Brief Report: Low Rates of Herpesvirus Detection in Blood of Individuals with Autism Spectrum Disorder and Controls.

Previous research indicates that infection, especially from viruses in the family Herpesviridae, may play a role in the etiology of some cases of autism spectrum disorder (ASD). Using a case-control design and the polymerase chain reaction with site-specific primers, we screened newborn and childhood blood samples for the presence of eight human herpesviruses. Herpesvirus DNA was detected in 4 of 225 ASD individuals and 2 of 235 controls, with the most frequently detected virus being HHV-6B. Although this study does not detect a significant ASD-Herpesviridae association, it is limited by the use of site-specific primers. We suggest that new techniques using bioinformatics to search next-generation sequencing databases will be more revealing of possible ASD-virus associations.

Common Genetic Variants Found in HLA and KIR Immune Genes in Autism Spectrum Disorder.

The "common variant-common disease" hypothesis was proposed to explain diseases with strong inheritance. This model suggests that a genetic disease is the result of the combination of several common genetic variants. Common genetic variants are described as a 5% frequency differential between diseased vs. matched control populations. This theory was recently supported by an epidemiology paper stating that about 50% of genetic risk for autism resides in common variants. However, rare variants, rather than common variants, have been found in numerous genome wide genetic studies and many have concluded that the "common variant-common disease" hypothesis is incorrect. One interpretation is that rare variants are major contributors to genetic diseases and autism involves the interaction of many rare variants, especially in the brain. It is obvious there is much yet to be learned about autism genetics. Evidence has been mounting over the years indicating immune involvement in autism, particularly the HLA genes on chromosome 6 and KIR genes on chromosome 19. These two large multigene complexes have important immune functions and have been shown to interact to eliminate unwanted virally infected and malignant cells. HLA proteins have important functions in antigen presentation in adaptive immunity and specific epitopes on HLA class I proteins act as cognate ligands for KIR receptors in innate immunity. Data suggests that HLA alleles and KIR activating genes/haplotypes are common variants in different autism populations. For example, class I allele (HLA-A2 and HLA-G 14 bp-indel) frequencies are significantly increased by more than 5% over control populations (Table 2). The HLA-DR4 Class II and shared epitope frequencies are significantly above the control populations (Table 2). Three activating KIR genes: 3DS1, 2DS1, and 2DS2 have increased frequencies of 15, 22, and 14% in autism populations, respectively. There is a 6% increase in total activating KIR genes in autism over control subjects. And, more importantly there is a 12% increase in activating KIR genes and their cognate HLA alleles over control populations (Torres et al., 2012a). These data suggest the interaction of HLA ligand/KIR receptor pairs encoded on two different chromosomes is more significant as a ligand/receptor complex than separately in autism.

Prenatal and Newborn Immunoglobulin Levels from Mother-Child Pairs and Risk of Autism Spectrum Disorders.

BACKGROUND: An etiological role for immune factors operating during early brain development in children with autism spectrum disorders (ASD) has not yet been established. A major obstacle has been the lack of early biologic specimens that can be linked to later diagnosis. In a prior study, we found lower risk of ASD associated with higher levels of maternally-derived total IgG and Toxoplasmosis gondii (Toxo) IgG in newborn blood spot specimens from children later diagnosed with ASD compared to population controls. METHODS: We obtained maternal mid-gestational serum specimens and newborn screening blood spots from the California Genetics Disease Screening Program (GDSP) for linked mother-baby pairs for 84 children with ASD and 49 children with developmental delay but not ASD (DD) identified from California Department of Developmental Services records and for 159 population controls sampled from birth certificates.Immunoglobulin levels in maternal and newborn specimens were measured by solid phase immunoassays and analyzed in logistic regression models for total IgG, total IgM, and Toxo IgG, and, for maternal specimens only, Toxo IgM. Correlations between maternal and newborn ranked values were evaluated. RESULTS: In both maternal and newborn specimens, we found significantly lower risk of ASD associated with higher levels of Toxo IgG. In addition, point estimates for all comparisons were < 1.0 suggesting an overall pattern of lower immunoglobulin levels associated with higher ASD risk but most did not reach statistical significance. We did not find differences in maternal or newborn specimens comparing children with DD to controls. DISCUSSION: These results are consistent with evidence from our prior study and other published reports indicating that immune factors during early neurodevelopment may be etiologically relevant to ASD. Lowered immunoglobulin levels may represent suboptimal function of the maternal immune system or reduced maternal exposure to common infectious agents. CONCLUSION: Patterns seen in these selected immunoglobulins may provide clues to mechanisms of early abnormalities in neurodevelopment contributing to ASD. We recommend further study of immunoglobulin profiles in larger samples of linked mother-baby pairs to evaluate possible etiologic relevance.

C4B null alleles are not associated with genetic polymorphisms in the adjacent gene CYP21A2 in autism.

BACKGROUND: Research indicates that the etiology of autism has a strong genetic component, yet so far the search for genes that contribute to the disorder, including several whole genome scans, has led to few consistent findings. However, three studies indicate that the complement C4B gene null allele (i.e. the missing or nonfunctional C4B gene) is significantly more frequent in individuals with autism. Due to the close proximity of the CYP21A2 gene to the C4B locus (3 kb) it was decided to examine samples from autistic subjects, including many with known C4B null alleles for common CYP21A2 mutations. METHODS: Samples from subjects diagnosed with autism and non-autistic controls (controls) previously typed for C4B null alleles were studied. Allele specific polymerase chain reaction (PCR) methods were used to determine 8 of the most common CYP21A2 genetic mutations, known to completely or partially inhibit 21-hydroxylase, the enzyme encoded by the CYP21A2 gene. RESULTS: Although the combined autism and control study subjects had 50 C4B null alleles only 15 CYP21A2 mutations were detected in over 2250 genotypes. Eight mutations were detected in the autistic samples and 7 in the controls. The frequency of CYP21A2 mutations was similar between the autism and control samples. Only one individual (autistic) carried a chromosome containing both C4B null allele and CYP21A2 mutations.

How relevant are GFAP autoantibodies in autism and Tourette Syndrome?

Controversy exists over the role of autoantibodies to central nervous system antigens in autism and Tourette Syndrome. We investigated plasma autoantibody titers to glial fibrillary acidic protein (GFAP) in children with classic onset (33) and regressive onset (26) autism, controls (25, healthy age- and gender-matched) and individuals with Tourette Syndrome (24) by enzyme-linked immunosorbent assays. We found a significant difference in autoantibody titers to GFAP, not accounted for by age, between the Tourette (significantly lower) and regressive autism groups. However, no differences were found between: classic/regressive; classic/controls; classic/Tourette; regressive/controls; or controls/Tourette. Autoantibody responses against GFAP are unlikely to play a pathogenic role in autism or Tourette Syndrome.

Are there enhanced MBP autoantibodies in autism?

Autoantibodies to central nervous system antigens, such as myelin basic protein (MBP), may play a role in autism. We measured autoantibody titers to MBP in children with autism, both classic onset and regressive onset forms, controls (healthy age- and gender-matched) and individuals with Tourette syndrome via enzyme-linked immunosorbent assays. We found a significant difference in autoantibody titers to MBP, not accounted for by age or medication, between Tourette and classic autism (both significantly lower) when compared to regressive autism, but not when compared to controls. Autoantibody responses against MBP are unlikely to play a pathogenic role in autism.

Are there altered antibody responses to measles, mumps, or rubella viruses in autism?

The role that virus infections play in autism is not known. Others have reported that antibodies against measles virus are higher in the sera/plasma of children with autism versus controls. The authors investigated antibody titers to measles, mumps, and rubella viruses and diphtheria toxoid in children with autism, both classic onset (33) and regressive onset (26) forms, controls (25, healthy age- and gender-matched) and individuals with Tourette's syndrome (24) via enzyme-linked immunosorbent assays. No significant differences in antibody titers to measles, mumps, and rubella viruses and diphtheria toxoid were found among the four groups. Additionally, there were no significant differences between the four groups for total immunoglobulin (Ig)G or IgM. Interestingly, the authors did find a significant number (15/59) of autism subjects (classic and regressive onset combined) who had a very low or no antibody titer against rubella virus, compared to a combine control/Tourette's group (2/49).

The association and linkage of the HLA-A2 class I allele with autism.

Previous research has revealed associations between autism and immune genes located in the human leukocyte antigen (HLA). To better understand which HLA genetic loci may be associated with autism, we compared the class I HLA-A and -B alleles in autistic probands with case control subjects from Caucasian families. The frequency of HLA-A2 alleles was significantly increased in autistic subjects compared with normal allelic frequencies from the National Marrow Donors Program (NMDP) (p = 0.0043 after allelic correction). The transmission disequilibrium test for the A2 allele revealed an increased frequency of inheritance for autistic children (p = 0.033). There were no significant associations of autism with HLA-B alleles; however, the A2-B44 and A2-B51 haplotypes were two times more frequent in autistic subjects. The association and linkage of the class I HLA-A2 allele with autism suggests its involvement in the etiology of autism. Possible roles are discussed for the HLA-A2 association in the presentation of microbial antigen within the central nervous system and/or in the establishment of synaptic and neuronal circuits in the developing brain.

Hyperserotoninemia and altered immunity in autism.

One of the most consistent biological findings in autism is elevated whole blood serotonin (5-HT) levels found in about 1/3 of cases. Immune abnormalities are also commonly observed in this disorder. Given 5-HT's role as an immunomodulator, possible connections between 5-HT and immune abnormalities in autism are explored in this review. Areas of focus include hyperserotoninemia and cellular immune function, autoantibodies to 5-HT receptors, and 5-HT's role in autoimmunity. Further research is needed to determine the interactions between neuropsychiatric and immune dysfunction in autism and related disorders.

Autistic disorder and viral infections.

Autistic disorder (autism) is a behaviorally defined developmental disorder with a wide range of behaviors. Although the etiology of autism is unknown, data suggest that autism results from multiple etiologies with both genetic and environmental contributions, which may explain the spectrum of behaviors seen in this disorder. One proposed etiology for autism is viral infection very early in development. The mechanism, by which viral infection may lead to autism, be it through direct infection of the central nervous system (CNS), through infection elsewhere in the body acting as a trigger for disease in the CNS, through alteration of the immune response of the mother or offspring, or through a combination of these, is not yet known. Animal models in which early viral infection results in behavioral changes later in life include the influenza virus model in pregnant mice and the Borna disease virus model in newborn Lewis rats. Many studies over the years have presented evidence both for and against the association of autism with various viral infections. The best association to date has been made between congenital rubella and autism; however, members of the herpes virus family may also have a role in autism. Recently, controversy has arisen as to the involvement of measles virus and/or the measles, mumps, rubella (MMR) vaccine in the development of autism. Biological assays lend support to the association between measles virus or MMR and autism whereas epidemiologic studies show no association between MMR and autism. Further research is needed to clarify both the mechanisms whereby viral infection early in development may lead to autism and the possible involvement of the MMR vaccine in the development of autism.

A pilot study of D-cycloserine in subjects with autistic disorder.

OBJECTIVE: The authors assessed the effects of D-cycloserine on the core symptom of social impairment in subjects with autism. METHOD: Following a 2-week, single-blind placebo lead-in phase, drug-free subjects with autistic disorder were administered three different doses of D-cycloserine during each of three 2-week periods. Measures used for subject ratings included the Clinical Global Impression (CGI) scale and Aberrant Behavior Checklist. RESULTS: Significant improvement was found on the CGI and social withdrawal subscale of the Aberrant Behavior Checklist. d-Cycloserine was well tolerated at most of the doses used in this study. CONCLUSIONS: In this pilot study, D-cycloserine treatment resulted in significant improvement in social withdrawal. Further controlled studies of D-cycloserine in autism appear warranted.

High nitric oxide production in autistic disorder: a possible role for interferon-gamma.

BACKGROUND: Neuroimmune regulation abnormalities have been implicated in the pathophysiology of autistic disorder. Nitric oxide (NO) is involved in immune reactivity and is known to affect brain neurodevelopmental processes. Recent evidence indicates that NO, and cytokines involved in NO production, may be high in children with autism. The purpose of this study was to verify that plasma NO is high in children with autism and determine whether this elevation is related to plasma levels of cytokines involved in NO production. METHODS: The metabolites of NO, nitrite, and nitrate (NOx), along with the cytokines interferon-gamma (IFN-gamma), tumor necrosis factor-alpha, and interleukin-1beta, were measured in plasma of 29 children with autism (mean age +/- SD = 6.1 +/- 2.8 years) and 27 age- and gender-matched healthy comparison subjects using commercially available assay kits. RESULTS: Plasma levels of NOx were significantly higher in the autistic subjects (p =.006); plasma levels of the cytokines did not differ between groups. NOx and IFN-gamma levels were positively correlated in the autistic subjects (r =.51; p =.005). CONCLUSIONS: These results confirm that plasma NO is high in some children with autism and suggest that this elevation may be related to IFN-gamma activity.

Brief report: autistic disorder in three children with cytomegalovirus infection.

Previous research has identified a relationship between autistic disorder (autism) and specific congenital infections. Three cases of congenital or perinatal cytomegalovirus (CMV) infection occurring in association with autism are described. Hypothetical mechanisms relating congenital infection, such as CMV, to the development of autism are discussed. A better understanding of the immunologic response to certain congenital infections may provide important information pertaining to the pathophysiology and etiology of autism in vulnerable individuals.

Increased prevalence of familial autoimmunity in probands with pervasive developmental disorders.

OBJECTIVES: Increased prevalence of familial autoimmune disease is a common finding among probands with various autoimmune disorders. Autistic disorder (autism) is a highly genetic disorder with known immune and immunogenetic abnormalities. Previous research has found an increased frequency of autoimmune disorders in families with autistic probands. We further investigated this association by determining the frequency of autoimmune disorders in families that have probands with pervasive developmental disorders (PDDs), including autism, compared with 2 control groups. METHODS: Three well-defined study groups, including 1) families that have a child with a PDD, 2) families that have a child with an autoimmune disorder, and 3) families with a healthy control child, constituted the sample. A questionnaire inquiring about which first- and second-degree family members had received a diagnosis of having specific autoimmune disorders was completed by 101 families in each group. RESULTS: The frequency of autoimmune disorders was significantly higher in families of the PDD probands compared with families of both the autoimmune and healthy control probands. Autoimmunity was highest among the parents of PDD probands compared with parents of the healthy control subjects. Hypothyroidism/Hashimoto's thyroiditis and rheumatic fever were significantly more common in families with PDD probands than in the healthy control families. CONCLUSIONS: Autoimmunity was increased significantly in families with PDD compared with those of healthy and autoimmune control subjects. These preliminary findings warrant additional investigation into immune and autoimmune mechanisms in autism.

High blood monocyte counts and neopterin levels in children with autistic disorder.

OBJECTIVE: Leukocyte counts and plasma neopterin levels were determined in autistic children and matched healthy comparison subjects. METHOD: Blood from 31 autistic children and 28 age- and gender-matched healthy comparison subjects was analyzed for numbers of neutrophils, eosinophils, basophils, lymphocytes, monocytes, and total leukocytes and for plasma neopterin levels. RESULTS: The monocyte count and neopterin level were significantly higher in the autistic children than in the comparison subjects. CONCLUSIONS: These results suggest that the immune system may be activated in some children with autism.

The amygdala and related structures in the pathophysiology of autism.

Autism is a neurodevelopmental disorder that is defined behaviorally by severe deficiencies in reciprocal social interaction, verbal and nonverbal communication, and restricted interests. The amygdala is involved in the regulation of social behaviors and may be an important site of pathology for the social dysfunction seen in autism. This review focuses on lesion, postmortem, and neuroimaging studies that investigate the amygdala and related structures in this disorder. Other brain regions potentially involved in the neuropathology of autism are also briefly discussed. Although supportive evidence exists for amygdala dysfunction in autism, the currently available data are inconsistent and additional research is needed.