Interactions between circulating inflammatory factors and autism spectrum disorder: a bidirectional Mendelian randomization study in European population.

Background: Extensive observational studies have reported an association between inflammatory factors and autism spectrum disorder (ASD), but their causal relationships remain unclear. This study aims to offer deeper insight into causal relationships between circulating inflammatory factors and ASD. Methods: Two-sample bidirectional Mendelian randomization (MR) analysis method was used in this study. The genetic variation of 91 circulating inflammatory factors was obtained from the genome-wide association study (GWAS) database of European ancestry. The germline GWAS summary data for ASD were also obtained (18,381 ASD cases and 27,969 controls). Single nucleotide polymorphisms robustly associated with the 91 inflammatory factors were used as instrumental variables. The random-effects inverse-variance weighted method was used as the primary analysis, and the Bonferroni correction for multiple comparisons was applied. Sensitivity tests were carried out to assess the validity of the causal relationship. Results: The forward MR analysis results suggest that levels of sulfotransferase 1A1, natural killer cell receptor 2B4, T-cell surface glycoprotein CD5, Fms-related tyrosine kinase 3 ligand, and tumor necrosis factor-related apoptosis-inducing ligand are positively associated with the occurrence of ASD, while levels of interleukin-7, interleukin-2 receptor subunit beta, and interleukin-2 are inversely associated with the occurrence of ASD. In addition, matrix metalloproteinase-10, caspase 8, tumor necrosis factor-related activation-induced cytokine, and C-C motif chemokine 19 were considered downstream consequences of ASD. Conclusion: This MR study identified additional inflammatory factors in patients with ASD relative to previous studies, and raised a possibility of ASD-caused immune abnormalities. These identified inflammatory factors may be potential biomarkers of immunologic dysfunction in ASD.

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OBJECTIVES: To investigate the levels of serum folate and vitamin B12 (VB12) and their association with the level of neurodevelopment in preschool children with autism spectrum disorder (ASD). METHODS: A total of 324 ASD children aged 2-6 years and 318 healthy children aged 2-6 years were recruited. Serum levels of folate and VB12 were measured using chemiluminescent immunoassay. The Social Responsiveness Scale and the Childhood Autism Rating Scale were used to assess the core symptoms of ASD children, and the Gesell Developmental Schedule was employed to evaluate the level of neurodevelopment. RESULTS: The levels of serum folate and VB12 in ASD children were significantly lower than those in healthy children (P<0.05). Serum folate levels in ASD children were positively correlated with gross and fine motor developmental quotients (P<0.05), and serum VB12 levels were positively correlated with adaptive behavior, fine motor, and language developmental quotients (P<0.05). In ASD children aged 2 to <4 years, serum folate levels were positively correlated with developmental quotients in all domains (P<0.05), and serum VB12 levels were positively correlated with language developmental quotient (P<0.05). In male ASD children, serum VB12 levels were positively correlated with language and personal-social developmental quotients (P<0.05). CONCLUSIONS: Serum folate and VB12 levels in preschool ASD children are lower than those in healthy children and are associated with neurodevelopmental levels, especially in ASD children under 4 years of age. Therefore, maintaining normal serum folate and VB12 levels may be beneficial for the neurodevelopment of ASD children, especially in ASD children under 4 years of age.

Detection of Circulating Serum microRNA/Protein Complexes in ASD Using Functionalized Chips for an Atomic Force Microscope.

MicroRNAs, which circulate in blood, are characterized by high diagnostic value; in biomedical research, they can be considered as candidate markers of various diseases. Mature microRNAs of glial cells and neurons can cross the blood-brain barrier and can be detected in the serum of patients with autism spectrum disorders (ASD) as components of macrovesicles, macromolecular protein and low-density lipoprotein particles. In our present study, we have proposed an approach, in which microRNAs in protein complexes can be concentrated on the surface of AFM chips with oligonucleotide molecular probes, specific against the target microRNAs. MicroRNAs, associated with the development of ASD in children, were selected as targets. The chips with immobilized molecular probes were incubated in serum samples of ASD patients and healthy volunteers. By atomic force microscopy (AFM), objects on the AFM chip surface have been revealed after incubation in the serum samples. The height of these objects amounted to 10 nm and 6 nm in the case of samples of ASD patients and healthy volunteers, respectively. MALDI-TOF-MS analysis of protein components on the chip surface allowed us to identify several cell proteins. These proteins are involved in the binding of nucleic acids (GBG10, RT24, RALYL), in the organization of proteasomes and nucleosomes (PSA4, NP1L4), and participate in the functioning of the channel of active potassium transport (KCNE5, KCNV2).

Alternations in the Cardiovascular Autonomic Regulation and Growth Factors in Autism.

Autism spectrum disorder (ASD) represents a serious neurodevelopmental disorder associated with autonomic nervous system dysregulation. The aim was to study complex cardiovascular autonomic regulation using heart rate variability (HRV) and systolic blood pressure variability (SBPV) linear/non-linear analysis at rest and during orthostasis, and to assess plasma levels of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) in autistic children. Twenty-five ASD boys and 25 age and gender-matched children at the age 7-15 years were examined. After venous blood taking, continuous ECG and blood pressure biosignals were recorded at rest and during orthostasis. Evaluated parameters: RR intervals, high- and low-frequency band of HRV spectral analysis (HF-HRV, LF-HRV), symbolic dynamics parameters 0V%, 1V%, 2LV%, 2UV%, low- and high-frequency band of SBPV (LF-SBPV, HF-SBPV), systolic, diastolic, mean blood pressure, EGF, VEGF plasma levels. RR intervals were significantly shortened and the HF-HRV, LF-SBPV, HF-SBPV parameters were significantly lower at rest, the HF-HRV and LF-SBPV remained lower during orthostasis in autistic children compared to controls (p<0.05). EGF plasma levels were significantly lower in ASD compared to controls (p=0.046). No significant differences were found in remaining parameters. Our study revealed tachycardia, cardiovagal underactivity, and blunted sympathetic vasomotor regulation at rest and during orthostasis in autistic children. Additionally, complex heart rate dynamics are similar in autistic children than controls. Furthermore, EGF was reduced in autistic children without significant correlations with any autonomic parameters. We suggest that the abnormal complex cardiovascular reflex control could contribute to understanding the pathway linking autonomic features and autism.

Cerebrospinal fluid and serum protein markers in autism: A co-twin study.

No robust biomarkers have yet been identified for autism spectrum disorder (ASD) or autistic traits. Familial factors likely influence biomarkers such as protein concentrations. Comparing twins with ASD or high autistic traits to the less affected co-twin allows estimating the impact of familial confounding. We measured 203 proteins in cerebrospinal fluid (n = 86) and serum (n = 127) in twins (mean age 14.2 years, 44.9% females) enriched for ASD and other neurodevelopmental conditions. Autistic traits were assessed by using the parent-report version of the Social Responsiveness Scale-2. In cerebrospinal fluid, autistic traits correlated negatively with three proteins and positively with one. In serum, autistic traits correlated positively with 15 and negatively with one. Also in serum, six were positively-and one negatively-associated with ASD. A pathway analysis of these proteins revealed immune system enrichment. In within twin pair analyses, autistic traits were associated with serum B-cell activating factor (BAFF) only, whereas Cystatin B (CSTB) remained significantly associated with ASD. These associations did not remain significant when only considering monozygotic twins. For the remainder, the within-pair analysis indicated familial confounding, including shared environment and genes, influencing both autism and protein levels. Our findings indicate proteins involved in immunity as putative biomarkers of autistic traits and ASD with partial genetic confounding. Although some results are in line with previous studies in general, further studies are needed for replication.

Reduction of essential amino acid levels and sex-specific alterations in serum amino acid concentration profiles in children with autism spectrum disorder.

BACKGROUND: Existing evidence has shown that metabolic disturbances may be involved in the pathological process of autism spectrum disorder(ASD). This study aimed to investigate the alterations of serum amino acid concentration profiles in Chinese Han children with ASD. METHODS: Serum amino acid levels were measured using tandem mass spectrometry in 60 children with ASD and 30 typically developing (TD) controls. The Chinese Wechsler Young Children Scale of Intelligence (C-WYCSI) was used to evaluate the ASD subjects' intelligence quotient (IQ). RESULTS: The serum levels of essential amino acids and some non-essential amino acids (glutamine, glycine, alanine, citrulline, cysteine, serine, tyrosine, and proline) in the ASD group were significantly lower than those in controls. The serum glutamate/glutamine (Glu/Gln) ratio was elevated in the ASD PIQ≥70 group, while serum levels of alanine, cysteine, phenylalanine, methionine and proline were significantly higher in male children with ASD than that in the female group. CONCLUSION: The study revealed that children with ASD exhibit alterations in the serum levels of certain amino acids, and the divergence can be sex-related or associated with different cognitive function, which might provide clues for further etiological research of ASD.

Peripheral blood neurotrophic factor levels in children with autism spectrum disorder: a meta-analysis.

Increasing evidence suggests that abnormal regulation of neurotrophic factors is involved in the etiology and pathogenesis of Autism Spectrum Disorder (ASD). However, clinical data on neurotrophic factor levels in children with ASD were inconsistent. Therefore, we performed a systematic review of peripheral blood neurotrophic factors levels in children with ASD, and quantitatively summarized the clinical data of peripheral blood neurotrophic factors in ASD children and healthy controls. A systematic search of PubMed and Web of Science identified 31 studies with 2627 ASD children and 4418 healthy controls to be included in the meta-analysis. The results of random effect meta-analysis showed that the peripheral blood levels of brain-derived neurotrophic factor (Hedges' g = 0.302; 95% CI = 0.014 to 0.591; P = 0.040) , nerve growth factor (Hedges' g = 0.395; 95% CI = 0.104 to 0.686; P = 0.008) and vascular endothelial growth factor (VEGF) (Hedges' g = 0.097; 95% CI = 0.018 to 0.175; P = 0.016) in children with ASD were significantly higher than that of healthy controls, whereas blood neurotrophin-3 (Hedges' g = - 0.795; 95% CI = - 1.723 to 0.134; P = 0.093) and neurotrophin-4 (Hedges' g = 0.182; 95% CI = - 0.285 to 0.650; P = 0.445) levels did not show significant differences between cases and controls. Taken together, these results clarified circulating neurotrophic factor profile in children with ASD, strengthening clinical evidence of neurotrophic factor aberrations in children with ASD.

Associations Between Sex Hormone Levels and Autistic Traits in Infertile Patients With Polycystic Ovary Syndrome and Their Offspring.

Objectives: 1) To explore the associations between autistic traits and sex hormone changes in patients with polycystic ovary syndrome (PCOS); 2) To explore the influence of PCOS parental autistic traits and related sex hormone changes on autistic traits in their offspring. Method: This is a case-control study that recruited two groups: polycystic ovarian syndrome-induced infertile subjects as the observation group and fallopian tube factor-induced infertility subjects as the control group. Both cohorts were patients undergoing infertility treatment in the Productive Medicine Center, Peking University People's Hospital. Two types of questionnaires were completed by patients between May 1st, 2015, and May 1st, 2016: 1. The autism-spectrum quotient (AQ) 2. Repetitive behavior scale-revised (RBS-r). Levels of sex hormones in serum were measured in patients. The correlations between the levels of these biochemical factors and scores of the autistic traits were analysed. From July 1st, 2020 to September 1st, 2021, these patients were followed up by telephone and asked to fill out a questionnaire online. The questionnaire included date of delivery, complications, medicine used and negative events during pregnancy (e.g., death of relatives, divorce, etc.), delivery condition, breastfeeding, AQ scale and Autism Behavior Checklist (ABC) of their children. Results: The patients in the PCOS group had significantly higher AQ scores than those in the control group. Levels of luteinizing hormone and testosterone were also higher in the PCOS group. No significant differences were found between the two groups in RBS-r levels, follicle-stimulating hormone, estradiol or progesterone. In the two combined groups, there were significantly positive correlations between the AQ scores and the luteinizing hormone concentration, as well as between scores of RBS-r and testosterone concentration. Moreover, there was a significantly negative correlation between the level of progesterone and the RBS-r score. According to the follow-up data, the AQ scores of offspring were positively correlated with the RBS-r scores of their mothers. The ABC scores of offspring were positively correlated with the RBS-r scores and the childbearing age of their mothers. No significant difference was found between the two groups in age of delivery, complications, special medication used, negative events during pregnancy, delivery situation, postpartum breastfeeding, age of children, or AQ scores or ABC scores of children. There were no significant correlations between the scale scores of children and the related sex hormone levels of mothers. This could indicate that the higher levels of luteinizing hormone and testosterone and the lower level of progesterone accompanied more pronounced autistic traits in PCOS. Furthermore, the higher delivery age and RBS-r score in mothers accompanied the higher AQ and ABC scores in children. Conclusion: Compared with the control group, PCOS patients had more autistic traits (especially social dysfunction). The autistic traits in PCOS patients might be related to the elevation in testosterone concentration and luteinizing hormone levels and the decline in progesterone level. Moreover, the autistic traits in the offspring of PCOS patients might be related to the parental high delivery age and high tendency to autism traits.

Identification of Amino Acid Dysregulation as a Potential Biomarker for Autism Spectrum Disorder in China.

Autism spectrum disorders (ASD) are increasingly common neurodevelopmental disorders accompanied by dysregulation of amino acid (AA) metabolism, and for which there are currently no reliable early diagnostic biomarkers. This study evaluated whether specific AAs can serve as biomarkers for screening ASD patients by analyzing the abundance 21 plasma AAs in 70 ASD patients and 70 control subjects by liquid chromatography-tandem mass spectrometry. We found significant differences between the two groups for eight of the AAs-namely, arginine, cysteine, homocysteine, histidine, methionine, serine, tyrosine, and valine. However, only homocysteine level was positively correlated with ASD symptom severity. Arginine, cysteine, histidine, and methionine were used to generate a predictive model in the Fisher discriminant analysis; cross-validation of this model showed that 88.6% of individuals were correctly segregated into ASD and healthy subject groups with a sensitivity of 85.5% and specificity of 92.2%. The area under the receiver operating characteristic curve was 0.959 (0.927-0.991). Thus, detection of a combination of AAs is an effective method for distinguishing ASD patients from healthy subjects, which may be useful for the early diagnosis of ASD.

Assessment of miR-181b-5p, miR-23a-3p, BCL-2, and IL-6 in Peripheral Blood Mononuclear Cells of Autistic Patients; Likelihood of Reliable Biomarkers.

Autism is a neurodevelopmental disorder that is recognized by stereotypic and repetitive behaviors after 2 years of old. Dysregulation of the immune system, especially inflammation which is mostly regulated by IL-6, imposes a deficit in CNS development. Along with this crucial biomarker, researchers have proposed BCL-2, micro RNA-23a-3p (miR-23a-3p), miR-181b-5p as other probable biomarkers involved in inflammation and apoptosis. The aim of the study was to evaluate the alteration in the expression of these biomarkers in a group of autism spectrum disorder (ASD) children. Peripheral blood mononuclear cells (PBMCs) were obtained from 37 autistic patients. After RNA extraction with precipitation method, the Syber green qReal-time Polymerase Chain Reaction (PCR) was performed in order to evaluate the possible alteration in the expression of IL-6, BCL-2, miR-181b-5p, and miR-23a-3p. The results were compared with healthy controls. IL-6 was significantly upregulated in ASD patients (p=0.003). On the other hand, miR-23a was upregulated and BCL-2 downregulated in ASD patients but the changes were not significant. In initial evaluations, expression changes of miR-181b-5p were not statistically significant. However, when Patients were divided into two groups of upregulated and downregulated, re-evaluation showed that both up- (p=0.005) and down-regulation (p=0.004) (i.e. changes regardless of the direction) of miR-181b were significant in autistic children. IL-6 and miR-181b-5p can have proper diagnostic values and are reliable biomarkers with high sensitivity and specificity. On the other hand, PBMC can be utilized for such studies and also evaluation of patients' condition instead of brain tissue as it is less accessible.

Blood biomarker levels of methylation capacity in autism spectrum disorder: a systematic review and meta-analysis.

OBJECTIVE: To compare the peripheral blood levels of methionine (Met), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and the SAM/SAH ratio (the most core and predictive indices of cellular methylation ability) between patients with autism spectrum disorder (ASD) and control subjects. METHODS: PubMed, Embase, PsycINFO, Web of Science, and Cochrane Library were searched from inception to August 2, 2019, without language restriction. The random-effects model was used to summarize effect sizes. RESULTS: We retrieved 1,493 records, of which 22 studies met inclusion criteria. Our overall analyses revealed that individuals with ASD had significantly decreased levels of Met (22 studies; Hedges' g = -0.62; 95% confidence interval [CI]: -0.89, -0.35), SAM (8 studies; Hedges' g = -0.60; 95% CI: -0.86, -0.34), and the SAM/SAH ratio (8 studies; Hedges' g = -0.98; 95% CI: -1.30, -0.66) and significantly increased levels of SAH (8 studies; Hedges' g = 0.69; 95% CI: 0.43, 0.94). The findings of the overall analyses were quite stable after being verified by sensitivity analyses and in agreement with the corresponding outcomes of subgroup analyses. Additionally, our results from meta-analytic techniques confirmed that the effect estimates of this meta-analysis did not originate from publication bias. CONCLUSION: Individuals with ASD have substantially aberrant peripheral blood levels of Met, SAM, SAH, and the SAM/SAH ratio, which supports the association between impaired methylation capacity and ASD. Therefore, further investigations into these indices as potential biomarkers for diagnosis and therapeutic targets of ASD are warranted.

Association of Cord Plasma Biomarkers of In Utero Acetaminophen Exposure With Risk of Attention-Deficit/Hyperactivity Disorder and Autism Spectrum Disorder in Childhood.

Importance: Prior studies have raised concern about maternal acetaminophen use during pregnancy and increased risk of attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) in their children; however, most studies have relied on maternal self-report. Objective: To examine the prospective associations between cord plasma acetaminophen metabolites and physician-diagnosed ADHD, ASD, both ADHD and ASD, and developmental disabilities (DDs) in childhood. Design, Setting, and Participants: This prospective cohort study analyzed 996 mother-infant dyads, a subset of the Boston Birth Cohort, who were enrolled at birth and followed up prospectively at the Boston Medical Center from October 1, 1998, to June 30, 2018. Exposures: Three cord acetaminophen metabolites (unchanged acetaminophen, acetaminophen glucuronide, and 3-[N-acetyl-l-cystein-S-yl]-acetaminophen) were measured in archived cord plasma samples collected at birth. Main Outcomes and Measures: Physician-diagnosed ADHD, ASD, and other DDs as documented in the child's medical records. Results: Of 996 participants (mean [SD] age, 9.8 [3.9] years; 548 [55.0%] male), the final sample included 257 children (25.8%) with ADHD only, 66 (6.6%) with ASD only, 42 (4.2%) with both ADHD and ASD, 304 (30.5%) with other DDs, and 327 (32.8%) who were neurotypical. Unchanged acetaminophen levels were detectable in all cord plasma samples. Compared with being in the first tertile, being in the second and third tertiles of cord acetaminophen burden was associated with higher odds of ADHD diagnosis (odds ratio [OR] for second tertile, 2.26; 95% CI, 1.40-3.69; OR for third tertile, 2.86; 95% CI, 1.77-4.67) and ASD diagnosis (OR for second tertile, 2.14; 95% CI, 0.93-5.13; OR for third tertile, 3.62; 95% CI, 1.62-8.60). Sensitivity analyses and subgroup analyses found consistent associations between acetaminophen buden and ADHD and acetaminophen burden and ASD across strata of potential confounders, including maternal indication, substance use, preterm birth, and child age and sex, for which point estimates for the ORs vary from 2.3 to 3.5 for ADHD and 1.6 to 4.1 for ASD. Conclusions and Relevance: Cord biomarkers of fetal exposure to acetaminophen were associated with significantly increased risk of childhood ADHD and ASD in a dose-response fashion. Our findings support previous studies regarding the association between prenatal and perinatal acetaminophen exposure and childhood neurodevelopmental risk and warrant additional investigations.

Phenotype-to-genotype approach reveals head-circumference-associated genes in an autism spectrum disorder cohort.

The genotype-first approach has been successfully applied and has elucidated several subtypes of autism spectrum disorder (ASD). However, it requires very large cohorts because of the extensive genetic heterogeneity. We investigate the alternate possibility of whether phenotype-specific genes can be identified from a small group of patients with specific phenotype(s). To identify novel genes associated with ASD and abnormal head circumference using a phenotype-to-genotype approach, we performed whole-exome sequencing on 67 families with ASD and abnormal head circumference. Clinically relevant pathogenic or likely pathogenic variants account for 23.9% of patients with microcephaly or macrocephaly, and 81.25% of those variants or genes are head-size associated. Significantly, recurrent pathogenic mutations were identified in two macrocephaly genes (PTEN, CHD8) in this small cohort. De novo mutations in several candidate genes (UBN2, BIRC6, SYNE1, and KCNMA1) were detected, as well as one new candidate gene (TNPO3) implicated in ASD and related neurodevelopmental disorders. We identify genotype-phenotype correlations for head-size-associated ASD genes and novel candidate genes for further investigation. Our results also suggest a phenotype-to-genotype strategy would accelerate the elucidation of genotype-phenotype relationships for ASD by using phenotype-restricted cohorts.

Dysregulation in IL-6 receptors is associated with upregulated IL-17A related signaling in CD4+ T cells of children with autism.

Autism spectrum disorder (ASD) is a heterogeneous syndrome characterized by dysregulations in speech and social interactions as well as repetitive and stereotypical behavioral patterns in which immune system plays a significant role. IL-6, an essential cytokine for polarization of Th0 cells into Th17 cells has been demonstrated to be crucial in the etiology of ASD in past studies both in humans and mice. Th17 cells are also believed to be central players in the pathogenesis of ASD through release of IL-17A. However, there is still insufficient data regarding identification of Th17 cells with respect to IL-6 signaling in ASD subjects. Therefore, this study explored IL-6 receptors (IL-6R/sIL-6R) and Th17 (p-STAT3/IL-17A/IL-23R) related markers comprehensively in the blood of typically-developing control (TDC, n = 35) and ASD children (n = 45). Our data show that there is enhanced sIL-6R levels in plasma and CD4+ T cells of ASD subjects as compared to TDC group. Increased sIL-6R signaling is associated with upregulated Th17 development in ASD subjects. Further, severe ASD subjects have higher inflammation in terms of IL-6/IL-17A related signaling as compared to moderate ASD patients. Furthermore, treatment of CD4 + T cells in vitro with IL-6 leads to much greater upregulation of p-STAT3, and IL-17A in ASD subjects than similarly treated CD4+ T cells in TDC group. Antagonism of IL-6 signaling by SC144 in vitro led to blockade of IL-6 mediated effects on CD4+ T cells. These data display unequivocally that IL-6 signaling components are dysregulated which play a crucial in enhancement of Th17 development in ASD subjects.

Peptides of neuron specific enolase as potential ASD biomarkers: From discovery to epitope mapping.

Autism spectrum disorder (ASD) is an important health issue and affects 1 in 59 children in the US. Prior studies determined that maternal autoantibody related (MAR) autism is thought to be associated with ~23% of ASD cases. We previously identified seven MAR-specific autoantigens including CRMP1, CRMP2, GDA, LDHA, LDHB, STIP1, and YBX1. We subsequently described the epitope peptide sequences recognized by maternal autoantibodies for each of the seven ASD-specific autoantigens. The aim of the current study was to expand upon our previous work and identify additional antigens recognized by the ASD-specific maternal autoantibodies, as well as to map the unique ASD-specific epitopes using microarray technology. Fetal Rhesus macaque brain tissues were separated by molecular weight and a fraction containing bands between 37 and 45 kDa was analyzed using 2-D gel electrophoresis, followed by peptide mass mapping using MALDI-TOF MS and TOF/TOF tandem MS/MS. Using this methodology, Neuron specific enolase (NSE) was identified as a target autoantigen and selected for epitope mapping. The full NSE sequence was translated into 15-mer peptides with an overlap of 14 amino acids onto microarray slides and probed with maternal plasma from mothers with an ASD child and from mothers with a Typically Developing child (TD) (ASD = 27 and TD = 21). The resulting data were analyzed by T-test. We found 16 ASD-specific NSE-peptide sequences for which four sequences were statistically significant (p < 0.05) using both the t-test and SAM t-test: DVAASEFYRDGKYDL (p = 0.047; SAM score 1.49), IEDPFDQDDWAAWSK (p = 0.049; SAM score 1.49), ERLAKYNQLMRIEEE (p = 0.045; SAM score 1.57), and RLAKYNQLMRIEEEL (p = 0.017; SAM score 1.82). We further identified 5 sequences that were recognized by both ASD and TD antibodies suggesting a large immunodominant epitope (DYPVVSIEDPFDQDDWAAW). While maternal autoantibodies against the NSE protein are present both in mothers with ASD and mothers of TD children, there are several ASD-specific epitopes that can potentially be used as MAR ASD biomarkers. Further, studies including analysis of NSE as a target protein in combination with the previously identified MAR ASD autoantigens are currently underway.

Early Second Trimester Maternal Serum Steroid-Related Biomarkers Associated with Autism Spectrum Disorder.

Epidemiologic studies link increased autism spectrum disorder (ASD) risk to obstetrical conditions associated with inflammation and steroid dysregulation, referred to as prenatal metabolic syndrome (PNMS). This pilot study measured steroid-related biomarkers in early second trimester maternal serum collected during the first and second trimester evaluation of risk study. ASD case and PNMS exposure status of index offspring were determined through linkage with autism registries and birth certificate records. ASD case (N = 53) and control (N = 19) groups were enriched for PNMS exposure. Higher estradiol and lower sex hormone binding globulin (SHBG) were significantly associated with increased ASD risk. Study findings provide preliminary evidence to link greater placental estradiol activity with ASD and support future investigations of the prenatal steroid environment in ASD.

Concentrations of Lead, Mercury, Arsenic, Cadmium, Manganese, and Aluminum in Blood of Romanian Children Suspected of Having Autism Spectrum Disorder.

Environmental exposure to lead (Pb), mercury (Hg), arsenic (As), cadmium (Cd), manganese (Mn), and aluminum (Al) has been associated with neurodevelopmental disorders including autism spectrum disorder (ASD). We conducted a pilot study during May 2015-May 2107 to estimate blood concentrations of six metals (Pb, Hg, As, Cd, Mn, and Al) and identify their associated factors for children with ASD or suspected of having ASD in Romania. Sixty children, age 2-8 years, were administered versions of ADOS or ADI-R translated from English to Romanian. After assessment, 2-3 mL of blood was obtained and analyzed for the concentrations of the six metals. The mean age of children was 51.9 months and about 90% were male. More than half (65%) of the children were born in Bucharest. Over 90% of concentrations of As and Cd were below limits of detection. Geometric mean concentrations of Pb, Mn, Al, and Hg were 1.14 µg/dL, 10.84 µg/L, 14.44 µg/L, and 0.35 µg/L, respectively. Multivariable linear regression analysis revealed that children who were female, had less educated parents, exhibited pica, and ate cold breakfast (e.g., cereal), watermelon, and lamb had significantly higher concentrations of Pb compared to their respective referent categories (all p < 0.05 except for eating lamb, which was marginally significant, p = 0.053). Although this is the first study that provides data on concentrations of the six metals for Romanian children with ASD, the findings from this study could be useful for designing future epidemiologic studies for investigating the role of these six metals in ASD in Romanian children.

Determination of neuroinflammatory biomarkers in autistic and neurotypical Saudi children.

To identify neuroinflammatory biomarkers in patients with various severity of autism spectrum disorder (ASD) increases the insight about the pathogenesis and pathophysiology of this neurodevelopmental disorder. The aim of the present study was to analyze the levels in plasma of TGFß2, Heat shock protein 70 (HSP70), and hematopoietic prostaglandin D2 synthase (H-PGDS) in Saudi ASD children and healthy age-matched neurotypical controls. Also, it was in the present study examined the correlation among these neuroinflammatory biomarkers and the sensory deficit exhibited by the ASD children. Blood samples from 38 Saudi children with ASD and 32 age-matched neurotypical controls were withdrawn after an overnight fast. For the blood taking 3 mL EDTA containing blood collection tubes was used. The samples were centrifuged for 20 min (4 °C; 3000×g) directly after the blood sampling. The harvested plasma was used for in vitro quantification of TGF-ß2, HSP70, and H-PGDS by using the sandwich enzyme immunoassay. Receiver operating characteristic (ROC) analysis and predictiveness curves showed that each of TGF-ß2, HSP70 or H-PGDS alone could not be used as a predictive neuroinflammatory biomarker for ASD. However, when TGF-ß2 and HSP70 were combined in one ROC curve, the AUC was increased to an appreciable value that makes them together robust predictors of variation between the ASD and neurotypical control groups. Overall, it was in the present study found significant differences for TGF-ß2 and HSP70 when the ASD and neurotypical control groups were compared, independently of the sensory deficit level. In conclusion, the present study highlights the usefulness of TGF-ß2, HSP70, and H-PGDS as diagnostic tools to differentiate between ASD and neurotypical control children, but not among subgroups of ASD children exhibiting different severity levels of sensory dysfunction. The presented data also suggest the effectiveness of ROC as a powerful statistical tool, which precisely can measure a combined effect of neuroinflammatory biomarkers intended for diagnostic purposes.

A meta-analysis of pro-inflammatory cytokines in autism spectrum disorders: Effects of age, gender, and latitude.

BACKGROUND: Autism spectrum disorders (ASD) occur in 1.5% of the general population worldwide. Studies suggest that ASD might have more costs than diabetes and attention deficit and hyperactivity disorder by 2025. Dysregulation of the cytokine system is well-documented in ASD. We conducted a meta-analysis of studies providing data on circulating concentrations of pro-inflammatory cytokines in people with ASD compared with control subjects without ASD. METHODS: We identified potentially eligible studies by systematically searching electronic databases from inception to February 2018. RESULTS: Thirty-eight studies with total of 2487 participants (1393 patients with ASD and 1094 control subjects) were included in the meta-analysis; 13 for interferon (IFN)-γ, 17 for interleukin (IL)-1ß, 22 for IL-6, 19 for tumor necrosis factor (TNF)-α, 4 for IL-1α, 6 for IL-2, 4 for IL-7, 8 for IL-8, 14 for IL-12, 3 for IL-15, 12 for IL-17, 3 for IL-18, 3 for IL-2 receptor, 3 for TNF-ß, and 3 for IL-23. We found medium increases in levels of plasma IFN-γ (standardized mean difference, SMD = 0.53) and serum IL-1ß (SMD = 0.56) and small increases in levels of blood IL-1ß (SMD = 0.35), serum IL-6 (SMD = 0.30) and serum TNF-α (SMD = 0.31) for patients with ASD. Meta-regression analyses identified latitude as a negative moderator of the effect size (ES) of difference in mean levels of IFN-γ (R2 = 0.26) and TNF-α (R2 = 0.74). Also, difference in the mean age between patients and controls had a negative interaction with the ES of difference in mean levels of IL-1ß. In contrast, there was a positive effect of the moderator of difference in the proportion of male subjects between patients and controls on the ES of difference in mean levels of IL-1ß. We found no significant alterations in peripheral levels of other pro-inflammatory cytokines including IL-1α, IL-2, IL-2R, IL-3, IL-7, IL-8, IL-12, IL-12p40, IL-12p70, IL-15, IL-17, IL-18, IL-23, TBF-ß, and TNFRI/II in patients with ASD. CONCLUSIONS: This meta-analysis provides evidence for higher concentration of pro-inflammatory cytokines IFN-γ, IL-1ß, IL-6, and TNF-α in autistic patents compared with control subjects. Also, meta-regression analyses point to the interaction of latitude, age, and gender with peripheral alterations of associated pro-inflammatory cytokines.

Evaluation of Platelet Parameters in Children with Autism Spectrum Disorder: Elongated Collagen-Adenosine Diphosphate and Collagen-Epinephrine Closure Times.

Changes related to the serotonin system play a key role in the etiology of autism spectrum disorder (ASD). Although we know that platelets are associated with the serotonin system, their relation to ASD has not yet been elucidated. In this study, we aim to investigate platelet parameters in children with ASD. Forty patients with ASD according to Diagnostic and Statistical Manual of Mental Disorders 5 (DSM-5) and 30 healthy controls were included in the study. A complete blood count was done to measure parameters relating to platelet morphology. Moreover, prothrombin time (PT) and activated partial thromboplastin time (aPTT) were evaluated. Lastly, platelet functions were assessed with a platelet functions analyzer 100 (PFA-100) device by measuring collagen-ADP and collagen-epinephrine (EPI) closure times. There was not a significant difference between the groups in terms of platelet count, mean platelet volume (MPV), platelet distribution width, plateletcrit, PT, or aPTT parameters for ASD patients when compared to the control group (P > 0.05). However, MPV in severe ASD, as quantified by the Childhood Autism Rating Scale, was found to be significantly lower when compared to mild to moderate ASD (P = 0.047). Moreover, in terms of platelet functions, the elongation in collagen-ADP and collagen-EPI closure times were significantly higher for the ASD group (P = 0.044). These results may suggest an impairment in platelet functions rather than in platelet morphology for children with ASD. Considering these results, further investigation of thrombocyte functions in the ASD may lead to a better understanding of the pathogenesis of ASD and to the development of our limited knowledge of this disorder. Autism Res 2019, 12: 1069-1076. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Serotonin is a chemical that is found in brain as wells as in blood cells that function in blood clotting in the human body. There are problems related to serotonin in brains of people who have autism. Thus, blood clotting cells may also be affected in people who have autism. In this study, we compare blood clotting functions of children with autism with that of healthy controls.