Transgenerational Effects and Heritability of Folate Receptor Alpha Autoantibodies in Autism Spectrum Disorder

Transgenerational Effects and Heritability of Folate Receptor Alpha Autoantibodies in Autism Spectrum Disorder | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Transgenerational Effects and Heritability of Folate Receptor Alpha Autoantibodies in Autism Spectrum Disorder Richard Frye, Ira Cohen, Jeffrey Sequeira, Zoë Hill, Alina Espinoza, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6755086/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Autism Spectrum Disorder (ASD) affects an estimated prevalence of 1 in 36 children but the cause in most cases is unknown. Human and animal studies have linked ASD to Folate Receptor Alpha Autoantibodies (FRAAs). Our previous studies demonstrated that FRAAs are more common, on average, in families with children with ASD. This study reanalyzed data from this previous study which included 82 children diagnosed with ASD, 53 unaffected siblings, 70 mothers and 65 fathers and 52 typically developing controls who did not have a history of ASD in their family. This study investigates the association of FRAA titers with ASD risk factors and explores the relationship of FRAA titers across generations. Several known risk factors for ASD, including multiplex ASD families, multiple birth pregnancies and increased maternal and paternal age at birth were related to offspring FRAA titers. Multiplex ASD families demonstrated higher FRAA titers and a significant correlation between maternal and offspring blocking FRAA titers. FRAA titers increased across generations, although the increase in blocking FRAAs was only seen in multiplex families. The proband with ASD demonstrated higher blocking but not binding titers compared to their non-affected siblings. Paternal FRAA titers are associated with several measures of offspring behavior and cognitive development. This research highlights the potential transgenerational transmission of FRAAs and their role in ASD, demonstrating that heritable non-genetic factors may be important in the etiology of ASD and that FRAAs may demonstrate anticipation (worsening across generations), especially in multiplex families. Disruption of immune regulation and susceptibility to autoimmune disease may underly disruption of brain development and function in ASD. Health sciences/Diseases/Psychiatric disorders Biological sciences/Physiology Health sciences/Biomarkers/Predictive markers anticipation autism spectrum disorder cerebral folate deficiency folate receptor alpha folate receptor alpha autoantibodies heritability Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Autism spectrum disorder (ASD) is a behaviorally defined neurodevelopmental disorder characterized by social-communication deficits with restrictive and repetitive behaviors and interests as outlined by the Diagnostic Statistical Manual of Mental Disorders Version 5 Text Revision. 1 The Centers for Disease Control and Prevention funded Autism and Developmental Disabilities Monitoring Network has reported a continued increase in the prevalence of ASD over the past two decades with a current estimated prevalence of 1 in 31 children. 2 Despite decades of ASD surveillance and research, the cause of ASD remains uncertain in most cases. 3 ASD is highly heritable with studies finding a heritability of about 50%. 4 Having one child with ASD increases the chances of having another child with ASD. For example, there is an 80% recurrent risk for identical twins and 20% for nonidentical siblings. 4 However, there are two types of ASD families, multiplex families who have multiple children with ASD and simplex families who only have one child with ASD. Most commonly, heritable factors are thought to be transmitted genetically. However, inherited defects in single genes are rare 5 with most single gene mutations being de novo , meaning that they are not inherited. 6 , 7 Older studies demonstrated low yields (~ 16%) of genetic defects when chromosomal microarray analysis and whole-exome sequencing were used, 8 , 9 but more advanced whole genome sequencing has increase the yield to between 33% and 50%. 10 Non-genetic factors are also heritable. For example, many physiological abnormalities without a purely genetic etiology are found in both children with ASD and their mothers. These include mitochondrial, 11 , 12 transmethylation/transsulfuration 13,14 and immune 15 abnormalities. This suggests that heritable transmission of these abnormalities may not be purely genetic. Older studies have estimated that shared environment accounts for approximately 58% of the ASD risk and suggested that ASD may mainly be driven by genetic-environmental interactions. 16 Indeed, ASD risk can be linked to the maternal environment 17 , 18 and exposure to environmental factors. 19 Consistent with this notion, the many physiological systems which are shared by the child with ASD and their mother are sensitive to environmental stressors. These include the immune system, 20 transmethylation/transsulfuration, 21 and mitochondrial metabolism. 22 Abnormal central folate metabolism is being recognized as a significant metabolic abnormality associated with ASD. Many children with ASD cannot efficiently transport folate into the brain. The primary transporter of folate into the brain is the folate receptor α (FRα), which can become nonfunctional for several reasons. One of the major causes of disrupted FRα function is the presence of one or two types of autoantibodies, known as the blocking and binding FRα autoantibodies (FRAAs). FRAAs bind to the FRα and block folate from binding to the FRα or interfere with FRα function and triggers local inflammation. 23 Interestingly Quadros et al 24 found that these antibodies were not only prevalent in the individuals with ASD but also in other family members as compared to typically developing controls from families without children with ASD, a finding that verified other previous reports. 25 Interestingly, the nuances of the relationships to ASD and to potential transgenerational transmission remain understudied. In this study the data from Quadros et al 24 is re-analyzed to determine whether FRAA titers are related to other ASD known risk factors such as being born in multiplex families, in a multiple birth pregnancy, or being related to maternal or paternal age. For example, a relationship between multiplex families and FRAAs could help examine the particular increased risk of ASD in these families as genetic inheritances does not seem to examine this this phenomenon. 26 For example, when sibling have causative genetic mutation, they are only the same 31% of the time, 9 suggesting that the primary etiology is not the genetic mutation itself a process driving the development of genetic mutations, such as a folate pathway abnormality. Additionally, it would be important to know whether titers increase across generations and whether parental FRAAs are related to the behavioral or cognitive outcome of the child with ASD. 2. Materials and Methods Data were derived from our previous published study on the family prevalence of the FRAA. 24 Participants were recruited as part of a study of ASD at the Institute for Basic Research in Developmental Disabilities (IBR), Staten Island, New York between the years 2000 and 2017. Diagnosis of ASD was established based on DSM-IV (1984); DSM-IV-TR (2000) and DSM-5 (2013) criteria using information from the Autism Diagnostic Interview -- Revised and Autism Diagnostic Observation Scale, the two gold standards for ASD diagnosis, and parental interview. The protocol was approved by the Institutional Review Board at IBR (Staten Island, New York). Parents of participants provided written informed consent. Data were then deidentified for analysis. All dates were transformed to age in days at the visit and personal health identifiers were removed to deidentify the data for further analysis. Thus, the final dataset was analyzed under 45 CFR 46 exemption. Titer measurement The FRAA assay was performed by the laboratory of Dr Quadros at SUNY Downstate. An in vitro functional blocking assay was used to measure blocking FRAAs while an enzyme-linked immunosorbent assay (ELISA) specific for binding IgG was used to measure binding FRAAs as previously described. 27 Developmental Assessments The Griffiths Scales of Child Development (1984) provide an overall measure of development for children from infancy to 8 years of age. 28 It includes subscales for assessing learning, language and communication, eye and hand coordination, personal, social and emotional function and gross motor function. It is used by a trained examiner interacting with the child. Age standardized scores (MA/CAX 100) are provided with lower scores representing worse development. The PDD Behavior Inventory (PDDBI; 2005) is a reliable and valid caregiver questionnaire assessment tool which measures both problem behaviors and social communication abilities. 29 It provides age-standardized T-scores based on a large sample of well diagnosed children with ASD. It is divided into two dimensions: Approach-Withdrawal (AWP) problems and Receptive Expressive Social Communication Abilities (REXSCA) along with an overall Autism Composite score. For the AWP dimension and the Autism Composite, higher scores indicate greater severity. For the REXSCA dimension, higher scores indicate greater competence. The Vineland Adaptive Behavior Social Subscale III (VABS) is a widely used standardized, well-validated assessment tool for children with developmental delays that measures functional abilities. It is a valid measures of social impairments in children with ASD. 30 The VABS relies on an informant (caretaker) to complete. Higher scores represent better development. Gross motor development was assessed with the PDDBI in a subset of parents by asking the parents to note at which age the child sat and walked without support as these are milestones that are typically remembered well by parents. Statistical Analysis Analyses were performed using PASW Statistics version 28.0.0.0 (IBM SPSS Statistics, Armonk, NY). Graphs were produced using Excel version 14.0 (Microsoft Corp, Redmond, WA). An alpha of 5% was used as a cutoff for significance. In general, mixed-model regression models with random effects of family (the shared variance among the parents and sibling) to control for repeated effects of family level mean and variance were used to examine the effects of dichotomous variables such as multiplex family, multiple birth, sex and continuous variables such as offspring, maternal and paternal age. Two-way Interactions between variables were included in the model. The final model was simplified to only significant variables and variables that were dependent on significant interactions. Cohen’s d’ was calculated to represent effect size.. Effects from model coefficients are provided for dicrotous variables with standard errors. 3. Results 3.1. Participant Characteristics 82 children diagnosed with ASD consisted of 65 boys and 17 girls ranging from 1.6 to 15 years of age [Mean (SD) 5.18 (2.6)]. 53 unaffected siblings (22 brothers; 29 sisters, and one half-brother and one half-sister) were tested [Mean (SD) 6.7 (4.2)], as were 70 mothers [Mean (SD) 31.5 years (5.1 y)] and 65 fathers [Mean (SD) 33.9y (5.6y)]. Unrelated, non-ASD controls comprising 24 boys and 28 girls, aged 3 to 17 years [Mean (SD) 11.8y (4.0y)] were recruited from a local pediatric practice. There were 17 (22%) multiplex families and 8 (11%) with multiple births. 3.2. Pregnancy and ASD Family Type Effects on FRAA Titers in Children with ASD and their Siblings The effect of pregnancy factors, ASD family type (simplex vs multiplex) and parental age on FRAA titers in the offspring (ASD and siblings combined) was analyzed. Controls were not included in these analyses. For probands and their siblings, multiplex families were found to have significantly higher blocking titers by 4.86 (1.13) pmol/ml as compared to simplex families with a large effect size [F(1,75) = 18.50, p < 0.001; Cohen’s d’ =1.1] with no significant effect found for sex, multiple birth pregnancy or age at antibody measurement. Older maternal [F(1,75) = 9.18, p < 0.01; Cohen’s d’ =0.61] and parental age [F(1,75) = 11.06, p = 0.001; Cohen’s d’ =0.67] at birth were related to higher blocking titers, both with medium effect sizes (See Fig. 1 A). For probands and their siblings, multiple birth pregnancy was found to have significantly higher binding titers as compared to single birth pregnancies with a medium-to-large effect size [F(1,109) = 3.88, p = 0.05, Cohen’s d’ =0.66] and binding titers decreased with age at which the binding antibody was measured with a medium effect size [F(1,109) = 8.546, p < 0.01; Cohen’s d’ =0.58] with no significant effect found for sex or multiplex families (Fig. 1 B). 3.3. Transgenerational Changes in FRAA Titers Offspring (ASD and Sibling) blocking titers were significantly related to maternal blocking titers [F(1,105) = 33.514, p < 0.001, Cohen’s d’ =1.16] (Fig. 2 ) but binding offspring FRAA titers were not found to be related to parental FRAA titers. The relationship between blocking autoantibodies in the offsprings and mothers was clearly driven by a group in which titers were high for both the offspring and mother (point in the oval in Fig. 2 ) and another group where the titers were only elevated in the offspring or the mother. As the mean titers were higher for the multiplex families, it was hypothesized that these double positives (offspring, mother) were driven by multiplex families. For the blocking FRAA, 53% of the multiplex families demonstrated positivity in both offspring and mother whereas this was only seen in 12% of the simplex families. This difference was highly significant with a large effect size [χ 2 = 24.47, p < 0.0001, Cohen’s d’ =1.14]. Comparing offspring to parental blocking titers, multiplex families (Fig. 3 A) manifest significantly higher titer than simplex families (Fig. 3 B) by 3.73 (0.73) pmol/ml with a large effect size [F(1,72) = 26.50, p < 0.001, Cohen’s d’ =0.82] and the offspring blocking titers were significantly greater than maternal [F(1,200) = 5.91, p < 0.05, Cohen’s d’ =0.39] and paternal [F(1,200) = 6.74, p < 0.001, Cohen’s d’ =0.65] blocking titers by 1.06 (0.44) and 1.78 (0.44) with a small and medium effect size, respectively. Binding titers were not significantly different across family members or between simplex or multiplex families (See Fig. 3 C). 3.4. Effect of ASD on Family Titers To determine the effect of ASD over and above the effect of being an offspring, a mixed-model with separate factors for offspring and ASD child was used to examine FRAA titers across generations. Blocking titers were significantly higher by 1.62 (0.50) [F(1,215) = 10.60, p < 0.001, Cohen d’ =0.46] in offspring as compared to parents and being affected by ASD increased the titer by 3.92 (1.08) [F(1,215) = 5.92, p < 0.05, Cohen d’ =0.40], both with a small-to-medium effect size. Multiplex families demonstrated titers higher by 3.44 (0.75) [F(1,74) = 21.09, p < 0.001, Cohen’s d’ =0.74] with a medium-to-large effect size. Multiple birth pregnancy interacted with the affected individual such that a child with ASD demonstrated a significantly higher titer if they were born from a multiple birth pregnancy [F(1,211) = 16.95, p < 0.001, Cohen’s d’ =0.67] with a medium effect size. Binding titers were significantly higher by 0.08 (0.04) [F(1,208) = 4.24, p < 0.05, Cohen’s d’ =0.34] in offspring as compared to parents, with a small effect size, but being affected by ASD did not significantly change the FRAA titer. 3.5. Familial FRAA Titers Compared to Controls Participant groups (proband, sibling, parent, controls) differed for blocking titers [F(3,272) = 10.51, p < 0.001, Cohen’s d’ =0.38] with a small-to-medium effect size and binding titers [F(3,278) = 3.93, p < 0.01, Cohen’s d’ =0.23] with a small effect size (Fig. 4 ). Post-hoc comparisons using 2-sided Dunnett t found that blocking titers were significantly higher in the families of children with ASD as compared to typically developing children. Binding titers were significantly higher in the children with ASD and their siblings as compared to typically developing children. 3.6. Behavior and Development The participants with ASD were assessed using several instruments to determine their development and ASD symptoms. A mixed model controlling for family unit was used to determine whether the presence of FRAAs (positive vs negative) in the mom or dad was associated with development and/or ASD symptoms. The presence of blocking and binding FRAAs in the child was included in the model to control for any effect of FRAAs in the child. The results are summarized in Table 1 . A positive FRAA status for the father was related to poorer development and greater ASD symptoms in the child, with medium to large effect sizes , as described below. Several of the Griffth’s Mental Development Scales, which were performed on the subset of children within the range of the test ( < = 8 years of age), were lower when the father was FRAA positive. This included total developmental score ( Cohen’s d’ = 0.66), motor development ( Cohen’s d’ = 0.69), speech and language development ( Cohen’s d’ = 0.85), coordination ( Cohen’s d’ = 0.66) and practical reasoning ( Cohen’s d’ = 0.64), all with medium effect sizes. The VABS demonstrated poorer development in communication ( Cohen’s d’ = 0.47), socialization ( Cohen’s d’ = 0.73), daily living skills ( Cohen’s d’ = 0.73) and motor skills ( Cohen’s d’ = 0.48) if the father was FRAA positive, with medium-to-large effect sizes. ASD symptoms were assessed using the PDDBI, which demonstrated more severe ASD symptoms if the father was FRAA positive, specifically, a positive FRAA in the father was related to worse overall PDDBI score (Cohen’s d’ =0.46) , Expressive Language (Cohen’s d’ =0.46) , Learning, Memory, and Receptive Language (Cohen’s d’ =0.45) , Expressive Social Communication Abilities Composite (Cohen’s d’ =0.40) and Receptive/Expressive Social Communication Composite (Cohen’s d’ =0.46). Positive FRAA in the mother was associated with worse Social Pragmatic Problems (Cohen d’ =0.61) , all with medium effect sizes. Table 1 Relationship between FRAA status in mom and dad and cognitive and behavior of the ASD offspring. Means and standard errors of the offspring’s cognitive and behavioral symptoms are given for positive and negative FRAA antibody status of the parents. Statistically significant values are bolded. The number of participants for each evaluation are given in the instrument name header. Mom Dad Griffth’s Mental Development Scales Neg N = 18 Pos N = 29 Neg N = 10 Pos N = 32 Total Scale (GQ) 69.1 (5.2) 75.4 (3.9) 85.2 (8.5)* 70.4 (3.4) Locomotor Development 83.9 (3.6) 86.6 (2.5) 93.5 (6.8)* 83.4 (1.9) Personal Social Development 64.6 (5.2) 65.3 (3.9) 74.0 (8.3) 63.0 (3.2) Hearing and speech 54.8 (6.9) 61.3 (6.9) 80.8 (15.0)** 51.9 (27.7) Hand and Eye Coordination 68.3 (5.7) 76.9 (4.1) 85.1 (7.8)* 71.2 (3.8) Performance 80.9 (6.4) 88.2 (4.0) 96.5 (7.4) 83.9 (4.2) Practical reasoning 63.2 (6.3) 69.0 (5.2) 81.2 (11.0)* 63.3 (4.2) PDD Behavior Inventory Neg N = 41 Pos N = 63 Neg N = 25 Pos N = 72 Autism Composite 49.1 (1.7) 51.0 (1.3) 45.9 (1.8)* 51.3 (1.2) Sensory/Perceptual Approach Behaviors 51.4 (1.5) 48.7 (1.2) 46.7 (1.7) 50.4 (1.2) Ritualisms/Resistance to Change 48.3 (1.3) 50.3 (1.3) 45.9 (1.6) 49.6 (1.2) Social Pragmatic Problems 47.0 (1.6)** 53.1 (1.1) 48.9 (1.7) 51.1 (1.2) Semantic/Pragmatic Problems 51.5 (2.1) 48.8 (1.1) 49.7 (2.2) 49.2 (1.1) Arousal Regulation Problems 52.3 (1.5) 50.3 (1.3) 49.9 (1.8) 51.1 (1.2) Specific Fears 48.8 (1.5) 50.7 (1.2) 47.7 (2.1) 50.7 (1.1) Aggressiveness 48.8 (1.3) 50.9 (1.6) 48.5 (2.5) 49.5 (1.1) Repetitive, Ritualistic, & Pragmatic Prob Comp 50.7 (1.8) 50.5 (1.2) 48.4 (1.9) 50.5 (1.2) Approach/Withdrawal Problems Composite 50.6 (1.9) 51.3 (1.3) 49.4 (2.3) 50.7 (1.3) Social Approach Behaviors 50.0 (1.8) 48.4 (1.2) 51.7 (2.0) 47.3 (1.2) Expressive Language 50.1 (1.6) 49.1 (1.2) 53.4 (1.9)* 47.8 (1.1) Learning, Memory, and Receptive Language 50.1 (1.6) 49.6 (1.1) 53.0 (2.0)* 48.2 (1.0) Expressive Social Comm Abilities Composite 50.0 (1.8) 49.6 (1.2) 52.7 (1.9)* 48.0 (1.2) Receptive/Expressive Social Comm Composite 50.0 (1.8) 49.7 (1.2) 52.7 (1.9)* 48.0 (1.2) Vineland Adaptive Behavior Scales Neg N = 29 Pos N = 44 Neg N = 19 Pos N = 49 Communication 71.0 (4.7) 63.7 (3.3) 77.5 (5.8)* 63.4 (3.3) Daily Living Skills 62.0 (3.2) 58.0 (2.2) 69.5 (4.7)** 56.5 (1.7) Socialization 66.1 (3.3) 60.3 (1.9) 72.1 (4.5)** 59.3 (1.8) Motor Skills 70.2 (4.0) 70.8 (2.4) 79.6 (5.2)* 67.7 (2.4) Developmental Gross Motor Milestones [N = 36] Neg N = 15 Pos N = 21 Neg N = 10 Pos N = 26 Age when sat without support 6.7 (0.3) 6.9 (0.3) 6.5 (0.2) 7.1 (0.3) Age when walking without support 13.4 (3.1) 14.0 (0.7) 12.8 (0.7) 14.4 (0.7) *p < = 0.05; **p < = 0.01; ***p < = 0.001 4. Discussion This paper examines the transgenerational heritability of folate receptor alpha autoantibodies (FRAAs) particularly with respect to autism spectrum disorder (ASD), one of the major disorders it has become associated with. Many interesting aspects of this study include the fact that the transgenerational heritability appears to be slightly different for the blocking and binding FRAAs. Of note, two previously unrecognized factors that affect the FRAA titers of the offspring, multiplex ASD and multiple birth, were found. Interestingly, the FRAA status of the father appears to have a relationship with the outcome of the ASD child. Although families with multiple births and multiplex families represented a minority of the sample, it was found that these families demonstrated a distinct relationship to FRAA titers. Families with multiple birth pregnancies manifested higher FRAA binding titers. Both of these factors are risk factors for ASD. Specifically multiple birth pregnancy is a risk factor for the offspring developing ASD, 31 possibly because of perinatal and neonatal complications which are also associated with ASD. 32 , 33 . Multiplex families were found to have an overall higher blocking titer as compared to simplex families, and the risk of having another child with ASD is higher in multiplex families, 34 suggesting that FRAAs may be involved in the biological mechanisms which increase ASD risk especially since multiplex families are less likely to have an identifiable genetic cause for ASD, suggesting other non-genetic factors, such as the FRAA, may be playing a role in these families. 26 The affected ASD child in the multiplex family demonstrated a significantly higher titers than the other typically developing family members, thus being a factor which distinguishes the ASD child from the typically developing family members. Interestingly, multiplex families having higher FRAA titers than simplex families is consistent with several studies which demonstrate that the members of multiplex families have subtle cognitive deficits. For example, non-ASD siblings in multiplex families, where FRAA titers were higher, tend to have lower cognitive function and adaptive behavior as compared to non-ASD siblings from simplex families 35 and family members in multiplex families exhibit more ASD characteristics than family members in simplex families. 36 – 39 Although the number of multiplex families was lower than the number of simplex families, the proportion of the samples of multiplex families was similar to other studies comparing multiplex and simplex families. 35 Nevertheless, these are compelling findings that need to be confirmed in larger samples. Another compelling finding is that the blocking titers increases with parental age, particularly the father. Such a finding is consistent with the association of advanced parental age as a risk factor for ASD. Advanced maternal age is well-known to be associated with several reproductive issues such as infertility and fetal malformations. FRAA have been proposed to be associated with pregnancy complication given the high concentration of folate receptors at the maternal-fetal interface 40 and the association between FRAAs and neural-tube defects, 41 – 43 preterm birth 44 and subfertility. 45 The association of ASD with advanced paternal age is somewhat unique for ASD. Studies suggest that this is due to decreased sperm quality and testicular function. Although the folate receptor alpha is not known to be located on the testes, one major role of the epididymis and vas deferens is to secrete a folate binding protein which adhere to spermatozoa. 46 Potential cross reaction between FRAA and the folate binding protein could effect on sperm quality. Blocking but not binding titers were correlated between mother and offspring. Closer examination of this relationship demonstrated that this effect was driven by cases in which both mother and offspring had high FRAAs. Interestingly these cases were overrepresented by multiplex families, suggesting that multiplex families are more likely to have maternal-offspring heritability of FRAAs, whereas simplex families tend to have positive blocking FRAAs in either mother or offspring but not both. Interestingly, both binding and blocking titers increased across generations. This phenomenon of increasing severity of a disease across generations has been termed anticipation in genetics where it is most commonly used to describe an increase in the severity of trinucleotide repeat disorders across generations. Similar to genetic anticipation in which the gene abnormality can become worse until it reaches a threshold to cause disease, FRAAs appear to increase in severity (titer concentration) across generations. It may very well be that the FRAA requires a specific level to cause disease, so ASD is not manifested until the titers reach a certain level. However, it is more likely that the ASD symptoms are less severe in family members with FRAAs such that they do not severely interfere with life. The mechanism by which titers increase across generations is not known and will require further research. We found several developmental characteristics related to paternal FRAA status, including measure of mental development, ASD behavior and adaptive functioning. As shown in Table 1 , the PDDBI data largely replicated the Griffiths and Vineland results and indicated that the primary effect was on the domains and composite scores assessing social communication competence (a core feature of ASD), rather than on the domains and composite scores assessing pragmatic problems, repetitive/ritualistic behaviors and arousal/anxiety problems. In addition, the PDDBI also emphasizes the language defect associated with FRAA status. As previously discussed, it is not clear how the paternal FRAA status affects the development of offspring, but there are several potential mechanisms. The FRα is secreated into the semen where it interacts with spermatozoa. 47 FRAAs binding of the FRα could disrupt this interaction. FRAAs in the semen could bind to JUNO (a pseudo-folate receptor known as FOLR4) on the oocyte during fertalization. 48 A glycolipid linked high-affinity folate binding protein has been found to be secreated from the epithelium of the epididymal and vas deferens where it associates with prostasome-like vesicles adherent to spermatozoa. 46 It has been hypotheized that this folate binding protein has a bacteriostatic function or facilitates folate transport into the spermatozoa. 46 FRAAs binding to this folate binding protein could possiblity disrupt spermatozoa health. Thus, it is not unreasonable to hypothesize that FRAAs could interfere with the development of high-quality sperm. 5. Conclusions This study examined the relationship between the FRAA in families to help better define its role in reproductive health and neurodevelopmental outcomes of the offspring. Several novel findings were found, including a relationship between FRAA titers and both multiplex families and multiple birth pregnancies, two factors that are known to increase the risk of ASD in the offspring. The blocking FRAA appears to be highly correlated between mother and offspring, thereby supporting the notion of a relationship across generations. Finally, the notion of anticipation of the FRAA across generations is compelling and may suggest an increased risk of ASD in parents that have high titers. This study is limited, mostly by its sample size. Larger studies will be needed to verify and further this research. Declarations Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki. The protocol was approved by the Institutional Review Board at Institute for Basic Research in Developmental Disabilities (Staten Island, New York). All dates were transformed to age in days at the visit and per-sonal health identifiers were removed to deidentify the data for further analysis. Thus, the final dataset was analyzed under 45 CFR 46 exemption 4. Informed Consent Statement : Parents of participants provided written informed consent. Data was then deidentified for analysis. Conflicts of Interest: EVQ is an inventor on a US patent for the detection of FRAAs issued to the Research Foundation of the State University of New York, USA. The remainder of the authors declare no conflict of interest. Funding: This work was supported by funding from the New York State OPWDD (ILC), research grant #8202 from Autism Speaks (EVQ), research grant RFGA2022-010-31 from the Arizona Department of Health Services and the XEL Foundation (Pittsburgh, PA). None of the sponsors were involved with the design or conduct of the study, collection, management, analysis or interpretation of the data; or preparation, approval of the manuscript or decision to submit the manuscript for publication. Author Contributions: Conceptualization, EVQ, IIC, REF; methodology, EVQ, IIC, REF, JMS; formal analysis, REF; investigation, EVQ, IIC, JMS, WTB, CM, EM, MF, ECJ, MTV; writing—original draft preparation, REF, EVQ; writing—review and editing, all authors; visualization, REF; funding acquisition, REF, EVQ. All authors have read and agreed to the published version of the manuscript.” Acknowledgments: The authors would like to thank all of the staff which were involved in making this study possible and the participants and their families who gave their time. Data Availability Statement: Data is available upon request. References Association, A.P. Diagnostic and Statistical Manual of Mental Disorders (DSM-5®) , (American Psychiatric Association Publishing, Washington, DC, 2013). Shaw, K.A. , et al. Prevalence and Early Identification of Autism Spectrum Disorder Among Children Aged 4 and 8 Years - Autism and Developmental Disabilities Monitoring Network, 16 Sites, United States, 2022. MMWR Surveill Summ 74 , 1-22 (2025). Maenner, M.J. , et al. Prevalence and Characteristics of Autism Spectrum Disorder Among Children Aged 8 Years - Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2020. MMWR Surveill Summ 72 , 1-14 (2023). Sandin, S. , et al. The familial risk of autism. Jama 311 , 1770-1777 (2014). Schaefer, G.B., Mendelsohn, N.J., Professional, P. & Guidelines, C. Clinical genetics evaluation in identifying the etiology of autism spectrum disorders: 2013 guideline revisions. Genet Med 15 , 399-407 (2013). O'Roak, B.J. , et al. Recurrent de novo mutations implicate novel genes underlying simplex autism risk. Nature communications 5 , 5595 (2014). Wang, L. , et al. Functional relationships between recessive inherited genes and genes with de novo variants in autism spectrum disorder. Molecular autism 11 , 75 (2020). Tammimies, K. , et al. Molecular Diagnostic Yield of Chromosomal Microarray Analysis and Whole-Exome Sequencing in Children With Autism Spectrum Disorder. Jama 314 , 895-903 (2015). Yuen, R.K. , et al. Whole-genome sequencing of quartet families with autism spectrum disorder. Nature medicine 21 , 185-191 (2015). Bar, O., Vahey, E., Mintz, M., Frye, R.E. & Boles, R.G. Reanalysis of Trio Whole-Genome Sequencing Data Doubles the Yield in Autism Spectrum Disorder: De Novo Variants Present in Half. Int J Mol Sci 25 (2024). Frye, R.E., McCarty, P.J., Werner, B.A., Rose, S. & Scheck, A.C. Bioenergetic signatures of neurodevelopmental regression. Front Physiol 15 , 1306038 (2024). Frye, R.E. , et al. Biomarkers of mitochondrial dysfunction in autism spectrum disorder: A systematic review and meta-analysis. Neurobiol Dis 197 , 106520 (2024). Hollowood, K. , et al. Maternal metabolic profile predicts high or low risk of an autism pregnancy outcome. Res Autism Spectr Disord 56 , 72-82 (2018). Zhu, Y. , et al. Expression Changes in Epigenetic Gene Pathways Associated With One-Carbon Nutritional Metabolites in Maternal Blood From Pregnancies Resulting in Autism and Non-Typical Neurodevelopment. Autism Res 14 , 11-28 (2021). Gardner, R.M., Brynge, M., Sjöqvist, H., Dalman, C. & Karlsson, H. Maternal immune activation and autism in the offspring-what is the evidence for causation? Biol Psychiatry (2024). Hallmayer, J. , et al. Genetic heritability and shared environmental factors among twin pairs with autism. Archives of general psychiatry 68 , 1095-1102 (2011). Frye, R.E. , et al. Physiological mediators of prenatal environmental influences in autism spectrum disorder. Bioessays 43 , e2000307 (2021). Frye, R.E. , et al. Mitochondria May Mediate Prenatal Environmental Influences in Autism Spectrum Disorder. J Pers Med 11 (2021). Rossignol, D.A., Genuis, S.J. & Frye, R.E. Environmental toxicants and autism spectrum disorders: a systematic review. Translational psychiatry 4 , e360 (2014). Hughes, H.K., Moreno, R.J. & Ashwood, P. Innate immune dysfunction and neuroinflammation in autism spectrum disorder (ASD). Brain Behav Immun 108 , 245-254 (2023). Howsmon, D.P., Kruger, U., Melnyk, S., James, S.J. & Hahn, J. Classification and adaptive behavior prediction of children with autism spectrum disorder based upon multivariate data analysis of markers of oxidative stress and DNA methylation. PLoS Comput Biol 13 , e1005385 (2017). Rose, S. , et al. Clinical and Molecular Characteristics of Mitochondrial Dysfunction in Autism Spectrum Disorder. Mol Diagn Ther 22 , 571-593 (2018). Ramaekers, V.T. , et al. Autoantibodies to folate receptors in the cerebral folate deficiency syndrome. N Engl J Med 352 , 1985-1991 (2005). Quadros, E.V. , et al. Folate receptor autoantibodies are prevalent in children diagnosed with autism spectrum disorder, their normal siblings and parents. Autism Res 11 , 707-712 (2018). Rossignol, D.A. & Frye, R.E. Cerebral Folate Deficiency, Folate Receptor Alpha Autoantibodies and Leucovorin (Folinic Acid) Treatment in Autism Spectrum Disorders: A Systematic Review and Meta-Analysis. J Pers Med 11 (2021). Sebat, J. , et al. Strong association of de novo copy number mutations with autism. Science 316 , 445-449 (2007). Sequeira, J.M., Ramaekers, V.T. & Quadros, E.V. The diagnostic utility of folate receptor autoantibodies in blood. Clin Chem Lab Med 51 , 545-554 (2013). Griffiths, R. The abilities of young children: A comprehensive system of mental measurement for the first eight years of life (Revised ed.) , (A.R.I.C.D. The Test Agency Limited., Bucks, UK, 1984). Cohen, I.L. & Sudhalter, V. The PDD Behavior Inventory , (Psychological Assessment Resources, Inc., Lutz, FL, 2005). Pine, E., Luby, J., Abbacchi, A. & Constantino, J.N. Quantitative assessment of autistic symptomatology in preschoolers. Autism : the international journal of research and practice 10 , 344-352 (2006). Gardener, H., Spiegelman, D. & Buka, S.L. Perinatal and neonatal risk factors for autism: a comprehensive meta-analysis. Pediatrics 128 , 344-355 (2011). Karmel, B.Z. , et al. Early medical and behavioral characteristics of NICU infants later classified with ASD. Pediatrics 126 , 457-467 (2010). Cohen, I.L. , et al. Neonatal brainstem function and 4-month arousal-modulated attention are jointly associated with autism. Autism Res 6 , 11-22 (2013). Ozonoff, S. , et al. Familial Recurrence of Autism: Updates From the Baby Siblings Research Consortium. Pediatrics 154 (2024). McDonald, N.M. , et al. Developmental Trajectories of Infants With Multiplex Family Risk for Autism: A Baby Siblings Research Consortium Study. JAMA Neurol 77 , 73-81 (2020). Virkud, Y.V., Todd, R.D., Abbacchi, A.M., Zhang, Y. & Constantino, J.N. Familial aggregation of quantitative autistic traits in multiplex versus simplex autism. Am J Med Genet B Neuropsychiatr Genet 150b , 328-334 (2009). Gerdts, J.A., Bernier, R., Dawson, G. & Estes, A. The broader autism phenotype in simplex and multiplex families. J Autism Dev Disord 43 , 1597-1605 (2013). Oerlemans, A.M. , et al. Recognition of facial emotion and affective prosody in children with ASD (+ADHD) and their unaffected siblings. Eur Child Adolesc Psychiatry 23 , 257-271 (2014). Oerlemans, A.M., Hartman, C.A., Franke, B., Buitelaar, J.K. & Rommelse, N.N. Does the cognitive architecture of simplex and multiplex ASD families differ? J Autism Dev Disord 46 , 489-501 (2016). Qin, X.Y., Ha, S.Y., Chen, L., Zhang, T. & Li, M.Q. Recent Advances in Folates and Autoantibodies against Folate Receptors in Early Pregnancy and Miscarriage. Nutrients 15 (2023). Rothenberg, S.P. , et al. Autoantibodies against folate receptors in women with a pregnancy complicated by a neural-tube defect. N Engl J Med 350 , 134-142 (2004). Cabrera, R.M. , et al. Autoantibodies to folate receptor during pregnancy and neural tube defect risk. J Reprod Immunol 79 , 85-92 (2008). Boyles, A.L. , et al. Association between inhibited binding of folic acid to folate receptor alpha in maternal serum and folate-related birth defects in Norway. Hum Reprod 26 , 2232-2238 (2011). Vo, H.D., Sequeira, J.M., Quadros, E.V., Schwarz, S.M. & Perenyi, A.R. The role of folate receptor autoantibodies in preterm birth. Nutrition 31 , 1224-1227 (2015). Berrocal-Zaragoza, M.I. , et al. Association between blocking folate receptor autoantibodies and subfertility. Fertil Steril 91 , 1518-1521 (2009). Malm, J. , et al. A minor fraction of a high-affinity folate binding protein from the epididymis is associated with membranous vesicles and spermatozoa in human semen. Int J Androl 28 , 267-274 (2005). Holm, J. & Hansen, S.I. Characterization of soluble folate receptors (folate binding proteins) in humans. Biological roles and clinical potentials in infection and malignancy. Biochim Biophys Acta Proteins Proteom 1868 , 140466 (2020). Díaz-Fuster, L., Sáez-Espinosa, P., Moya, I., Peinado, I. & Gómez-Torres, M.J. Updating the Role of JUNO and Factors Involved in Its Function during Fertilization. Cells Tissues Organs , 1-16 (2025). Additional Declarations Yes EVQ is an inventor on a US patent for the detection of FRAAs issued to the Research Foundation of the State University of New York, USA. The remainder of the authors declare no conflict of interest. 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Jenkins4 Jenkins","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Edmund","middleName":"C. Jenkins4","lastName":"Jenkins","suffix":""},{"id":465842064,"identity":"0619c37c-a6a8-4c2a-84c5-a236ac26681f","order_by":10,"name":"Milen Velinov","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Milen","middleName":"","lastName":"Velinov","suffix":""},{"id":465842065,"identity":"f968e3c0-93f5-4a6f-9c5b-fe4681c27096","order_by":11,"name":"Edward Quadros","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Edward","middleName":"","lastName":"Quadros","suffix":""}],"badges":[],"createdAt":"2025-05-27 03:55:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6755086/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6755086/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84338003,"identity":"1516832b-1d88-48bd-bf78-500248e974c5","added_by":"auto","created_at":"2025-06-10 18:05:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":123279,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationship between age and FRAA titers. (A) Both the maternal and paternal age were related to the blocking autoantibodies such that higher blocking autoantibodies in the offspring were associated with higher parental age. (B) Binding autoantibodies decreased with older offspring age.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6755086/v1/9c5b4ecc1dbef2b7e982a7e2.png"},{"id":84339170,"identity":"83825a90-b485-4315-96f3-637eaf690344","added_by":"auto","created_at":"2025-06-10 18:13:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":76740,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationship between offspring and maternal blocking FRAA titers. The oval represents the group of cases where both maternal and offspring titers were positive. These cases disproportionally are from multiplex families.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6755086/v1/5815313bcde835ac528f175a.png"},{"id":84338004,"identity":"27bd0832-2747-41c3-9ab1-4169ba9e7844","added_by":"auto","created_at":"2025-06-10 18:05:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":68156,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship of FRAA titers between parents and offspring. The change in blocking titers across generations was significantly higher in (A) multiplex as compared to (B) simplex families. For both multiplex and simplex families, offspring blocking titers were significantly higher than parental blocking titers. (B) The titers for the offspring, mother and father were similar in simplex families. (C) Binding titers were not significantly difference across family members.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6755086/v1/e4d7c2c94eb1786256735e62.png"},{"id":84338005,"identity":"a62b6f51-ee66-466f-83ec-5e678360cc30","added_by":"auto","created_at":"2025-06-10 18:05:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":77675,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of (A) Blocking and (B) Binding FRAA titers in proband families to typically developing controls.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6755086/v1/dafce2ee6ea9b2129027deee.png"},{"id":85755899,"identity":"6c42dafb-8b4e-4118-aba7-01acd85dcd69","added_by":"auto","created_at":"2025-07-01 10:46:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1290046,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6755086/v1/238813b5-be38-49a9-9b20-a8dbb0340ea8.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e\nEVQ is an inventor on a US patent for the detection of FRAAs issued to the Research Foundation of the State University of New York, USA. The remainder of the authors declare no conflict of interest.","formattedTitle":"Transgenerational Effects and Heritability of Folate Receptor Alpha Autoantibodies in Autism Spectrum Disorder","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAutism spectrum disorder (ASD) is a behaviorally defined neurodevelopmental disorder characterized by social-communication deficits with restrictive and repetitive behaviors and interests as outlined by the Diagnostic Statistical Manual of Mental Disorders Version 5 Text Revision.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e The Centers for Disease Control and Prevention funded Autism and Developmental Disabilities Monitoring Network has reported a continued increase in the prevalence of ASD over the past two decades with a current estimated prevalence of 1 in 31 children.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Despite decades of ASD surveillance and research, the cause of ASD remains uncertain in most cases.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eASD is highly heritable with studies finding a heritability of about 50%.\u003csup\u003e4\u003c/sup\u003e Having one child with ASD increases the chances of having another child with ASD. For example, there is an 80% recurrent risk for identical twins and 20% for nonidentical siblings.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e However, there are two types of ASD families, multiplex families who have multiple children with ASD and simplex families who only have one child with ASD. Most commonly, heritable factors are thought to be transmitted genetically. However, inherited defects in single genes are rare\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e with most single gene mutations being \u003cem\u003ede novo\u003c/em\u003e, meaning that they are not inherited.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Older studies demonstrated low yields (~\u0026thinsp;16%) of genetic defects when chromosomal microarray analysis and whole-exome sequencing were used,\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e but more advanced whole genome sequencing has increase the yield to between 33% and 50%.\u003csup\u003e10\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eNon-genetic factors are also heritable. For example, many physiological abnormalities without a purely genetic etiology are found in both children with ASD and their mothers. These include mitochondrial,\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e transmethylation/transsulfuration\u003csup\u003e13,14\u003c/sup\u003e and immune\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e abnormalities. This suggests that heritable transmission of these abnormalities may not be purely genetic. Older studies have estimated that shared environment accounts for approximately 58% of the ASD risk and suggested that ASD may mainly be driven by genetic-environmental interactions.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Indeed, ASD risk can be linked to the maternal environment\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and exposure to environmental factors.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Consistent with this notion, the many physiological systems which are shared by the child with ASD and their mother are sensitive to environmental stressors. These include the immune system,\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e transmethylation/transsulfuration,\u003csup\u003e21\u003c/sup\u003e and mitochondrial metabolism.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eAbnormal central folate metabolism is being recognized as a significant metabolic abnormality associated with ASD. Many children with ASD cannot efficiently transport folate into the brain. The primary transporter of folate into the brain is the folate receptor α (FRα), which can become nonfunctional for several reasons. One of the major causes of disrupted FRα function is the presence of one or two types of autoantibodies, known as the blocking and binding FRα autoantibodies (FRAAs). FRAAs bind to the FRα and block folate from binding to the FRα or interfere with FRα function and triggers local inflammation.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eInterestingly Quadros et al\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e found that these antibodies were not only prevalent in the individuals with ASD but also in other family members as compared to typically developing controls from families without children with ASD, a finding that verified other previous reports.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Interestingly, the nuances of the relationships to ASD and to potential transgenerational transmission remain understudied. In this study the data from Quadros et al\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e is re-analyzed to determine whether FRAA titers are related to other ASD known risk factors such as being born in multiplex families, in a multiple birth pregnancy, or being related to maternal or paternal age. For example, a relationship between multiplex families and FRAAs could help examine the particular increased risk of ASD in these families as genetic inheritances does not seem to examine this this phenomenon.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e For example, when sibling have causative genetic mutation, they are only the same 31% of the time,\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e suggesting that the primary etiology is not the genetic mutation itself a process driving the development of genetic mutations, such as a folate pathway abnormality. Additionally, it would be important to know whether titers increase across generations and whether parental FRAAs are related to the behavioral or cognitive outcome of the child with ASD.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eData were derived from our previous published study on the family prevalence of the FRAA.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Participants were recruited as part of a study of ASD at the Institute for Basic Research in Developmental Disabilities (IBR), Staten Island, New York between the years 2000 and 2017. Diagnosis of ASD was established based on DSM-IV (1984); DSM-IV-TR (2000) and DSM-5 (2013) criteria using information from the Autism Diagnostic Interview -- Revised and Autism Diagnostic Observation Scale, the two gold standards for ASD diagnosis, and parental interview.\u003c/p\u003e\u003cp\u003e The protocol was approved by the Institutional Review Board at IBR (Staten Island, New York). Parents of participants provided written informed consent. Data were then deidentified for analysis. All dates were transformed to age in days at the visit and personal health identifiers were removed to deidentify the data for further analysis. Thus, the final dataset was analyzed under 45 CFR 46 exemption.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTiter measurement\u003c/span\u003e\u003c/p\u003e\u003cp\u003eThe FRAA assay was performed by the laboratory of Dr Quadros at SUNY Downstate. An \u003cem\u003ein vitro\u003c/em\u003e functional blocking assay was used to measure blocking FRAAs while an enzyme-linked immunosorbent assay (ELISA) specific for binding IgG was used to measure binding FRAAs as previously described.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eDevelopmental Assessments\u003c/span\u003e\u003c/p\u003e\u003cp\u003eThe Griffiths Scales of Child Development (1984) provide an overall measure of development for children from infancy to 8 years of age.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e It includes subscales for assessing learning, language and communication, eye and hand coordination, personal, social and emotional function and gross motor function. It is used by a trained examiner interacting with the child. Age standardized scores (MA/CAX 100) are provided with lower scores representing worse development.\u003c/p\u003e\u003cp\u003eThe PDD Behavior Inventory (PDDBI; 2005) is a reliable and valid caregiver questionnaire assessment tool which measures both problem behaviors and social communication abilities.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e It provides age-standardized T-scores based on a large sample of well diagnosed children with ASD. It is divided into two dimensions: Approach-Withdrawal (AWP) problems and Receptive Expressive Social Communication Abilities (REXSCA) along with an overall Autism Composite score. For the AWP dimension and the Autism Composite, higher scores indicate greater severity. For the REXSCA dimension, higher scores indicate greater competence.\u003c/p\u003e\u003cp\u003eThe Vineland Adaptive Behavior Social Subscale III (VABS) is a widely used standardized, well-validated assessment tool for children with developmental delays that measures functional abilities. It is a valid measures of social impairments in children with ASD.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e The VABS relies on an informant (caretaker) to complete. Higher scores represent better development.\u003c/p\u003e\u003cp\u003e Gross motor development was assessed with the PDDBI in a subset of parents by asking the parents to note at which age the child sat and walked without support as these are milestones that are typically remembered well by parents.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eStatistical Analysis\u003c/span\u003e\u003c/p\u003e\u003cp\u003eAnalyses were performed using PASW Statistics version 28.0.0.0 (IBM SPSS Statistics, Armonk, NY). Graphs were produced using Excel version 14.0 (Microsoft Corp, Redmond, WA). An alpha of 5% was used as a cutoff for significance.\u003c/p\u003e\u003cp\u003eIn general, mixed-model regression models with random effects of family (the shared variance among the parents and sibling) to control for repeated effects of family level mean and variance were used to examine the effects of dichotomous variables such as multiplex family, multiple birth, sex and continuous variables such as offspring, maternal and paternal age. Two-way Interactions between variables were included in the model. The final model was simplified to only significant variables and variables that were dependent on significant interactions. Cohen\u0026rsquo;s \u003cem\u003ed\u0026rsquo;\u003c/em\u003e was calculated to represent effect size.. Effects from model coefficients are provided for dicrotous variables with standard errors.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Participant Characteristics\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e82 children diagnosed with ASD consisted of 65 boys and 17 girls ranging from 1.6 to 15 years of age [Mean (SD) 5.18 (2.6)]. 53 unaffected siblings (22 brothers; 29 sisters, and one half-brother and one half-sister) were tested [Mean (SD) 6.7 (4.2)], as were 70 mothers [Mean (SD) 31.5 years (5.1 y)] and 65 fathers [Mean (SD) 33.9y (5.6y)]. Unrelated, non-ASD controls comprising 24 boys and 28 girls, aged 3 to 17 years [Mean (SD) 11.8y (4.0y)] were recruited from a local pediatric practice. There were 17 (22%) multiplex families and 8 (11%) with multiple births.\u003c/p\u003e \u003cp\u003e \u003cem\u003e3.2. Pregnancy and ASD Family Type Effects on FRAA Titers in Children with ASD and their Siblings\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe effect of pregnancy factors, ASD family type (simplex vs multiplex) and parental age on FRAA titers in the offspring (ASD and siblings combined) was analyzed. Controls were not included in these analyses.\u003c/p\u003e \u003cp\u003eFor probands and their siblings, multiplex families were found to have significantly higher blocking titers by 4.86 (1.13) pmol/ml as compared to simplex families with a large effect size [F(1,75)\u0026thinsp;=\u0026thinsp;18.50, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Cohen\u0026rsquo;s \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=1.1] with no significant effect found for sex, multiple birth pregnancy or age at antibody measurement. Older maternal [F(1,75)\u0026thinsp;=\u0026thinsp;9.18, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Cohen\u0026rsquo;s \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=0.61] and parental age [F(1,75)\u0026thinsp;=\u0026thinsp;11.06, p\u0026thinsp;=\u0026thinsp;0.001; Cohen\u0026rsquo;s \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=0.67] at birth were related to higher blocking titers, both with medium effect sizes (See Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eFor probands and their siblings, multiple birth pregnancy was found to have significantly higher binding titers as compared to single birth pregnancies with a medium-to-large effect size [F(1,109)\u0026thinsp;=\u0026thinsp;3.88, p\u0026thinsp;=\u0026thinsp;0.05, Cohen\u0026rsquo;s \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=0.66] and binding titers decreased with age at which the binding antibody was measured with a medium effect size [F(1,109)\u0026thinsp;=\u0026thinsp;8.546, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Cohen\u0026rsquo;s \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=0.58] with no significant effect found for sex or multiplex families (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Transgenerational Changes in FRAA Titers\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOffspring (ASD and Sibling) blocking titers were significantly related to maternal blocking titers [F(1,105)\u0026thinsp;=\u0026thinsp;33.514, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Cohen\u0026rsquo;s \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=1.16] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) but binding offspring FRAA titers were not found to be related to parental FRAA titers.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe relationship between blocking autoantibodies in the offsprings and mothers was clearly driven by a group in which titers were high for both the offspring and mother (point in the oval in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and another group where the titers were only elevated in the offspring or the mother. As the mean titers were higher for the multiplex families, it was hypothesized that these double positives (offspring, mother) were driven by multiplex families. For the blocking FRAA, 53% of the multiplex families demonstrated positivity in both offspring and mother whereas this was only seen in 12% of the simplex families. This difference was highly significant with a large effect size [χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;24.47, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Cohen\u0026rsquo;s \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=1.14].\u003c/p\u003e \u003cp\u003eComparing offspring to parental blocking titers, multiplex families (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) manifest significantly higher titer than simplex families (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) by 3.73 (0.73) pmol/ml with a large effect size [F(1,72)\u0026thinsp;=\u0026thinsp;26.50, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Cohen\u0026rsquo;s \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=0.82] and the offspring blocking titers were significantly greater than maternal [F(1,200)\u0026thinsp;=\u0026thinsp;5.91, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Cohen\u0026rsquo;s \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=0.39] and paternal [F(1,200)\u0026thinsp;=\u0026thinsp;6.74, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Cohen\u0026rsquo;s \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=0.65] blocking titers by 1.06 (0.44) and 1.78 (0.44) with a small and medium effect size, respectively. Binding titers were not significantly different across family members or between simplex or multiplex families (See Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Effect of ASD on Family Titers\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo determine the effect of ASD over and above the effect of being an offspring, a mixed-model with separate factors for offspring and ASD child was used to examine FRAA titers across generations.\u003c/p\u003e \u003cp\u003eBlocking titers were significantly higher by 1.62 (0.50) [F(1,215)\u0026thinsp;=\u0026thinsp;10.60, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Cohen \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=0.46] in offspring as compared to parents and being affected by ASD increased the titer by 3.92 (1.08) [F(1,215)\u0026thinsp;=\u0026thinsp;5.92, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Cohen \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=0.40], both with a small-to-medium effect size. Multiplex families demonstrated titers higher by 3.44 (0.75) [F(1,74)\u0026thinsp;=\u0026thinsp;21.09, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Cohen\u0026rsquo;s \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=0.74] with a medium-to-large effect size. Multiple birth pregnancy interacted with the affected individual such that a child with ASD demonstrated a significantly higher titer if they were born from a multiple birth pregnancy [F(1,211)\u0026thinsp;=\u0026thinsp;16.95, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Cohen\u0026rsquo;s \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=0.67] with a medium effect size.\u003c/p\u003e \u003cp\u003eBinding titers were significantly higher by 0.08 (0.04) [F(1,208)\u0026thinsp;=\u0026thinsp;4.24, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Cohen\u0026rsquo;s \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=0.34] in offspring as compared to parents, with a small effect size, but being affected by ASD did not significantly change the FRAA titer.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Familial FRAA Titers Compared to Controls\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eParticipant groups (proband, sibling, parent, controls) differed for blocking titers [F(3,272)\u0026thinsp;=\u0026thinsp;10.51, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Cohen\u0026rsquo;s \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=0.38] with a small-to-medium effect size and binding titers [F(3,278)\u0026thinsp;=\u0026thinsp;3.93, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Cohen\u0026rsquo;s \u003cem\u003ed\u0026rsquo;\u003c/em\u003e=0.23] with a small effect size (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Post-hoc comparisons using 2-sided Dunnett t found that blocking titers were significantly higher in the families of children with ASD as compared to typically developing children. Binding titers were significantly higher in the children with ASD and their siblings as compared to typically developing children.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Behavior and Development\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe participants with ASD were assessed using several instruments to determine their development and ASD symptoms. A mixed model controlling for family unit was used to determine whether the presence of FRAAs (positive vs negative) in the mom or dad was associated with development and/or ASD symptoms. The presence of blocking and binding FRAAs in the child was included in the model to control for any effect of FRAAs in the child. The results are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eA positive FRAA status for the father was related to poorer development and greater ASD symptoms in the child, \u003cem\u003ewith medium to large effect sizes\u003c/em\u003e, as described below. Several of the Griffth\u0026rsquo;s Mental Development Scales, which were performed on the subset of children within the range of the test (\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;8 years of age), were lower when the father was FRAA positive. This included total developmental score (\u003cem\u003eCohen\u0026rsquo;s\u003c/em\u003e d\u0026rsquo;\u003cem\u003e=\u003c/em\u003e0.66), motor development (\u003cem\u003eCohen\u0026rsquo;s\u003c/em\u003e d\u0026rsquo;\u003cem\u003e=\u003c/em\u003e0.69), speech and language development (\u003cem\u003eCohen\u0026rsquo;s\u003c/em\u003e d\u0026rsquo;\u003cem\u003e=\u003c/em\u003e0.85), coordination (\u003cem\u003eCohen\u0026rsquo;s\u003c/em\u003e d\u0026rsquo;\u003cem\u003e=\u003c/em\u003e0.66) and practical reasoning (\u003cem\u003eCohen\u0026rsquo;s\u003c/em\u003e d\u0026rsquo;\u003cem\u003e=\u003c/em\u003e0.64), all with medium effect sizes. The VABS demonstrated poorer development in communication (\u003cem\u003eCohen\u0026rsquo;s\u003c/em\u003e d\u0026rsquo;\u003cem\u003e=\u003c/em\u003e0.47), socialization (\u003cem\u003eCohen\u0026rsquo;s\u003c/em\u003e d\u0026rsquo;\u003cem\u003e=\u003c/em\u003e0.73), daily living skills (\u003cem\u003eCohen\u0026rsquo;s\u003c/em\u003e d\u0026rsquo;\u003cem\u003e=\u003c/em\u003e0.73) and motor skills (\u003cem\u003eCohen\u0026rsquo;s\u003c/em\u003e d\u0026rsquo;\u003cem\u003e=\u003c/em\u003e0.48) if the father was FRAA positive, with medium-to-large effect sizes.\u003c/p\u003e \u003cp\u003eASD symptoms were assessed using the PDDBI, which demonstrated more severe ASD symptoms if the father was FRAA positive, \u003cem\u003especifically, a positive FRAA in the father was related to worse overall PDDBI score (Cohen\u0026rsquo;s\u003c/em\u003e d\u0026rsquo;\u003cem\u003e=0.46)\u003c/em\u003e, Expressive Language \u003cem\u003e(Cohen\u0026rsquo;s\u003c/em\u003e d\u0026rsquo;\u003cem\u003e=0.46)\u003c/em\u003e, Learning, Memory, and Receptive Language \u003cem\u003e(Cohen\u0026rsquo;s\u003c/em\u003e d\u0026rsquo;\u003cem\u003e=0.45)\u003c/em\u003e, Expressive Social \u003cem\u003eCommunication\u003c/em\u003e Abilities Composite \u003cem\u003e(Cohen\u0026rsquo;s\u003c/em\u003e d\u0026rsquo;\u003cem\u003e=0.40) and\u003c/em\u003e Receptive/Expressive Social \u003cem\u003eCommunication\u003c/em\u003e Composite \u003cem\u003e(Cohen\u0026rsquo;s\u003c/em\u003e d\u0026rsquo;\u003cem\u003e=0.46). Positive FRAA in the mother was associated with worse\u003c/em\u003e Social Pragmatic Problems \u003cem\u003e(Cohen\u003c/em\u003e d\u0026rsquo;\u003cem\u003e=0.61)\u003c/em\u003e, all with medium effect sizes.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRelationship between FRAA status in mom and dad and cognitive and behavior of the ASD offspring. Means and standard errors of the offspring\u0026rsquo;s cognitive and behavioral symptoms are given for positive and negative FRAA antibody status of the parents. Statistically significant values are bolded. The number of participants for each evaluation are given in the instrument name header.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMom\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eDad\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGriffth\u0026rsquo;s Mental Development Scales\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eNeg\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003ePos\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eNeg\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003ePos\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Scale (GQ)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e69.1 (5.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e75.4 (3.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e85.2 (8.5)*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e70.4 (3.4)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocomotor Development\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83.9 (3.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86.6 (2.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e93.5 (6.8)*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e83.4 (1.9)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePersonal Social Development\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64.6 (5.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65.3 (3.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74.0 (8.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63.0 (3.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHearing and speech\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54.8 (6.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.3 (6.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e80.8 (15.0)**\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e51.9 (27.7)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHand and Eye Coordination\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68.3 (5.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76.9 (4.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e85.1 (7.8)*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e71.2 (3.8)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePerformance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80.9 (6.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e88.2 (4.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.5 (7.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e83.9 (4.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePractical reasoning\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63.2 (6.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69.0 (5.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e81.2 (11.0)*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e63.3 (4.2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePDD Behavior Inventory\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eNeg\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003ePos\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eNeg\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003ePos\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAutism Composite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49.1 (1.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51.0 (1.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e45.9 (1.8)*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e51.3 (1.2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSensory/Perceptual Approach Behaviors\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.4 (1.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.7 (1.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e46.7 (1.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50.4 (1.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRitualisms/Resistance to Change\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48.3 (1.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.3 (1.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.9 (1.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e49.6 (1.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSocial Pragmatic Problems\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e47.0 (1.6)**\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e53.1 (1.1)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48.9 (1.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51.1 (1.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSemantic/Pragmatic Problems\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.5 (2.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.8 (1.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49.7 (2.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e49.2 (1.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArousal Regulation Problems\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.3 (1.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.3 (1.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49.9 (1.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51.1 (1.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecific Fears\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48.8 (1.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.7 (1.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47.7 (2.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50.7 (1.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAggressiveness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48.8 (1.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.9 (1.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48.5 (2.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e49.5 (1.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRepetitive, Ritualistic, \u0026amp; Pragmatic Prob Comp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50.7 (1.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.5 (1.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48.4 (1.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50.5 (1.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eApproach/Withdrawal Problems Composite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50.6 (1.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51.3 (1.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49.4 (2.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50.7 (1.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSocial Approach Behaviors\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50.0 (1.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.4 (1.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.7 (2.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e47.3 (1.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExpressive Language\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50.1 (1.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49.1 (1.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e53.4 (1.9)*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e47.8 (1.1)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLearning, Memory, and Receptive Language\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50.1 (1.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49.6 (1.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e53.0 (2.0)*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e48.2 (1.0)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExpressive Social Comm Abilities Composite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50.0 (1.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49.6 (1.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e52.7 (1.9)*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e48.0 (1.2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReceptive/Expressive Social Comm Composite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50.0 (1.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49.7 (1.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e52.7 (1.9)*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e48.0 (1.2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVineland Adaptive Behavior Scales\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eNeg\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003ePos\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eNeg\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003ePos\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCommunication\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e71.0 (4.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e63.7 (3.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e77.5 (5.8)*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e63.4 (3.3)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDaily Living Skills\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.0 (3.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58.0 (2.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e69.5 (4.7)**\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e56.5 (1.7)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSocialization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.1 (3.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60.3 (1.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e72.1 (4.5)**\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e59.3 (1.8)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMotor Skills\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70.2 (4.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70.8 (2.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e79.6 (5.2)*\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e67.7 (2.4)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDevelopmental Gross Motor Milestones [N\u0026thinsp;=\u0026thinsp;36]\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eNeg\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003ePos\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eNeg\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003ePos\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge when sat without support\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.7 (0.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.9 (0.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.5 (0.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.1 (0.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge when walking without support\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.4 (3.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.0 (0.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.8 (0.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.4 (0.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e*p\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.01; ***p\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.001\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThis paper examines the transgenerational heritability of folate receptor alpha autoantibodies (FRAAs) particularly with respect to autism spectrum disorder (ASD), one of the major disorders it has become associated with. Many interesting aspects of this study include the fact that the transgenerational heritability appears to be slightly different for the blocking and binding FRAAs. Of note, two previously unrecognized factors that affect the FRAA titers of the offspring, multiplex ASD and multiple birth, were found. Interestingly, the FRAA status of the father appears to have a relationship with the outcome of the ASD child.\u003c/p\u003e \u003cp\u003eAlthough families with multiple births and multiplex families represented a minority of the sample, it was found that these families demonstrated a distinct relationship to FRAA titers. Families with multiple birth pregnancies manifested higher FRAA binding titers. Both of these factors are risk factors for ASD. Specifically multiple birth pregnancy is a risk factor for the offspring developing ASD,\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e possibly because of perinatal and neonatal complications which are also associated with ASD.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e. Multiplex families were found to have an overall higher blocking titer as compared to simplex families, and the risk of having another child with ASD is higher in multiplex families,\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e suggesting that FRAAs may be involved in the biological mechanisms which increase ASD risk especially since multiplex families are less likely to have an identifiable genetic cause for ASD, suggesting other non-genetic factors, such as the FRAA, may be playing a role in these families.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e The affected ASD child in the multiplex family demonstrated a significantly higher titers than the other typically developing family members, thus being a factor which distinguishes the ASD child from the typically developing family members. Interestingly, multiplex families having higher FRAA titers than simplex families is consistent with several studies which demonstrate that the members of multiplex families have subtle cognitive deficits. For example, non-ASD siblings in multiplex families, where FRAA titers were higher, tend to have lower cognitive function and adaptive behavior as compared to non-ASD siblings from simplex families\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e and family members in multiplex families exhibit more ASD characteristics than family members in simplex families.\u003csup\u003e\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e Although the number of multiplex families was lower than the number of simplex families, the proportion of the samples of multiplex families was similar to other studies comparing multiplex and simplex families.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e Nevertheless, these are compelling findings that need to be confirmed in larger samples.\u003c/p\u003e \u003cp\u003eAnother compelling finding is that the blocking titers increases with parental age, particularly the father. Such a finding is consistent with the association of advanced parental age as a risk factor for ASD. Advanced maternal age is well-known to be associated with several reproductive issues such as infertility and fetal malformations. FRAA have been proposed to be associated with pregnancy complication given the high concentration of folate receptors at the maternal-fetal interface\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and the association between FRAAs and neural-tube defects,\u003csup\u003e\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e preterm birth\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e and subfertility.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e The association of ASD with advanced paternal age is somewhat unique for ASD. Studies suggest that this is due to decreased sperm quality and testicular function. Although the folate receptor alpha is not known to be located on the testes, one major role of the epididymis and vas deferens is to secrete a folate binding protein which adhere to spermatozoa.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e Potential cross reaction between FRAA and the folate binding protein could effect on sperm quality.\u003c/p\u003e \u003cp\u003eBlocking but not binding titers were correlated between mother and offspring. Closer examination of this relationship demonstrated that this effect was driven by cases in which both mother and offspring had high FRAAs. Interestingly these cases were overrepresented by multiplex families, suggesting that multiplex families are more likely to have maternal-offspring heritability of FRAAs, whereas simplex families tend to have positive blocking FRAAs in either mother or offspring but not both.\u003c/p\u003e \u003cp\u003eInterestingly, both binding and blocking titers increased across generations. This phenomenon of increasing severity of a disease across generations has been termed anticipation in genetics where it is most commonly used to describe an increase in the severity of trinucleotide repeat disorders across generations. Similar to genetic anticipation in which the gene abnormality can become worse until it reaches a threshold to cause disease, FRAAs appear to increase in severity (titer concentration) across generations. It may very well be that the FRAA requires a specific level to cause disease, so ASD is not manifested until the titers reach a certain level. However, it is more likely that the ASD symptoms are less severe in family members with FRAAs such that they do not severely interfere with life. The mechanism by which titers increase across generations is not known and will require further research.\u003c/p\u003e \u003cp\u003e \u003cem\u003eWe found several developmental characteristics related to paternal FRAA status, including measure of mental development, ASD behavior and adaptive functioning.\u003c/em\u003e As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the PDDBI data largely replicated the Griffiths and Vineland results and indicated that the primary effect was on the domains and composite scores assessing social communication competence (a core feature of ASD), rather than on the domains and composite scores assessing pragmatic problems, repetitive/ritualistic behaviors and arousal/anxiety problems. In addition, the PDDBI also emphasizes the language defect associated with FRAA status.\u003c/p\u003e \u003cp\u003eAs previously discussed, it is not clear how the paternal FRAA status affects the development of offspring, but there are several potential mechanisms. The FRα is secreated into the semen where it interacts with spermatozoa.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e FRAAs binding of the FRα could disrupt this interaction. FRAAs in the semen could bind to JUNO (a pseudo-folate receptor known as FOLR4) on the oocyte during fertalization.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e A glycolipid linked high-affinity folate binding protein has been found to be secreated from the epithelium of the epididymal and vas deferens where it associates with prostasome-like vesicles adherent to spermatozoa.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e It has been hypotheized that this folate binding protein has a bacteriostatic function or facilitates folate transport into the spermatozoa.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e FRAAs binding to this folate binding protein could possiblity disrupt spermatozoa health. Thus, it is not unreasonable to hypothesize that FRAAs could interfere with the development of high-quality sperm.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis study examined the relationship between the FRAA in families to help better define its role in reproductive health and neurodevelopmental outcomes of the offspring. Several novel findings were found, including a relationship between FRAA titers and both multiplex families and multiple birth pregnancies, two factors that are known to increase the risk of ASD in the offspring. The blocking FRAA appears to be highly correlated between mother and offspring, thereby supporting the notion of a relationship across generations. Finally, the notion of anticipation of the FRAA across generations is compelling and may suggest an increased risk of ASD in parents that have high titers. This study is limited, mostly by its sample size. Larger studies will be needed to verify and further this research.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eInstitutional Review Board Statement\u003c/h2\u003e \u003cp\u003e The study was conducted in accordance with the Declaration of Helsinki. The protocol was approved by the Institutional Review Board at Institute for Basic Research in Developmental Disabilities (Staten Island, New York). All dates were transformed to age in days at the visit and per-sonal health identifiers were removed to deidentify the data for further analysis. Thus, the final dataset was analyzed under 45 CFR 46 exemption 4.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eInformed Consent\u003c/strong\u003e \u003cp\u003e\u003cb\u003eStatement\u003c/b\u003e: Parents of participants provided written informed consent. Data was then deidentified for analysis.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eEVQ is an inventor on a US patent for the detection of FRAAs issued to the Research Foundation of the State University of New York, USA. The remainder of the authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis work was supported by funding from the New York State OPWDD (ILC), research grant #8202 from Autism Speaks (EVQ), research grant RFGA2022-010-31 from the Arizona Department of Health Services and the XEL Foundation (Pittsburgh, PA). None of the sponsors were involved with the design or conduct of the study, collection, management, analysis or interpretation of the data; or preparation, approval of the manuscript or decision to submit the manuscript for publication.\u003c/p\u003e\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e \u003cp\u003eConceptualization, EVQ, IIC, REF; methodology, EVQ, IIC, REF, JMS; formal analysis, REF; investigation, EVQ, IIC, JMS, WTB, CM, EM, MF, ECJ, MTV; writing\u0026mdash;original draft preparation, REF, EVQ; writing\u0026mdash;review and editing, all authors; visualization, REF; funding acquisition, REF, EVQ. All authors have read and agreed to the published version of the manuscript.\u0026rdquo;\u003c/p\u003e\u003ch2\u003eAcknowledgments:\u003c/h2\u003e \u003cp\u003eThe authors would like to thank all of the staff which were involved in making this study possible and the participants and their families who gave their time.\u003c/p\u003e\u003ch2\u003eData Availability Statement:\u003c/h2\u003e \u003cp\u003eData is available upon request.\u003c/p\u003e "},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAssociation, A.P. \u003cem\u003eDiagnostic and Statistical Manual of Mental Disorders (DSM-5\u0026reg;)\u003c/em\u003e, (American Psychiatric Association Publishing, Washington, DC, 2013).\u003c/li\u003e\n\u003cli\u003eShaw, K.A.\u003cem\u003e, et al.\u003c/em\u003e Prevalence and Early Identification of Autism Spectrum Disorder Among Children Aged 4 and 8 Years - Autism and Developmental Disabilities Monitoring Network, 16 Sites, United States, 2022. \u003cem\u003eMMWR Surveill Summ\u003c/em\u003e \u003cstrong\u003e74\u003c/strong\u003e, 1-22 (2025).\u003c/li\u003e\n\u003cli\u003eMaenner, M.J.\u003cem\u003e, et al.\u003c/em\u003e Prevalence and Characteristics of Autism Spectrum Disorder Among Children Aged 8 Years - Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2020. \u003cem\u003eMMWR Surveill Summ\u003c/em\u003e \u003cstrong\u003e72\u003c/strong\u003e, 1-14 (2023).\u003c/li\u003e\n\u003cli\u003eSandin, S.\u003cem\u003e, et al.\u003c/em\u003e The familial risk of autism. \u003cem\u003eJama\u003c/em\u003e \u003cstrong\u003e311\u003c/strong\u003e, 1770-1777 (2014).\u003c/li\u003e\n\u003cli\u003eSchaefer, G.B., Mendelsohn, N.J., Professional, P. \u0026amp; Guidelines, C. Clinical genetics evaluation in identifying the etiology of autism spectrum disorders: 2013 guideline revisions. \u003cem\u003eGenet Med\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 399-407 (2013).\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Roak, B.J.\u003cem\u003e, et al.\u003c/em\u003e Recurrent de novo mutations implicate novel genes underlying simplex autism risk. \u003cem\u003eNature communications\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 5595 (2014).\u003c/li\u003e\n\u003cli\u003eWang, L.\u003cem\u003e, et al.\u003c/em\u003e Functional relationships between recessive inherited genes and genes with de novo variants in autism spectrum disorder. \u003cem\u003eMolecular autism\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 75 (2020).\u003c/li\u003e\n\u003cli\u003eTammimies, K.\u003cem\u003e, et al.\u003c/em\u003e Molecular Diagnostic Yield of Chromosomal Microarray Analysis and Whole-Exome Sequencing in Children With Autism Spectrum Disorder. \u003cem\u003eJama\u003c/em\u003e \u003cstrong\u003e314\u003c/strong\u003e, 895-903 (2015).\u003c/li\u003e\n\u003cli\u003eYuen, R.K.\u003cem\u003e, et al.\u003c/em\u003e Whole-genome sequencing of quartet families with autism spectrum disorder. \u003cem\u003eNature medicine\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 185-191 (2015).\u003c/li\u003e\n\u003cli\u003eBar, O., Vahey, E., Mintz, M., Frye, R.E. \u0026amp; Boles, R.G. Reanalysis of Trio Whole-Genome Sequencing Data Doubles the Yield in Autism Spectrum Disorder: De Novo Variants Present in Half. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e(2024).\u003c/li\u003e\n\u003cli\u003eFrye, R.E., McCarty, P.J., Werner, B.A., Rose, S. \u0026amp; Scheck, A.C. Bioenergetic signatures of neurodevelopmental regression. \u003cem\u003eFront Physiol\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 1306038 (2024).\u003c/li\u003e\n\u003cli\u003eFrye, R.E.\u003cem\u003e, et al.\u003c/em\u003e Biomarkers of mitochondrial dysfunction in autism spectrum disorder: A systematic review and meta-analysis. \u003cem\u003eNeurobiol Dis\u003c/em\u003e \u003cstrong\u003e197\u003c/strong\u003e, 106520 (2024).\u003c/li\u003e\n\u003cli\u003eHollowood, K.\u003cem\u003e, et al.\u003c/em\u003e Maternal metabolic profile predicts high or low risk of an autism pregnancy outcome. \u003cem\u003eRes Autism Spectr Disord\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e, 72-82 (2018).\u003c/li\u003e\n\u003cli\u003eZhu, Y.\u003cem\u003e, et al.\u003c/em\u003e Expression Changes in Epigenetic Gene Pathways Associated With One-Carbon Nutritional Metabolites in Maternal Blood From Pregnancies Resulting in Autism and Non-Typical Neurodevelopment. \u003cem\u003eAutism Res\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 11-28 (2021).\u003c/li\u003e\n\u003cli\u003eGardner, R.M., Brynge, M., Sj\u0026ouml;qvist, H., Dalman, C. \u0026amp; Karlsson, H. Maternal immune activation and autism in the offspring-what is the evidence for causation? \u003cem\u003eBiol Psychiatry\u003c/em\u003e (2024).\u003c/li\u003e\n\u003cli\u003eHallmayer, J.\u003cem\u003e, et al.\u003c/em\u003e Genetic heritability and shared environmental factors among twin pairs with autism. \u003cem\u003eArchives of general psychiatry\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 1095-1102 (2011).\u003c/li\u003e\n\u003cli\u003eFrye, R.E.\u003cem\u003e, et al.\u003c/em\u003e Physiological mediators of prenatal environmental influences in autism spectrum disorder. \u003cem\u003eBioessays\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, e2000307 (2021).\u003c/li\u003e\n\u003cli\u003eFrye, R.E.\u003cem\u003e, et al.\u003c/em\u003e Mitochondria May Mediate Prenatal Environmental Influences in Autism Spectrum Disorder. \u003cem\u003eJ Pers Med\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e(2021).\u003c/li\u003e\n\u003cli\u003eRossignol, D.A., Genuis, S.J. \u0026amp; Frye, R.E. Environmental toxicants and autism spectrum disorders: a systematic review. \u003cem\u003eTranslational psychiatry\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, e360 (2014).\u003c/li\u003e\n\u003cli\u003eHughes, H.K., Moreno, R.J. \u0026amp; Ashwood, P. Innate immune dysfunction and neuroinflammation in autism spectrum disorder (ASD). \u003cem\u003eBrain Behav Immun\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 245-254 (2023).\u003c/li\u003e\n\u003cli\u003eHowsmon, D.P., Kruger, U., Melnyk, S., James, S.J. \u0026amp; Hahn, J. Classification and adaptive behavior prediction of children with autism spectrum disorder based upon multivariate data analysis of markers of oxidative stress and DNA methylation. \u003cem\u003ePLoS Comput Biol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, e1005385 (2017).\u003c/li\u003e\n\u003cli\u003eRose, S.\u003cem\u003e, et al.\u003c/em\u003e Clinical and Molecular Characteristics of Mitochondrial Dysfunction in Autism Spectrum Disorder. \u003cem\u003eMol Diagn Ther\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 571-593 (2018).\u003c/li\u003e\n\u003cli\u003eRamaekers, V.T.\u003cem\u003e, et al.\u003c/em\u003e Autoantibodies to folate receptors in the cerebral folate deficiency syndrome. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e352\u003c/strong\u003e, 1985-1991 (2005).\u003c/li\u003e\n\u003cli\u003eQuadros, E.V.\u003cem\u003e, et al.\u003c/em\u003e Folate receptor autoantibodies are prevalent in children diagnosed with autism spectrum disorder, their normal siblings and parents. \u003cem\u003eAutism Res\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 707-712 (2018).\u003c/li\u003e\n\u003cli\u003eRossignol, D.A. \u0026amp; Frye, R.E. Cerebral Folate Deficiency, Folate Receptor Alpha Autoantibodies and Leucovorin (Folinic Acid) Treatment in Autism Spectrum Disorders: A Systematic Review and Meta-Analysis. \u003cem\u003eJ Pers Med\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e(2021).\u003c/li\u003e\n\u003cli\u003eSebat, J.\u003cem\u003e, et al.\u003c/em\u003e Strong association of de novo copy number mutations with autism. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e316\u003c/strong\u003e, 445-449 (2007).\u003c/li\u003e\n\u003cli\u003eSequeira, J.M., Ramaekers, V.T. \u0026amp; Quadros, E.V. The diagnostic utility of folate receptor autoantibodies in blood. \u003cem\u003eClin Chem Lab Med\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e, 545-554 (2013).\u003c/li\u003e\n\u003cli\u003eGriffiths, R. \u003cem\u003eThe abilities of young children: A comprehensive system of mental measurement for the first eight years of life (Revised ed.)\u003c/em\u003e, (A.R.I.C.D. The Test Agency Limited., Bucks, UK, 1984).\u003c/li\u003e\n\u003cli\u003eCohen, I.L. \u0026amp; Sudhalter, V. \u003cem\u003eThe PDD Behavior Inventory\u003c/em\u003e, (Psychological Assessment Resources, Inc., Lutz, FL, 2005).\u003c/li\u003e\n\u003cli\u003ePine, E., Luby, J., Abbacchi, A. \u0026amp; Constantino, J.N. Quantitative assessment of autistic symptomatology in preschoolers. \u003cem\u003eAutism : the international journal of research and practice\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 344-352 (2006).\u003c/li\u003e\n\u003cli\u003eGardener, H., Spiegelman, D. \u0026amp; Buka, S.L. Perinatal and neonatal risk factors for autism: a comprehensive meta-analysis. \u003cem\u003ePediatrics\u003c/em\u003e \u003cstrong\u003e128\u003c/strong\u003e, 344-355 (2011).\u003c/li\u003e\n\u003cli\u003eKarmel, B.Z.\u003cem\u003e, et al.\u003c/em\u003e Early medical and behavioral characteristics of NICU infants later classified with ASD. \u003cem\u003ePediatrics\u003c/em\u003e \u003cstrong\u003e126\u003c/strong\u003e, 457-467 (2010).\u003c/li\u003e\n\u003cli\u003eCohen, I.L.\u003cem\u003e, et al.\u003c/em\u003e Neonatal brainstem function and 4-month arousal-modulated attention are jointly associated with autism. \u003cem\u003eAutism Res\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 11-22 (2013).\u003c/li\u003e\n\u003cli\u003eOzonoff, S.\u003cem\u003e, et al.\u003c/em\u003e Familial Recurrence of Autism: Updates From the Baby Siblings Research Consortium. \u003cem\u003ePediatrics\u003c/em\u003e \u003cstrong\u003e154\u003c/strong\u003e(2024).\u003c/li\u003e\n\u003cli\u003eMcDonald, N.M.\u003cem\u003e, et al.\u003c/em\u003e Developmental Trajectories of Infants With Multiplex Family Risk for Autism: A Baby Siblings Research Consortium Study. \u003cem\u003eJAMA Neurol\u003c/em\u003e \u003cstrong\u003e77\u003c/strong\u003e, 73-81 (2020).\u003c/li\u003e\n\u003cli\u003eVirkud, Y.V., Todd, R.D., Abbacchi, A.M., Zhang, Y. \u0026amp; Constantino, J.N. Familial aggregation of quantitative autistic traits in multiplex versus simplex autism. \u003cem\u003eAm J Med Genet B Neuropsychiatr Genet\u003c/em\u003e \u003cstrong\u003e150b\u003c/strong\u003e, 328-334 (2009).\u003c/li\u003e\n\u003cli\u003eGerdts, J.A., Bernier, R., Dawson, G. \u0026amp; Estes, A. The broader autism phenotype in simplex and multiplex families. \u003cem\u003eJ Autism Dev Disord\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 1597-1605 (2013).\u003c/li\u003e\n\u003cli\u003eOerlemans, A.M.\u003cem\u003e, et al.\u003c/em\u003e Recognition of facial emotion and affective prosody in children with ASD (+ADHD) and their unaffected siblings. \u003cem\u003eEur Child Adolesc Psychiatry\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 257-271 (2014).\u003c/li\u003e\n\u003cli\u003eOerlemans, A.M., Hartman, C.A., Franke, B., Buitelaar, J.K. \u0026amp; Rommelse, N.N. Does the cognitive architecture of simplex and multiplex ASD families differ? \u003cem\u003eJ Autism Dev Disord\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, 489-501 (2016).\u003c/li\u003e\n\u003cli\u003eQin, X.Y., Ha, S.Y., Chen, L., Zhang, T. \u0026amp; Li, M.Q. Recent Advances in Folates and Autoantibodies against Folate Receptors in Early Pregnancy and Miscarriage. \u003cem\u003eNutrients\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e(2023).\u003c/li\u003e\n\u003cli\u003eRothenberg, S.P.\u003cem\u003e, et al.\u003c/em\u003e Autoantibodies against folate receptors in women with a pregnancy complicated by a neural-tube defect. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e350\u003c/strong\u003e, 134-142 (2004).\u003c/li\u003e\n\u003cli\u003eCabrera, R.M.\u003cem\u003e, et al.\u003c/em\u003e Autoantibodies to folate receptor during pregnancy and neural tube defect risk. \u003cem\u003eJ Reprod Immunol\u003c/em\u003e \u003cstrong\u003e79\u003c/strong\u003e, 85-92 (2008).\u003c/li\u003e\n\u003cli\u003eBoyles, A.L.\u003cem\u003e, et al.\u003c/em\u003e Association between inhibited binding of folic acid to folate receptor alpha in maternal serum and folate-related birth defects in Norway. \u003cem\u003eHum Reprod\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 2232-2238 (2011).\u003c/li\u003e\n\u003cli\u003eVo, H.D., Sequeira, J.M., Quadros, E.V., Schwarz, S.M. \u0026amp; Perenyi, A.R. The role of folate receptor autoantibodies in preterm birth. \u003cem\u003eNutrition\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 1224-1227 (2015).\u003c/li\u003e\n\u003cli\u003eBerrocal-Zaragoza, M.I.\u003cem\u003e, et al.\u003c/em\u003e Association between blocking folate receptor autoantibodies and subfertility. \u003cem\u003eFertil Steril\u003c/em\u003e \u003cstrong\u003e91\u003c/strong\u003e, 1518-1521 (2009).\u003c/li\u003e\n\u003cli\u003eMalm, J.\u003cem\u003e, et al.\u003c/em\u003e A minor fraction of a high-affinity folate binding protein from the epididymis is associated with membranous vesicles and spermatozoa in human semen. \u003cem\u003eInt J Androl\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 267-274 (2005).\u003c/li\u003e\n\u003cli\u003eHolm, J. \u0026amp; Hansen, S.I. Characterization of soluble folate receptors (folate binding proteins) in humans. Biological roles and clinical potentials in infection and malignancy. \u003cem\u003eBiochim Biophys Acta Proteins Proteom\u003c/em\u003e \u003cstrong\u003e1868\u003c/strong\u003e, 140466 (2020).\u003c/li\u003e\n\u003cli\u003eD\u0026iacute;az-Fuster, L., S\u0026aacute;ez-Espinosa, P., Moya, I., Peinado, I. \u0026amp; G\u0026oacute;mez-Torres, M.J. Updating the Role of JUNO and Factors Involved in Its Function during Fertilization. \u003cem\u003eCells Tissues Organs\u003c/em\u003e, 1-16 (2025).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"anticipation, autism spectrum disorder; cerebral folate deficiency, folate receptor alpha, folate receptor alpha autoantibodies; heritability","lastPublishedDoi":"10.21203/rs.3.rs-6755086/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6755086/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAutism Spectrum Disorder (ASD) affects an estimated prevalence of 1 in 36 children but the cause in most cases is unknown. Human and animal studies have linked ASD to Folate Receptor Alpha Autoantibodies (FRAAs). Our previous studies demonstrated that FRAAs are more common, on average, in families with children with ASD. This study reanalyzed data from this previous study which included 82 children diagnosed with ASD, 53 unaffected siblings, 70 mothers and 65 fathers and 52 typically developing controls who did not have a history of ASD in their family. This study investigates the association of FRAA titers with ASD risk factors and explores the relationship of FRAA titers across generations. Several known risk factors for ASD, including multiplex ASD families, multiple birth pregnancies and increased maternal and paternal age at birth were related to offspring FRAA titers. Multiplex ASD families demonstrated higher FRAA titers and a significant correlation between maternal and offspring blocking FRAA titers. FRAA titers increased across generations, although the increase in blocking FRAAs was only seen in multiplex families. The proband with ASD demonstrated higher blocking but not binding titers compared to their non-affected siblings. Paternal FRAA titers are associated with several measures of offspring behavior and cognitive development. This research highlights the potential transgenerational transmission of FRAAs and their role in ASD, demonstrating that heritable non-genetic factors may be important in the etiology of ASD and that FRAAs may demonstrate anticipation (worsening across generations), especially in multiplex families. Disruption of immune regulation and susceptibility to autoimmune disease may underly disruption of brain development and function in ASD.\u003c/p\u003e","manuscriptTitle":"Transgenerational Effects and Heritability of Folate Receptor Alpha Autoantibodies in Autism Spectrum Disorder","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-10 18:04:57","doi":"10.21203/rs.3.rs-6755086/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2cbe4e3c-6629-412c-83ad-4b88abd89d9c","owner":[],"postedDate":"June 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49454829,"name":"Health sciences/Diseases/Psychiatric disorders"},{"id":49454830,"name":"Biological sciences/Physiology"},{"id":49454831,"name":"Health sciences/Biomarkers/Predictive markers"}],"tags":[],"updatedAt":"2025-07-01T10:38:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-10 18:04:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6755086","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6755086","identity":"rs-6755086","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}