of them compared mothers of individuals with autism with controls in case-control study designs and none of them carried out a more direct test such as the maternal transmission/disequilibrium test (TDT)1,2,1820 that could document the presence of a maternal allele acting during pregnancy to contribute to the autism phenotype. Because HLA-DR4 had been associated with both individuals with autism and the mothers of affected children compared with controls, we tested the association of these alleles with autism using a case-parent study design with mothers of children with autism and maternal grandparents and analysis by standard TDT.21 Finding the action of HLA-DR4 during pregnancy to contribute to autism in offspring could be important because the DR4 data for autism show one of the highest odds ratios (OR), 4.20, of any marker associated with autism so far.17 Also, the association of DR4 with autism would raise the possibility of an autoimmune component to autism and thus the possibility for therapy. Identifying HLA-DR4 as a teratogenic allele for autism would be important because pregnancy may be the earliest opportunity for therapeutic intervention in autism.

Increased frequency of an allele in both cases and mothers in a case-control study could also occur if the allele were imprinted in the mothers and acted in the cases. Therefore, as a secondary hypothesis, we tested for maternal imprinting of these alleles.


Thirty-one families with mothers and maternal grandparents as well as probands and fathers were ascertained with the help of the Center for Outreach and Services for the Autism Community, a New Jersey autism support group. All but 1 of the families were also part of a separate study of GSTP1.22 Family members were genotyped for HLA-DR by the Center for Blood Research Laboratories and BioSciences Research Associates at Harvard University. The HLA-DR alleles were determined by polymerase chain reaction amplification of genomic DNA and dot-blot analysis using sequence-specific oligonucleotide probes.23 All probands were tested using the Autism Diagnostic Observation Schedule–Western Psychological Services (ADOS-WPS) and Autism Diagnostic Interview, Revised (ADI-R). Using “mother of child with autism” as the affected phenotype, we applied the standard TDT, comparing the transmissions and nontransmissions of HLA-DR4 against the binned set of all other alleles. Overtransmission of an hypothesized risk allele to mothers would suggest that the allele genuinely is a risk allele for autism that acts in mothers.

An additional explanation for the observed increased allele frequency in mothers and probands, but not fathers, in families with a child with autism would be that the allele is imprinted in the mothers but acts in probands, both mothers and probands being enriched for that allele. To examine this possibility, as a secondary test we tested for maternal imprinting with the method of Weinberg24 and also applied the TDT to the offspring with autism.21 Because these additional tests were secondary tests, a correction for multiple comparisons was not applied; even if a correction had been applied, the maternal TDT data would have remained statistically significant.

At the observed allele frequencies in founders, the study had sufficient power to detect large effects, namely 80% power to detect an OR of 3.6. The study was approved by the institutional review board of UMDNJ–Robert Wood Johnson Medical School and informed consent was obtained from the participants.


Clinical diagnoses by ADOS-WPS and ADI-R were autistic disorder for 30 of the probands and pervasive developmental disorder not otherwise specified (PDD-NOS) for 1. In 25 families, both parents were non-Hispanic white; in the other 6 families, 1 parent in each was non-Hispanic white and the other was non-Hispanic Asian (3), Hispanic white (2), or non-Hispanic black (1).

In the 31 families with an autistic child genotyped for HLA-DRβ1, statistically significant transmission disequilibrium for HLA-DR4 was seen by TDT (OR, 4.67; 95% CI, 1.34-16.24; P = .008) (Table) for transmissions to mothers of individuals with autism from maternal grandparents, supporting a role for HLA-DR4 as a risk factor for autism acting in the mothers in this group of families. There were 14 copies transmitted and 3 untransmitted, as opposed to 35 and 46, respectively, for other alleles. The mother of the child with PDD-NOS had an HLA-DR4 transmission; when that family was dropped, the result remained statistically significant (OR, 4.33; 95% CI, 1.23-15.21; P = .01).

Image not available
Transmission Disequilibrium Testing at the DRβ1 Locus for Mothers of Probands With Autism and, as a Secondary Test, for Probands With Autism

To examine an alternative possible explanation for the significantly increased frequency of HLA-DR4 reported in mothers of individuals with autism, that the HLA-DR4 allele is a risk allele in the child through maternal imprinting and that the mothers are, of necessity, enriched for this allele, we followed up with a secondary test in the children with autism and their parents, again using the standard TDT along with a test for maternal imprinting. Statistically significant transmission disequilibrium was not seen for transmissions from parents to individuals with autism themselves (OR, 1.33; 95% CI, 0.56-3.18; P = .39) (Table), nor from mothers specifically (OR, 1.00; 95% CI, 0.34-2.97; P = .99) (Table). The Weinberg test for maternal imprinting was also not statistically significant (P = .79). These findings did not support action of HLA-DR4 as a risk factor for autism acting in children with autism themselves either directly or through imprinting.


As discussed above, earlier case-control studies suggested the possibility of a maternal effect in autism originating from HLA-DR4, but they did not clarify what sort of a maternal effect, if any, might be operating. Nor did they use a more direct study design to prove this.

The present study describes the action of HLA-DR4 during pregnancy in mothers of children with autism that contributes to autism in their offspring. The known possible reasons for increased frequency of an allele repeatedly observed in mothers of affected individuals include (1) the allele is a teratogenic allele, (2) the allele acts by imprinting and is imprinted in the mother, (3) it acts in the affected individual and hence will have increased frequency in the parents—sometimes by chance the allele will have increased frequency in mothers but not fathers, and (4) the allele is a mitochondrial allele and hence is transmitted only by mothers to affected individuals; therefore, it has increased frequency in mothers. Our study excluded all of these possibilities except the first. Our tests for imprinting were negative. Case TDT was not supported. Mitochondrial alleles do not show segregation, but DR4 did show segregation in transmissions from maternal grandparents to mothers; in any case, DR4 is known not to be the product of a mitochondrial gene. The study has several novel or unusual features. First, it provides an answer to the long-standing question of what sort of maternal effect originating from HLA-DR4 contributes to autism. Second, it supports the possibility of an immune component to autism pathogenesis acting in mothers during pregnancy. Third, it adds to the small number of gene alleles shown to act in mothers, probably during pregnancy, to contribute to a disorder in their offspring. Because nearly all of these disorders have turned out to be neurodevelopmental,1,2 it is possible that this could be a mechanism of more general importance for these disorders, especially if it were more widely known and more frequently looked for. The genetic architecture of autism can not be adequately understood if maternally acting genes are not looked for. Fourth, our findings encourage additional studies to address the pathogenesis of autism that could ultimately contribute to prevention or therapy for the disorder.

Our study was a small one, but was not underpowered for this purpose. At the observed allele frequencies among founders, the study had sufficient power to detect large effects, namely 80% power to detect an OR of 3.6. The effect that we found for HLA-DR4 gave an OR of 4.67 (95% CI, 1.34-16.24; P = .008). This OR is comparable with those in other studies of the effect that did not determine the genetic mechanism. Participants in the present study were all families from New Jersey, and the number of families was similar to those of geographically defined areas in the earlier studies of autism and HLA-DR4. This report should encourage larger studies to confirm this effect in autism. Unlike the case-control study design, the case-parent study design is resistant to population stratification,21 which was, in any case, minimal in our families.

The possible mode of action of DR4 in mothers is unknown. Maternal DR4 could contribute to a subset of autism cases by interacting with other risk alleles for autism and with environmental factors to perturb pathways affecting brain development in autism. A possible environmental factor could be the maternal infections during pregnancy (urinary tract, respiratory, and vaginal) described previously as more common in the mothers of children with autism than controls, although that increase was not statistically significant.25 The HLA-DR4 gene (along with DR3) is a risk allele for type I diabetes mellitus and appears to modulate the humoral immune response to enterovirus antigens.26

Interestingly, in mice, maternal immune stimulation during gestation may affect the developmental outcome of offspring. For example, maternal immune stimulation reportedly ameliorated malformations induced by chemical teratogens,27 perhaps through maternal immune regulation of fetal gene expression including cell cycle and/or apoptotic genes.28 Maternal stimulation with interferon gamma decreased the severity of fetal cleft palate caused by urethane, while stimulation with Freund's complete adjuvant reduced both the incidence and severity of the lesion.29 Maternal immune stimulation inducing inflammation increased fetal brain cytokine response, decreased the number of reelin-immunoreactive cells in certain areas of postnatal brain in offspring, and altered behavior in adult offspring.30 Reelin gene polymorphisms have been associated with autism,31,32 and reelin protein levels are decreased in autism in blood33 and cerebellum.34

Immune responses to infections generate products of oxidative stress, which could be a contributing factor to altered brain development in autism. We recently reported that a biomarker of oxidative stress is elevated postnatally in children with autism,35 a finding that has been confirmed.36 Interestingly, recent work suggests that the immune system participates in the shaping of brain synaptic circuits during childhood development.37

The present findings do not exclude the possibility of a contribution to autism susceptibility by an allele of HLA-DQβ1, a major histocompatibility complex locus closely linked to HLA-DRβ1, or alleles at other loci within a conserved extended haplotype containing HLA-DR4.38 Further studies are needed to confirm and extend the present findings, including a larger maternal TDT study, a study of other DR alleles, a study of fine structure subtypes of DR4, and a study of whether HLA-DQβ1 alleles contribute to autism.

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Article Information

Correspondence: William G. Johnson, MD, University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854 (

Accepted for Publication: October 24, 2008.

Author Contributions: Dr Johnson had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Johnson, Stenroos, Williams, and Lambert. Acquisition of data: Johnson, Mars, Stenroos, Williams, Stein, and Lambert. Analysis and interpretation of data: Johnson, Buyske, Sreenath, Stenroos, Williams, and Lambert. Drafting of the manuscript: Johnson, Buyske, Stein, and Lambert. Critical revision of the manuscript for important intellectual content: Johnson, Buyske, Mars, Sreenath, Stenroos, Williams, and Lambert. Statistical analysis: Buyske. Obtained funding: Johnson and Lambert. Administrative, technical, and material support: Sreenath, Stenroos, Williams, Stein, and Lambert. Study supervision: Johnson.

Financial Disclosure: Drs Johnson and Mars report being members of the Professional Advisory Board of The New Jersey Center for Outreach and Services for the Autism Community (COSAC).

Funding/Support: This study was supported by grants R21-44244 and K25-AA015346 from the National Institutes of Health (NIH); grant 2R25 GM55145 from the Initiative to Maximize Student Diversity in Biomedical Sciences (Dr Williams); The New Jersey Governor's Council; the National Alliance for Autism Research; and grants P50-ES09598 and P01-ES11256 from the NIH United States Environmental Protection Agency Center of Excellence.

Previous Presentations: These data were presented as a poster at the International Meeting for Autism Research; May 16, 2008; London, England; and the Child Neurology Society; November 6, 2008; Santa Clara, California.

Additional Contributions: The authors are grateful for the participation of the individuals with autism and their family members and for the assistance of COSAC (Ewing, New Jersey), which helped with recruitment of participants. The authors wish to thank Zuheir Awdeh, PhD, of CBR Laboratories for reading the manuscript.

Johnson  WG Teratogenic alleles and neurodevelopmental disorders. Bioessays 2003;25 (5) 464- 477
Johnson  WGSreenath  MBuyske  SStenroos  ES Teratogenic alleles in autism and other neurodevelopmental disorders. Zimmerman  AAutism Current Theories and Evidence. Totewa, NJ Humana Press2008;41- 68
American Psychiatric Association, Diagnostic and Statistical Manual of Mental Disorders. 4th ed. Washington, DC American Psychiatric Association1994;
Rapin  I Autism. N Engl J Med 1997;337 (2) 97- 104
Muhle  RTrentacoste  SVRapin  I The genetics of autism. Pediatrics 2004;113 (5) e472- e486
Szatmari  P The causes of autism spectrum disorders. BMJ 2003;326 (7382) 173- 174
Lawler  CPCroen  LAGrether  JKVan de Water  J Identifying environmental contributions to autism: provocative clues and false leads. Ment Retard Dev Disabil Res Rev 2004;10 (4) 292- 302
Rodier  PMIngram  JLTisdale  BNelson  SRomano  J Embryological origin for autism: developmental anomalies of the cranial nerve motor nuclei. J Comp Neurol 1996;370 (2) 247- 261
Strömland  KNordin  VMiller  MAkerström  BGillberg  C Autism in thalidomide embryopathy: a population study. Dev Med Child Neurol 1994;36 (4) 351- 356
Piven  JO'Leary  D Neuroimaging in autism. Child Adolesc Psychiatr Clin N Am 1999;6305- 323
Casanova  MFBuxhoeveden  DGomez  J Disruption in the inhibitory architecture of the cell minicolumn: implications for autism. Neuroscientist 2003;9 (6) 496- 507
Casanova  MFBuxhoeveden  DPSwitala  AERoy  E Minicolumnar pathology in autism. Neurology 2002;58 (3) 428- 432
Warren  RPSingh  VKCole  P  et al.  Possible association of the extended MHC haplotype B44-SC30-DR4 with autism. Immunogenetics 1992;36 (4) 203- 207
Daniels  WWWarren  RPOdell  JD  et al.  Increased frequency of the extended or ancestral haplotype B44-SC30-DR4 in autism. Neuropsychobiology 1995;32 (3) 120- 123
Warren  RPOdell  JDWarren  WL  et al.  Strong association of the third hypervariable region of HLA-DR beta 1 with autism. J Neuroimmunol 1996;67 (2) 97- 102
Torres  ARMaciulis  AStubbs  EGCutler  AOdell  D The transmission disequilibrium test suggests that HLA-DR4 and DR13 are linked to autism spectrum disorder. Hum Immunol 2002;63 (4) 311- 316
Lee  LCZachary  AALeffell  MS  et al.  HLA-DR4 in families with autism. Pediatr Neurol 2006;35 (5) 303- 307
Mitchell  LE Differentiating between fetal and maternal genotypic effects, using the transmission test for linkage disequilibrium. Am J Hum Genet 1997;60 (4) 1006- 1007
Johnson  WG The DNA polymorphism-diet-cofactor-development hypothesis and the gene-teratogen model for schizophrenia and other developmental disorders. Am J Med Genet 1999;88 (4) 311- 323
Doolin  MTBarbaux  S McDonnell  MHoess  KWhitehead  ASMitchell  LE Maternal genetic effects, exerted by genes involved in homocysteine remethylation, influence the risk of spina bifida. Am J Hum Genet 2002;71 (5) 1222- 1226
Spielman  RS McGinnis  REEwens  WJ Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM). Am J Hum Genet 1993;52 (3) 506- 516
Williams  TAMars  AEBuyske  SG  et al.  Risk of autistic disorder in affected offspring of mothers with a glutathione S-transferase P1 haplotype. Arch Pediatr Adolesc Med 2007;161 (4) 356- 361
Zachary  AAedTeresi  GAed Typing HLA. ASHI Laboratory Manual. New York, NY American Society for Histocompatibility and Immunogenetics1990;195
Weinberg  CR Methods for detection of parent-of-origin effects in genetic studies of case-parents triads. Am J Hum Genet 1999;65 (1) 229- 235
Comi  AMZimmerman  AWFrye  VHLaw  PAPeeden  JN Familial clustering of autoimmune disorders and evaluation of medical risk factors in autism. J Child Neurol 1999;14 (6) 388- 394
Sadeharju  KKnip  MHiltunen  MAkerblom  HKHyoty  H The HLA-DR phenotype modulates the humoral immune response to enterovirus antigens. Diabetologia 2003;46 (8) 1100- 1105
Holladay  SDSharova  LVPunareewattana  K  et al.  Maternal immune stimulation in mice decreases fetal malformations caused by teratogens. Int Immunopharmacol 2002;2 (2-3) 325- 332
Sharova  LSura  PSmith  BJ  et al.  Nonspecific stimulation of the maternal immune system II: effects on gene expression in the fetus. Teratology 2000;62 (6) 420- 428
Holladay  SDSharova  LSmith  BJGogal  RM  JrWard  DLBlaylock  BL Nonspecific stimulation of the maternal immune system I: effects on teratogen-induced fetal malformations. Teratology 2000;62 (6) 413- 419
Meyer  UNyffeler  MEngler  A  et al.  The time of prenatal immune challenge determines the specificity of inflammation-mediated brain and behavioral pathology. J Neurosci 2006;26 (18) 4752- 4762
Serajee  FJZhong  HMahbubul Huq  AH Association of Reelin gene polymorphisms with autism. Genomics 2006;87 (1) 75- 83
Skaar  DAShao  YHaines  JL  et al.  Analysis of the RELN gene as a genetic risk factor for autism. Mol Psychiatry 2005;10 (6) 563- 571
Fatemi  SHStary  JMEgan  EA Reduced blood levels of reelin as a vulnerability factor in pathophysiology of autistic disorder. Cell Mol Neurobiol 2002;22 (2) 139- 152
Fatemi  SHStary  JMHalt  ARRealmuto  GR Dysregulation of Reelin and Bcl-2 proteins in autistic cerebellum. J Autism Dev Disord 2001;31 (6) 529- 535
Ming  XStein  TPBrimacombe  MJohnson  WGLambert  GHWagner  GC Increased excretion of a lipid peroxidation biomarker in autism. Prostaglandins Leukot Essent Fatty Acids 2005;73 (5) 379- 384
Yao  YWalsh  WJ McGinnis  WRPratico  D Altered vascular phenotype in autism: correlation with oxidative stress. Arch Neurol 2006;63 (8) 1161- 1164
Stevens  BAllen  NJVazquez  LE  et al.  The classical complement cascade mediates CNS synapse elimination. Cell 2007;131 (6) 1164- 1178
Alper  CALarsen  CEDubey  DPAwdeh  ZLFici  DAYunis  EJ The haplotype structure of the human major histocompatibility complex. Hum Immunol 2006;67 (1-2) 73- 84