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Institute, Davis, CA, USA3Department of Medical Microbiology, University of California, Davis, Davis, CA, USA4Department of Psychiatry and the Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA5Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, USACorrespondence: Dr DB Campbell, 213 Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA. Health Sciences Dr., Suite 6510; University of California Davis; Davis, CA 95616, USA. Some mothers of children with ASD have autoantibodies that react to fetal brain proteins, raising the possibility that a subset of ASD cases may be associated with a maternal antibody response during gestation. The mechanism by which the maternal immune system breaks tolerance has not been addressed. We hypothesized that the mechanism may involve decreased expression of the MET receptor tyrosine kinase, an ASD risk gene that also serves as a key negative regulator of immune responsiveness. In a sample of 365 mothers, including 202 mothers of children with ASD, the functional MET promoter variant rs1858830 C allele was strongly associated with the presence of an ASD specific 37 band pattern of maternal autoantibodies to fetal brain proteins (P To determine the mechanism of this genetic association, we measured MET protein and cytokine production in freshly prepared peripheral blood mononuclear cells from 76 mothers of ASD and typically developing children. The MET rs1858830 C allele was significantly associated with MET protein expression (P Moreover, decreased expression of the regulatory cytokine IL 10 was associated with both the MET gene C allele (P and reduced MET protein levels (P These results indicate genetic distinction among mothers who produce ASD associated antibodies to fetal brain proteins, and suggest a potential mechanism for how a genetically determined decrease in MET protein production may lead to a reduction in immune regulation. Keywords: autism; gene; immune; maternal; cytokine Top of pageIntroductionAutism spectrum disorders (ASDs) are a complex, behaviorally defined group of developmental disorders characterized by social deficits, language impairments and repetitive behaviors with restricted interests. Previous studies of twins and sibling recurrence risk have suggested that ASD is among the most heritable of neuropsychiatric disorders, encouraging extensive research into the genetic basis of ASD risk.1, 2, 3 However, a more recent twin study reported that susceptibility to ASD has a much lower genetic heritability component, while the more substantial component of risk was explained by environmental factors in a shared twin environment.4 This would indicate that the gestational environment, susceptible to genetic regulation by both the fetus and the mother, might be a major factor in determining ASD risk. The genetic risk may lie in (a) the direct impact of genetic regulation on brain development, (b) altering sensitivities to environmental factors and (c) disruption of autonomic and peripheral organ development and function that may influence brain development. In this regard, there is a growing body of evidence that, among risk factors, immune system dysregulation may be a feature in this neurodevelopmental disorder.5, 6, 7 Growing evidence indicates that maternal immune dysfunction during gestation may contribute to ASD risk. In animal models, maternal immune activation during gestation leads to behavioral changes in subsequent offspring.8 In humans, epidemiologic analyses reported that a family history of autoimmune disorders was more common in families of children with ASD,9, 10 and that immune mediated disorders (psoriasis, maternal asthma and allergy) occurred more often around the time of pregnancy in the mothers of children with ASD compared with the mothers with typically developing (TD) children.11 One early report demonstrated that serum from a mother of a child with ASD showed IgG antibody reactivity to rodent Purkinje cells; this same serum was then injected into gestating mice and shown to elicit behavioral deficits in the offspring.12 In another study, 11 mothers of children with ASD and their affected children were found to have consistent patterns of antibody reactivity against rat prenatal brain proteins.13 Further studies have highlighted that this reactivity is to fetal and not adult brain proteins.14 Our recent studies have demonstrated reactivity against fetal brain proteins at 37 and 73 in plasma from 7 of 61 (11.5 mothers of children with ASD but not in 102 mothers of TD children.15 Injection of rhesus monkeys with human IgG from mothers of children with ASD (who were positive for fetal brain antibodies) during gestation resulted in increased stereotypical behavior and hyperactivity in the offspring when compared with the offspring of rhesus monkeys injected with IgG from mothers of TD children.16 Evidence of a potential role for these antibodies with respect to changes in neurodevelopment was also noted in a recent murine model.17 Although the preponderance of evidence points to an immune mediated etiology in some cases of ASD, the biological mechanism of the maternal immune dysfunction remains unclear. In an independent line of study, we described robust genetic association with ASD of a functional variant in oakley half jacket 2.0 xl nose pads the 5 promoter of the gene encoding the MET receptor tyrosine kinase.18 The MET promoter variant, rs1858830, is a common G to C single nucleotide polymorphism (SNP); the allele is inherited by individuals with ASD more often than predicted by chance and is more common in individuals with ASD than in a sample of the general population.18, 19, 20 Stratifying ASD subpopulations demonstrated that association of the MET allele is enriched in individuals with co occurring ASD and gastrointestinal conditions,21 and in the social and communication domains of ASD.22 Independent evidence indicates decreased expression of the ligand for the MET receptor, hepatocyte growth factor (HGF), in serum of individuals with co occurring ASD and gastrointestinal conditions.23 In addition to its roles in brain development and gastrointestinal repair, the MET receptor tyrosine kinase is a key negative regulator of immune responsiveness. Ligation of the MET receptor by its HGF ligand prevents lupus nephritis in chronic graft versus host disease,24 ameliorates the progression of experimental autoimmune myocarditis25 and suppresses collagen induced arthritis in mice.26 MET also induces morphogenic changes in antigen presenting cells. Importantly, MET signaling induces a tolerogenic phenotype in antigen presenting cells through the induction of IL 10, without affecting their antigen presenting capabilities.27, 28 This tolerogenic phenotype induces T regulatory cells in response to antigen that would otherwise have broken tolerance and potentially lead to autoimmunity.28 MET protein expression is decreased in postmortem brain of individuals with the MET allele.29 If the same genotype correlation is observed in the immune system, then one prediction is that disrupted MET signaling may confer susceptibility to immune dysregulation. We hypothesized that MET disruption would be associated with markers of immune dysregulation in mothers of children with ASD. To test this hypothesis, we examined the MET promoter variant rs1858830 allele in the context of ASD specific maternal antibodies to fetal brain proteins, as well as the cytokine profile of peripheral blood cells following immune challenge. Top of pageMaterials and methodsSubjectsAll mothers, (n including those of children with an ASD (n and those of TD control children (n participated in the Childhood Autism Risks from Genetics and the Environment (CHARGE) study.30 Those children enrolled in the study met the following criteria: (a) they were between the ages of 24 and 60 months at the time of enrollment, (b) lived with at least one biologic parent, (c) had a parent who spoke either English or Spanish, (d) were born in the state of California and (e) resided in the catchment areas of a specified list of regional centers in Northern California. The CHARGE study is an ongoing population based case study. This study protocol followed the ethical guidelines of the most recent oakley flak jacket xlj how to change nose pads Declaration of Helsinki, and was approved by the institutional review boards at the University of California, Davis, The State of California Department of Developmental Services, and the University of Southern California. Informed consent was obtained before participation. Institute. The Social Communication Questionnaire was used to screen for behavioral and developmental characteristics of ASD among TD controls; children who scored above the screening cutoff score of 15 were fully assessed using the ADI and ADOS. Those controls that met criteria for autistic disorder were classified as ASD. Controls who did not meet criteria for ASD were classified as TD. AutoantibodiesWestern blot data collected and analyzed in a previous study was used for the association analyses described herein. Western blots were performed as previously described using fetal monkey brain extract.15, 38 Genotyping the ASD associated MET promoter variant rs1858830As the rs1858830 SNP falls within a highly GC rich region, indirect genotyping methods fail when using genomic DNA. A 652 bp fragment containing the rs1858830 SNP was amplified from 15 genomic DNA with primers 5 (Forward) and 5 (Reverse). Cycling conditions were: 95 for 5 followed by 35 cycles of 95 for 30 61 for 30 and 72 for 1 Specific amplification of the 652 bp product was confirmed by agarose gel electrophoresis. Each PCR product was subjected to direct re sequencing using an ABI 3730xl using Big Dye Terminator chemistry. Genotype at the MET rs1858830 locus was determined from the sequencing result using Sequencher software (Gene Codes, Ann Arbor, MI, USA). Cell culture and stimulationCHARGE is an ongoing study for which we have collected and banked both plasma and DNA that allowed us to retroactively analyze subjects for both autoantibody production and genotype at the MET rs1858830 locus. However, cytokine production in response to immune challenge must be done on freshly isolated peripheral blood mononuclear cells (PBMCs). Therefore, a random subset of 76 mothers (22 C 33 C and 21 G prospective to study initiation, was used to assess the functional cytokine response in relation to MET rs1858830 genotype. Whole blood was collected through venipuncture into yellow top citrate tubes (BD, Franklin Lakes, NJ, USA) according to study protocol, and centrifuged at 900 for 10 to pellet cells. The cell pellet was then adjusted to appropriate density with HBSS and layered over Histopaque density gradient (Sigma, St Louis, MO, USA) followed by centrifugation at 600 for 30 The PBMC layer was removed and washed two additional times at 900 for 10 at which point cells were counted with a hemocytometer, and 300 cells per well were plated into 96 well round bottom plates. Determination of cytokine levelsAs a measure of innate immune reactivity following lipopolysaccharide stimulation, cell culture supernatants were analyzed for seven different cytokine and chemokines, including IL 6, GM CSF, IL 1, IL 12p40, TNF MIP 1 and IL 10. Levels were determined using a commercially available multiplex bead based kit and run according to manufacturer's instructions (Millipore, Billerica, MA, USA). Briefly, 25 of cell culture supernatant was incubated with anti cytokine conjugated beads in a 96 well filter bottom plate on a plate shaker overnight at 4 The beads were then washed using a vacuum manifold, and biotin conjugated detection antibodies were added for a 1 h incubation, followed by the subsequent addition of streptavidin PE for 30 The plates were then read on a Bio Plex 100 (BioRad, Hercules, CA, USA), and analyzed using Bio Plex Manager software using a 5 point standard curve. Reference samples were run on each plate to determine assay consistency. Determination of MET protein levelsAfter the supernatant was extracted, the cultured cells were used to determine the expression of the membrane bound MET protein. The cell pellet was resuspended in 60 Novagen Widescreen protein extraction reagent. Protein assays were performed with the Luminex xMAP technology with a bead based MET (HGF Receptor) ELISA kit (Novagen, Rockland, MA, USA), by manufacturer's protocol. Briefly, the isolated protein was mixed with capture beads containing primary antibody directed to MET (HGF Receptor; Novagen), followed by overnight shaking incubation at 4 Protein was measured on the Bio Plex Suspension Array System (Bio Rad, Hercules, CA, USA) at the Beckman Center Core at the USC Norris Cancer Center. Data acquisition and analysis were performed using the Bio Plex Manager software (Bio Rad). Statistical analysesThe was used to analyze the association of MET rs1858830 genotype with the presence of maternal antibody. Allelic results are presented; genotypic results are similar. Genotype dependent differences in MET protein and cytokine production were determined by analysis of variance followed by post hoc Tukey tests. Linear regression was used to determine correlation between MET protein expression and cytokine levels. Bonferroni correction for multiple comparisons was used to account for the analysis of multiple cytokines. Top of pageResultsMaternal antibody patterns associated with ASD riskIn a previous report with the CHARGE sample,30 association with ASD risk was demonstrated for a 37 band pattern of maternal antibodies reactive to fetal brain proteins in 61 mothers of children with ASD but not 102 mothers of TD children.15 Data for the current study were derived from an expanded study to include a total of 202 mothers of children with an ASD and 163 mothers of TD children.38 Specificity of the combined 37 band pattern with ASD risk was maintained in this larger sample. The 37 band pattern (as illustrated in conjunction with Table 1) was present in 19 of 202 (9 mothers of children with an ASD, and none of the 163 mothers of TD children (Table 1). Among the 19 mothers who were positive for the 37 band pattern, 11 (58 were homozygous for the ASD associated MET rs1858830 C allele, 7 (37 were C heterozygotes and one (5 was homozygous for the G allele (Table 2). Among the 183 mothers of children with ASD who were negative for the 37 band pattern, 51 (28 were C homozygous, 79 (43 were C heterozygous and 53 (29 were G homozygous (Table 2). This MET C allele frequency of 76 in the 19 mothers with the 37 band pattern was significantly higher than the 49 C allele frequency observed in the 183 mothers of children with ASD who did not exhibit the 37 band pattern (P Table 2). Among 346 mothers of both ASD and TD children who were negative for the 37 band pattern, 101 (29 were C 154 (45 were C and 91 (26 were G (Table 2). The 76 frequency of the MET C allele in mothers with the 37 band pattern was also significantly higher than the 51 C allele frequency observed in all 346 mothers lacking the band pattern (P Table 2). As anticipated, results were similar to those observed in postmortem brain where the expression of MET protein was significantly reduced. In mothers with the MET rs1858830 C genotype, MET protein expression was reduced 1.4 fold in PBMCs compared with mothers with the G genotype (P Figure 1). MET protein was also significantly decreased in mothers with the C genotype compared with the G genotype (P Figure 1). Production of the regulatory cytokine IL 10 was significantly associated with MET rs1858830 genotype and MET protein levels. The production of IL 10 was decreased 1.8 fold in mothers of genotype C compared with mothers of genotype G (P Figure 2a). As expected by the association of MET genotype to MET protein levels, the production of IL 10 was also positively correlated with the amount of MET protein (P Figure 2b). It is important to note that the significance of both of these results survive Bonferroni correction for analysis of seven analytes. The effect of MET genotype was specific to IL 10 as there was no correlation to expression of the other six cytokines tested. Full figure and legend (82K) Top of pageDiscussionThe results of this study suggest that the functional MET promoter variant rs1858830 C allele is associated with the presence of ASD associated maternal antibodies to fetal brain proteins. The convergence of these two previously distinct associations with ASD risk (that of maternal antibodies to fetal brain proteins and of the MET C allele) provides the first link between an ASD susceptibility gene and a functional immune related outcome to that gene. MET is a receptor tyrosine kinase, serving different functions in distinct cell types, including proliferation, motogenesis, survival and differentiation. MET is best understood as an oncogene involved in the metastasis of several cancers: somatic gain of function mutations lead to dramatically increased signaling via the MET receptor, and uncontrolled cell proliferation and migration.39 MET is also involved in mediating HGF signaling in a number of developmental, physiological and repair processes, including liver regeneration,40 gastrointestinal repair,41, 42, 43 and in serving as a part of a negative feedback loop in immune responses.27, 44, 45 The MET C allele decreases transcription and results in reduced MET receptor tyrosine kinase protein in postmortem brain samples.18, 29

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