Final Report: Role of Developmental Neurotoxicology in Autism

EPA Grant Number: R824758
Title: Role of Developmental Neurotoxicology in Autism
Investigators: Rodier, Patricia M.
Institution: University of Rochester
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1995 through September 30, 1998
Project Amount: $412,372
RFA: Human Health Risk Assessment (1995) RFA Text |  Recipients Lists
Research Category: Health Effects , Human Health , Human Health Risk Assessment , Health

Objective:

Autism spectrum disorders (ASDs) are among the most common congenital anomalies, occurring at a rate of at least 1.6 /1000 births. Little is known about the causes, and even less about the nature of the CNS injury underlying the symptoms. Family studies indicate that unknown genetic factors account for about 90% of the variance and environmental factors are also involved. In 1994, it was discovered that exposure of the closing neural tube of the human embryo to thalidomide, a well-known teratogen, could produce autism at a high rate. Valproic acid (VPA) and ethanol have been implicated as other teratogens that increase the risk of autism.

The critical period for induction of autism by thalidomide was determined from the somatic defects of the autistic patients, each of whom had malformed ears and hearing deficits. This stage of development - days 20-24 of gestation - is much earlier than the periods usually considered in studies of neuroteratology. Only a few neurons of the brain stem form so early. Most neurons forming during the closure of the neural tube are motor neurons for cranial nerve nuclei. The thalidomide cases had neurological dysfunctions of the abducens, facial, and oculomotor nerves, confirming that the development of those neurons had been disturbed. Further, the literature on neurological and physical anomalies in idiopathic cases of autism indicates that many cases, not just those induced by thalidomide, have cranio-facial anomalies that could only arise during neural tube closure. For example, abnormalities of eye movement, peripheral hearing deficits, and malformed ears are more frequent in children with autism than in controls. Lack of facial expression is one of the diagnostic behaviors of the ASDs. The association of facial palsy with autism is also clear when patients with facial diplegia are tested for autism. People with Moebius syndrome, a congenital diplegia of facial muscles and eye abductors, have a 30% rate of autism. The cranio-facial symptoms have been ignored in the autism literature, because they seem trivial in comparison to the disabling behavioral symptoms, but they speak directly to the embryological origin of the disorder.

Because we had been working on the same period of brain development in studies of Fetal Alcohol Syndrome and pituitary development, the thalidomide results told us many things that might be useful in studying the etiology of autism. For example, they suggested that it should be possible to model the initiating injury in animals, to demonstrate brain stem anomalies in the brains of human cases, and to find somatic evidence of early injury in patients with autism. Our objective was to test these predictions.

Summary/Accomplishments (Outputs/Outcomes):

Neuroanatomy and somatic anatomy. We began our studies of autism by creating an animal model of the underlying brain injury (Rodier et al., 1996a; Ingram et al., 1996; Rodier et al. 1996b; Rodier, 1997), by exposing rats to VPA at various time points during neural tube closure. Comparison of cranial nerve nuclei and other early-forming neuron populations by cell counts from matched sections indicated that the cranial nerve abnormalities characteristic of the thalidomide cases can be reproduced by exposure to VPA and that the exposed animals survive to adulthood. Total counts of the abducens nucleus in animals exposed on day 12 exhibited variable reductions in neuron number, averaging 41%. A preliminary evaluation of the cerebellum suggested that it was reduced in both size and cell number. A larger study of the cerebellum has just been completed. Whereas mean brain and body weights are reduced by 18% and 25%, respectively in VPA exposed rats, the reduction of the cerebellum is much greater. Its volume is reduced by 45%, with a corresponding decrease of Purkinje cell number that is greatest in the hemispheres, and less severe in the vermis (Ingram, 1999a). This is the same pattern reported in human cases of autism.

We had the opportunity to analyze the dysmorphology and physical measures data from the largest prevalence study of autism spectrum disorders. The results confirmed the significant association of ear anomalies, especially posterior rotation, with ASDs, and suggested two new physical characteristics: esotropia and large hands/small feet (Rodier et al. 1997b), both of which are related to the symptoms of thalidomide embryopathy.

In a human case of autism we found several brain stem lesions that were predicted by both the animal model and the thalidomide cases (Rodier et al., 1996a). Most strikingly, the brain stem was shortened, as though a band of tissue were missing in the region derived from the fifth rhombomere. Such an anomaly could arise only during neural tube closure. This finding provided additional evidence supporting the period of neural tube closure as a stage of development critical for the initiation of autism.

Family studies. While our work on autism was designed to reveal how toxic agents might cause the lesion underlying the disorder, it has turned out to provide important clues about the genetic causes. The neuroanatomic abnormalities in our human brain were virtually identical to those observed by other investigators in Hoxa1 knockout mice. This gene is active only for a short period in brain development (the same period when teratogens alter the brain stem) and it controls developmental events in the region of the fifth rhombomere (an area injured in our human case and animal model). Null-mutations of Hoxb1, the paralog of Hoxa1 are known to cause reduction of facial neurons and facial diplegia. The similarity of phenotypes in the transgenic animals, the human case, and the thalidomide cases, as well as the timing and localization of expression suggested HOXA1 and HOXB1 as strong candidates for genes likely to be abnormal in familial cases of autism.

No polymorphic form of either gene had ever been detected in mammals. However, data collected on this grant allowed us to apply for funds to test this hypothesis by searching the candidate genes for deviations from the published sequence in genomic DNA from human cases of ASDs and controls. Data from about 50 families with evidence of familial autism indicate that a variant of HOXA1 is strongly associated with autism, a variant of HOXB1 shows a trend of association, and the effects of the two variants are additive. The distribution of the two variant alleles is significantly increased in probands, whether compared to historical controls or parent controls (controls constructed of the untransmitted alleles of the parents of probands). Other affected family members also differ significantly from the two control groups in frequency of the newly-discovered alleles. A transmission disequalibrium test of linkage of the candidate genes with autism is highly significant, and concordance for the alleles in probands and their affected relatives is significant at p<.001. The odds ratio is 2.7 and the fraction of cases attributable to the variants is calculated at 48%. A third variant of HOXA1 is now being studied. Several preliminary reports of the data have been published (Rodier et al., 1997; Ingram et al., 1997; Rodier, 1998a; Rodier, 1998b; Stodgell et al., 1998)), and a full report of the first two allelic variants discovered has been submitted (Ingram et al., 1998).

Mechanism of VPA effects on the brain stem. The results of the family studies confirmed the earlier hypothesis that allelic variants of HOXA1 and HOXB1 are involved in ASDs, but why do exposures to teratogens mimic the effects of genetic factors? Neither thalidomide nor VPA have known mechanisms of action. We wondered whether the early developmental genes might be involved in the teratologic induction of autism spectrum disorders as well as the inherited susceptibility to the disorder. To test whether known teratogens associated with the disorder influence the expression of the candidate genes, we exposed rats at the critical period of development to VPA, or retinoic acid (RA), which produces many of the same physical malformations over the same critical period, and is known to drive the expression of the Hox genes. Using RT-PCR, we quantified the RNA expression of Hoxa1 after exposure to the two teratogens. The result is that VPA alters the expression of the gene to the same degree as retinoic acid, suggesting that the effect of VPA on hindbrain and cranio-facial development may be mediated through a disturbance of the Hox cascade, just as RA?s effects are thought to be (Ingram and Rodier, 1998, Ingram, 1999b).

Conclusions:

Thus far, each kind of evidence we have collected supports the hypothesis that the autism spectrum disorders are initiated by brain stem injury during early development. An animal model in which embryos are exposed to valproic acid during neural tube closure reproduces the cranial nerve anomalies predicted by the neurological dysfunctions of thalidomide-induced autism. It also parallels the cerebellar anomalies reported in human histology and MRI. A study of physical measures and minor congenital anomalies in children with autism shows a significant association of several craniofacial dysmorphologies with the disorder. Postmortem examination of the brain of a person with autism demonstrates near-absence of the facial nucleus and other changes consistent with an insult to the closing neural tube resulting in failure of the fifth rhombomere. Two candidate genes selected for the resemblance of the brains of knockout mice to the brains of human cases and the VPA model have now been found to have polymorphisms associated with autism. A study of the expression of Hoxa1 in embryos exposed to VPA or RA confirms that VPA has strong effects on expression of Hoxa1, suggesting that genetic and environmental causes of autism may both depend on disturbance of Hox gene function in the early embryo.


Journal Articles on this Report : 8 Displayed | Download in RIS Format

Other project views: All 15 publications 9 publications in selected types All 8 journal articles
Type Citation Project Document Sources
Journal Article Ingram JL, Stodgell CJ, Hyman SL, Figlewicz DA, Weitkamp LR, Rodier PM. Discovery of allelic variants of HOXA1 and HOXB1: genetic susceptibility to autism spectrum disorders. Teratology 2000;62(6):393-405. R824758 (Final)
  • Abstract from PubMed
  • Abstract: Wiley
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  • Journal Article Ingram JL, Peckham SM, Tisdale B, Rodier PM. Prenatal exposure of rats to valproic acid reproduces the cerebellar anomalies associated with autism. Neurotoxicology and Teratology 2000;22(3):319-324. R824758 (Final)
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  • Full-text: Science Direct
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  • Abstract: Science Direct
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  • Other: Science Direct PDF
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  • Journal Article Rodier PM. Developing brain as a target of toxicity. Environmental Health Perspectives 1995;103(Suppl 6):73-76. R824758 (Final)
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  • Abstract from PubMed
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  • Full-text: EHP
  • Abstract: EHP
  • Journal Article Rodier PM, Ingram JL, Tisdale B, Nelson S, Romano J. Embryological origin for autism: developmental anomalies of the cranial nerve motor nuclei. Journal of Comparative Neurology 1996;370(2):247-261. R824758 (Final)
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  • Abstract: Wiley
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  • Journal Article Rodier PM. Animal model of autism based on developmental data. Mental Retardation and Developmental Disabilities Research Reviews 1996;2(4):249-256. R824758 (Final)
  • Abstract: Wiley
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  • Journal Article Rodier PM, Ingram JL, Tisdale B, Croog VJ. Linking etiologies in humans and animal models: studies of autism. Reproductive Toxicology 1997;11(2-3):417-422. R824758 (Final)
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  • Abstract: Science Direct
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  • Journal Article Rodier PM, Bryson SE, Welsh JP. Minor malformations and physical measures in autism: data from Nova Scotia. Teratology 1997;55(5):319-325. R824758 (Final)
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  • Abstract: Wiley
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  • Journal Article Rodier PM, Hyman SL. Early environmental factors in autism. Mental Retardation and Developmental Disabilities Research Reviews 1998;4(2):121-128. R824758 (Final)
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  • Supplemental Keywords:

    Health, Scientific Discipline, Toxicology, Health Risk Assessment, Physiology, Risk Assessments, Biochemistry, Molecular Biology/Genetics, birth defects, neurotoxic, neural tube defects, cranial nerve motor nuclei, developmental neurotoxicology, thalamus, animal model, developmental effects, somatosensory relay neurons, human exposure, medulla, autism, phaseolus vulgaris leukoagglutinin, immunocytochemical, human health risk

    Relevant Websites:

    http://www.urmc.rochester.edu/smd/obgyn/autism/index.htmlExit EPA icon

    Progress and Final Reports:

    Original Abstract
  • 1996
  • 1997