The Association Between Gestational Vitamin D Levels and Offspring Vitamin D Receptor Polymorphisms in Autism-Associated Traits

The Association Between Gestational Vitamin D Levels and Offspring Vitamin D Receptor Polymorphisms in Autism-Associated Traits | 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 The Association Between Gestational Vitamin D Levels and Offspring Vitamin D Receptor Polymorphisms in Autism-Associated Traits Fazlić Esma, Pasco Julie, Williams Lana, Hyde Natalie This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9216765/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract The relationship between gestational vitamin D status and offspring autism remains inconsistent, potentially due to genetic differences in vitamin D receptor (VDR) interactions. This exploratory study examined associations between maternal serum vitamin D concentrations, offspring VDR genotypes (BsmI, Fok1, ApaI, TaqI), and autism symptomatology using the Australian Scale for Autism Spectrum Conditions (ASASC) in 192 mother–child pairs at 11-years follow-up. No association was found between gestational vitamin D levels and total ASASC scores, nor between VDR genotype and total ASASC scores. However, secondary subscale analyses revealed that higher early vitamin D concentrations were associated with decreased fact-orienting scores in males (β = − 0.05, 95% CI − 0.08, − 0.01, p = 0.02). Additionally, the homozygous recessive TaqI genotype was associated with a 2.5-unit increase in fact-orienting scores compared to the homozygous dominant genotype (β = 2.52, 95% CI 0.36, 4.69, p = 0.02), though no interaction with gestational vitamin D was observed. While overall autism traits were unrelated to gestational vitamin D or VDR genotype, these exploratory findings suggest that specific autism-related traits may be influenced by maternal vitamin D levels and offspring genetic variation. This highlights the potential role of vitamin D gene-environment interactions in autism. Health sciences/Diseases Biological sciences/Genetics Health sciences/Medical research Biological sciences/Neuroscience Autism Vitamin D Polymorphisms Gestation Neurodevelopment 1. Introduction Vitamin D is a fat-soluble vitamin recognised for its role in musculoskeletal function, maintaining bone through calcium regulation ( 1 ). Vitamin D occurs in two forms: D2 and D3. Vitamin D2 is plant-derived, produced primarily by fungi and yeast ( 2 ). Vitamin D3 is synthesised in the skin by UV irradiation, further transported to the liver where both D2 and D3 are metabolised into the final circulating form, 25-hydroxyvitamin D (25(OH)D) ( 3 , 4 ). While critical in calcium metabolism, additional physiological processes are regulated by vitamin D, notably, brain function ( 1 ). Vitamin D function in the brain is associated with neuroprotective and neurodevelopmental processes, where lower concentrations are associated with elevated risk of dementia in adults ( 5 , 6 ). Vitamin D is evidenced in the modulation of neurodevelopmental processes, regulating several neurotropic and growth factors to influence neuronal growth, differentiation, maturation and survival ( 7 – 10 ). Alterations to such processes are implied in the pathogenesis of neurodevelopmental conditions, namely autism ( 11 ). Autism is a neurodevelopmental condition characterised by differences in social interaction and communication, combined with the presence of repetitive behaviours, activities, or interests ( 12 ). Potential associations with vitamin D are evident in several studies, where deficient maternal vitamin D status is associated with increased offspring autism risk ( 13 – 16 ). Vitamin D function is contingent on receptor binding. The vitamin D receptor (VDR) is expressed in the mammalian brain, including the hypothalamus, hippocampal formation, amygdala, stria terminalis and neocortex, and is located on chromosome 12q13.1 ( 17 , 18 ). Crucial in the regulation of cellular response to vitamin D, changes to VDR structure and function influence response quality. Such changes include genetic variability in the form of polymorphisms ( 19 ). Most reported single nucleotide polymorphisms include: Fok1 (rs2228570, adenine>guanine), Bsml (rs1544410 adenine>guanine), Apal (rs7975232 adenine>cytosine), and Taql (rs731236 cytosine>thymine), all resulting in altered functionality ( 20 ). The Taql polymorphism can modify VDR protein structure and vitamin D binding specificity ( 21 ). Fok1 is evidenced to alter VDR structure, delivering a shortened receptor associated with elevated transcriptional activity, in comparison with the longer protein ( 22 , 23 ). Bsml and Apal potentially influence VDR expression through strong linkage disequilibrium with a microsatellite repeat in the 3´untranslated region ( 24 ). The outlined polymorphisms are additionally implied in attenuated vitamin D response. One systematic review reported that variants of the Taql and Fok1 polymorphisms resulted in a better response to vitamin D supplementation ( 25 ). Another study observed lowered risk of maternal and neonatal vitamin D deficiency associated with the Fok1 polymorphism ( 26 ). In relation to autism, the transcriptionally less active Fok1 allele was associated with increased autism risk in one study, while another study observed reduced risk ( 27 , 28 ). Alleles of Taql and Apal were further associated with decreased autism incidence in a third study ( 29 ). Interestingly, VDR polymorphisms may attune the effect of deficient vitamin D in utero, as suggested by Morley et al., 2009, indicating altered birth weight in association with deficient gestational vitamin D was modulated by offspring VDR genotype ( 30 ). However, the effect of VDR polymorphisms in relation to maternal vitamin D status and correlation with autism is unknown. In this exploratory study, we investigated the association between vitamin D levels in gestation and four different VDR polymorphisms (Apal, Bsml, Taql, Fok1) in relation to offspring autism traits. We hypothesise that vitamin D in pregnancy will be associated with ASASC scores and that associations may be modified by child VDR genotype. 2. Methods 2.1 Vitamin D in Pregnancy Study Participants were selected as part of the vitamin D in pregnancy (VIP) study, which aimed to investigate maternal health during gestation and offspring developmental outcomes ( 31 ). Conducted in south-eastern Australia, 475 pregnant women were recruited from 2002 to 2004 from the antenatal clinic at Barwon Health, Geelong, Australia. Data for 192 mother-child pairs were available at 11 years follow-up. Participants were excluded if they had multiple pregnancies, diabetes, renal disease, sarcoidosis, substance use, or had significant maternal illness or disability. Participant inclusion and exclusion criteria are outlined in Table 1 . Maternal venous blood samples were collected at two points; during recruitment (< 16 weeks' gestation), and at 28–32 weeks' gestation. Serum samples were centrifuged within two hours and stored at − 70°C. 25(OH)D levels were detected in plasma via radioimmunoassay (Immunodiagnostic Systems, Tyne and Wear, UK). This study was approved by Barwon Health Human Research Ethics Committee 01/42_E2 (11/02/2013) and acknowledged by Deakin University 2013 − 116 (17/05/2013). All methods were performed in accordance with the Australian National Statement on Ethical Conduct in Human Research (2005) and Declaration of Helsinki. All participants provided full informed written consent on behalf of themselves and their children. At the 11-year follow-up, all child participants provided optional assent. All data were de-identified presented as aggregate data to ensure participant anonymity Table 1 Vitamin D in Pregnancy Study Participant Inclusion and Exclusion Criteria Inclusion criteria Exclusion criteria Singleton pregnancy Evidence of insulin-dependent diabetes, anticonvulsant therapy, renal disease Aged over 18 years Sarcoidosis or familial hypocalciuric hypercalcemia Gestational age of less than 16 weeks Substance use/abuse Unlikely to leave the area within the foreseeable future Maintenance systematic or high-dose glucocorticoid therapy Significant maternal illness and/or disability Dark skinned and/or veiled (as of known elevated risk for deficiency and usually screened and treated) Inclusion and exclusion criteria were designed to ensure a homogeneous study population and minimize confounding variables related to vitamin D metabolism and absorption. 2.2 Offspring Data Offspring DNA was extracted from bloodspot samples on Guthrie cards obtained shortly after birth using Chelex reagent (BioRad, Hercules, CA, USA). Genotyping was performed on 10 ng by standard restriction fragment length polymorphism analysis (overnight digestion) to identify the following VDR polymorphisms: Apal, Bsml, Fok1 and Taql. At 11 years follow-up, the Australian Scale for Asperger's Syndrome (ASASC) was administered to identify behaviours and capabilities characteristic of autism during childhood (age 5–19 years). With established test–retest reliability and internal consistency, the ASASC consists of 44 items within five dimensions: understand and express emotion, fact orientation, sensory sensitivity, social communication, and rigidity ( 32 ). Each dimension contains several questions or statements accompanied by a rating scale from “1” to “5”. Individual dimension and total scores were compared to groups including neurotypical, autism, and those with a diagnosis other than autism such as anxiety disorder or depression. The internal reliability of the scale was good (α = .87). Parents provided responses to the ASASC questionnaire on behalf of their child. Clinical measures and demographic information were obtained by an independent researcher. 2.3 Statistical Analysis Summary cohort characteristics were presented as median (IQR) if nonparametric or mean (± SD) if parametric. The Shapiro-wilk test was employed to assess normality. Binary data such as sex and total ASASC score were analysed using the Kruskal-Wallis Test. Where data were not distributed, normally appropriate transformations were made. Several confounding variables suggested to potentially influence maternal vitamin D levels and offspring autism susceptibility were selected. Included covariates included maternal age, body mass index, smoking status, season of serum collection and offspring weight, all of which were adjusted for in a backwards stepwise elimination approach. Potential effect modification by offspring VDR genotype and gestational vitamin D was tested in the models predicting ASASC scores as described above. Significant interactions were stratified by genotype and sex to explore potential effect modification. All analyses were conducted using STATA 18.0 (StataCorp LLC, College Station, TX). 3. Results This study included 192 mother-child pairs. Mean (± SD) maternal age during gestation was 29.7 (± 4.72) years. The median (IQR) maternal 25(OH)D level was 56 (42–73) nmol/L and 57 (44–74) nmol/L at the early (< 16 weeks' gestation) and late (28–32 weeks' gestation) timepoints. At birth, 53.7% of offspring were males. Participant characteristics are outlined in Table 2 . Table 2 Participant Characteristics and Genotype Frequencies Characteristic All Males Females Maternal n = 192 Age (yr) 29.7 (± 4.72) Height (cm) 166.1 (± 6.74) Weight (kg) 73.12 (± 16.7) BMI 26.4 (± 5.54) 25(OH)D Recruitment (nmol/L) 56 (42–73) 25(OH)D 28–32 weeks (nmol/L) 57 (44–74) Smoking during pregnancy (%) 19.6 Offspring , n (%) n = 192 n = 103 (53.65%) n = 89 (46.35%) Birth weight (kg) 3.52 (± 0.53) 3.58 (± 0.56) 3.47 (± 0.50) Follow up height (cm) 148.69 (± 7.62) 148.51 (± 7.89) 148.87 (± 7.37) Follow up weight (kg) 42.41 (± 10.19) 42.02 (± 10.55) 42.79 (± 9.85) VDR genotypes (%) Bsml BB 33.52 36 30.38 Bb 53.07 50 56.96 bb 13.41 14 12.66 Apal AA 31.84 34 29.11 Aa 52.51 49 56.96 aa 15.64 17 13.92 Taql TT 29.05 31 26.58 Tt 55.87 52 60.76 tt 15.08 17 12.66 Fok1 FF 40.78 35 48.10 Ff 44.69 50 37.97 ff 14.53 15 13.92 Data are presented as median (IQR) or mean (± SD). 25(OH)D represents serum 25-hydroxyvitamin D (measured in nmol/L). Neither early nor late pregnancy 25(OH)D levels were associated with total offspring ASASC scores (early β: -0.07, 95% CI: -0.19–0.04, p = 0.20; late β: -0.02, 95% CI: -0.13–0.09, p = 0.70). Total ASASC scores were additionally unaffected by offspring VDR genotype (Bsml β: -0.30, 95% CI: -4.63–4.05, p = 0.90; Fok1 β: -0.41, 95% CI: -4.50–3.68, p = 0.84; Apal β: 0.81, 95% CI: -3.51–5.13; p = 0.71; Taql β: -1.13, 95% CI: -5.53–3.27, p = 0.61). In secondary analyses of ASASC subscales (understanding emotion, fact orientation, sensory sensitivity, social communication, and rigidity), the Taql polymorphism was associated with elevated fact orientation scores (β: 1.07, 95% CI: 0.02–2.13, p = 0.05). Stratification by genotype identified the homozygous recessive genotype was associated with a 2.5-unit score increase over the homozygous dominant genotype (β: 2.52, 95% CI 0.36–4.69, p = 0.02). However, this finding was not significant in the adjusted model (p > 0.05). Table 3 displays a full distribution of ASASC results by genotype status. No other ASASC subscale was affected in either sex (all p > 0.05). No interaction was observed with gestational 25(OH)D with any genotype (all p > 0.05). Table 3 Beta Coefficients (95% CI) for Associations Between Gestational 25(OH)D, Offspring Genotype, and ASASC Total and Subscale Scores. Early pregnancy 25(OH)D Understanding Emotion Fact Orientation Sensory Sensitivity Social Communication Rigidity Total ASASC Score β: -0.01, 95% CI: -0.04–0.02, p = 0.51 β: -0.02, 95% CI: -0.05–0.00, p = 0.11 β: -0.02, 95% CI: -0.05–0.01, p = 0.22 β: 0.00, 95% CI: -0.03–0.03, p = 0.95 β: -0.02, 95% CI: -0.05–0.01 p = 0.24 β: -0.07, 95% CI: -0.19–0.04, p = 0.20 Late pregnancy 25(OH)D β: -0.02, 95% CI: -0.05–0.01, p = 0.19 β: 0.01, 95% CI: -0.02 – 0.03, p = 0.59 β: 0.00, 95% CI: -0.03–0.03, p = 0.90 β: -0.02, 95% CI: -0.05–0.01, p = 0.22 β: 0.00. 95% CI: -0.03–0.03 p = 0.84 β: -0.02, 95% CI: -0.13–0.09, p = 0.70 Bsml β: -0.20, 95% CI: -1.33–0.93, p = 0.73 β: -0.71, 95% CI: -1.77 – 0.34, p = 0.18 β: 0.12, 95% CI: -0.98–1.22, p = 0.83 β: 0.29, 95% CI: -0.93–1.50, p = 0.64 β: 0.27, 95% CI: -0.90–1.44 p = 0.65 β: -0.30, 95% CI: -4.63 – 4.05, p = 0.90 Fok1 β: 0.27, 95% CI: -0.79–1.33, p = 0.61 β: -0.05, 95% CI: -1.04 – 0.95, p = 0.93 β: -0.21, 95% CI: -1.25–0.82, p = 0.69 β: -0.19, 95% CI: -1.35–0.95, p = 0.74 β: -0.14, 95% CI: -1.24–0.96 p = 0.80 Fok1 β: -0.41, 95% CI: -4.50–3.68, p = 0.84 Apal β: -0.23, 95% CI: -1.33–0.88, p = 0.69 β: -0.10, 95% CI: -1.14 – 0.94, p = 0.85 β: 0.51, 95% CI: -0.56–1.58, p = 0.35 β: 0.68, 95% CI: -0.51–1.87, p = 0.26 β: 0.34, 95% CI: -0.81–1.49, p = 0.56 Apal β: 0.81, 95% CI: -3.51–5.13, p = 0.71 Taql β: -0.00, 95% CI: -1.14–1.14, p = 1.00 β: 1.07, 95% CI: 0.02–2.13 p = 0.05* β: -0.59, 95% CI: -1.70–0.51, p = 0.29 β: -1.03, 95% CI: -2.26–0.19, p = 0.10 β: -0.88,95% CI: -2.06–0.23 p = 0.14 Taql β: -1.13, 95% CI: -5.53–3.27, p = 0.61 Beta coefficients (β), 95% confidence intervals (CI), and p-values represent the associations between gestational 25-hydroxyvitamin D (25(OH)D) concentrations, offspring vitamin D receptor (VDR) genotypes (Bsml, Fok1, Apal, Taql), and Australian Scale for Autism Spectrum Conditions (ASASC) total and subscale scores. While overall early and late pregnancy 25(OH)D levels were not associated with any subscales (Table 3 ), interaction analyses identified higher early 25(OH)D levels were associated with decreased fact orientation scores in males in both adjusted and crude models (Crude β: -0.05, 95% CI -0.08 – -0.01, p = 0.02, Adjusted β: -0.05, 95% CI -0.09 – -0.01, p = 0.02). No significant association was observed in females (p = 0.88). No further interaction was observed in both early and late 25(OH)D and ASASC total scores or subscale scores in either sex (all p > 0.05). 4. Discussion This longitudinal study did not detect an association between gestational vitamin D concentrations at any timepoint and offspring ASASC scores. This is in contrast with studies which have reported associations in cases of diagnosed autism ( 13 – 15 ). VDR genotype additionally did not affect total ASASC scores in offspring. A combination of factors may account for the null relationship between total ASASC scores, maternal vitamin D concentration, and offspring VDR genotype. The total ASASC score represents a composite of diverse behavioural domains, where trait-specific effects may have been diluted as suggested by the subscale-specific findings at present. The lack of a direct comparison with diagnosed cases of autism in the broader literature may complicate findings when evaluating separate outcome measures. Further, our relatively small sample size may not reflect changes in vitamin D concentrations large enough to influence ASASC scores across the cohort. Low variability in vitamin D concentration was also observed across our cohort, where concentrations were generally sufficient. This in turn may obscure the true effect of VDR polymorphisms which are possibly contingent on low/deficient vitamin D exposure, as main-effect modelling may fail to capture these conditional associations. While secondary analyses of ASASC subscales, polymorphisms and maternal vitamin D concentrations revealed null associations, limited evidence presented an association between maternal vitamin D, the Taql VDR polymorphism and autism traits in offspring. Elevated early, 25(OH)D levels were associated with decreased fact orientation scores in males in both adjusted and crude models, leaning towards neurotypical scores in accordance with ASASC guidelines. Formal autism diagnosis is based on key core characteristics in accordance with the Diagnostic and Statistical Manual of Mental Disorders 5th Edition (DSM-5), or the International Classification of Diseases 11th Revision (ICD-11) ( 12 , 33 ). Core characteristics include communication and social interaction differences, and the presence of restrictive and repetitive behaviours and interests ( 12 , 33 ). While the ASASC scale does not constitute an autism diagnosis, items within the fact-orienting dimension can be mapped onto the DSM-5 criterion of restricted, repetitive patterns of behaviour, interests or activities (RRBs) ( 12 ). Behaviours characteristic of this criterion include highly restricted, fixated interests that are atypical in intensity or focus, further broken down into an insistence on sameness, inflexible adherence to routines, or ritualized patterns of verbal or nonverbal behaviour ( 12 ). Several neural processes and mechanisms are suggested to influence RRBs, including executive function, and upstream neural dysregulation in the cortico-striatal-thalamo-cortical circuit and cerebellum ( 34 – 36 ). While vitamin D deficiency is associated with structural and functional brain abnormalities, its effect on mechanistic drivers of RRBs remains largely unexplored ( 37 ). However, animal studies have linked behaviours characteristic of RRBs to deficient vitamin D in utero ( 38 ). Increased self-grooming in vitamin D deficient rat pups exposed to low vitamin D in utero was observed in one study, potentially overlapping with RRBs observed in autism ( 38 , 39 ). In a subsequent study, male mice exposed to vitamin D deficiency in utero exhibited increased repetitive behaviours and disrupted social interaction ( 39 ). In human studies of vitamin D supplementation, a 13% reduction in stereotypical behaviours (categorised as RRBs) was observed in children diagnosed with autism in comparison to control participants ( 40 ). A meta-analysis of children with autism additionally reported significant improvement in stereotypical behaviours following vitamin D supplementation ( 41 ). However, this effect has not been researched in relation to maternal vitamin D status. The potential of vitamin D supplementation to ameliorate autism symptoms is additionally not consistently reported, likely owing to varying ages, populations and questionnaires utilised across studies ( 42 ). Reduced fact orienting scores were further only observed in early pregnancy, a critical period in neurodevelopment particularly sensitive to changes in gestational vitamin D ( 43 , 44 ). Reduced fact-orientation scores were additionally only observed in male offspring. The male to female ratio of autism prevalence is close to 3:1 and is influenced by societal and biological factors ( 45 ). Autism criteria were initially developed on symptoms presented primarily in males, resulting in missed or inapplicable diagnostic and symptomatic measures in females ( 46 ). Sex differences in symptom presentation are further observed, namely across RRBs which are less predictive of autism in females ( 47 ). Additionally, presentation of autism symptoms, including RRBs vary based on sex, age, cognitive level and co-occurring mental health conditions ( 48 , 49 ). The homozygous recessive (tt) genotype of the Taql polymorphism was associated with elevated fact orientation scores in the crude model, suggesting the presence of traits consistent with autism. Interestingly, Coşkun et al., 2016 reported the frequency of the same genotype was higher in children with autism ( 50 ). Cieślińska et al., 2017 additionally reported decreased autism incidence in association with the dominant allele of the Taql VDR polymorphism ( 29 ). Taken together, this pattern may be consistent with the dominant allele’s exertion of a neuroprotective effect when compared with the recessive variant ( 29 , 50 ). While research relating to the influence of Taql and neurodevelopment is sparse, the Taql variant is evidenced to confer both elevated or decreased risk of neurodegenerative disease, suggesting the role of Taql both as a risk and protective factor in neurological processes ( 51 – 54 ). Variants of the Taql polymorphism are associated with increased mRNA stability, potentially enhancing vitamin D signalling ( 55 ). Activation of VDR by its ligand is necessary for mediation of neurogenic and synaptic activity of vitamin D in the brain ( 56 ). The efficacy of this response may be dictated by VDR genotype, where Taql heterozygous and recessive genotypes were associated with a better response to vitamin D supplementation in one study ( 25 ). However, the effect observed in this study occurred irrespective of maternal vitamin D status, similar to two studies associating Taql with autism where offspring vitamin D concentration was controlled ( 29 , 50 ). As such, the exact Taql genotypes and variants broadly associated with increased autism risk or symptomatology remain unclear. However, known Taql associations may be the result of linkage disequilibrium with other VDR polymorphisms known to influence VDR expression and activity ( 57 , 58 ). Our finding was once again only observed in the fact orientation ASASC subscale, providing limited evidence of singular genetic factors in the influence phenotypic expression of autism, as has been previously observed in RRBs ( 59 – 61 ). Further research into the molecular pathways exerted by the Taql polymorphism is warranted to underpin the genotypes and variants associated with autism risk/symptomatology. Several limitations present in the current study. While the ASASC does not provide an autism diagnosis, its subscales provide insight into autism traits, symptom variability, and their relationship to vitamin D and VDR polymorphisms. The continuous nature of the scale reflects characteristics also present in the general population, such as RRBs which present in other neurodevelopmental conditions ( 62 ). As such, ASASC outcomes may be applied beyond the autism population. However, the utility of the present findings in relation to autism risk should be interpreted with caution in the absence of diagnostic information. Parental report in the assessment of autism traits relies on subjective interpretation of behavioural traits, introducing variability in the precision of reported behaviours. Further, the use of the ASASC as a measure of autism symptomatology carries unique outcomes and measures based on the Australian population ( 32 ). Variations in results across studies may be attributed to differences in utilised scales and questionnaires, given that use of the ASASC in the literature is limited. The limited sample size in the current study may restrict our ability to detect associations between maternal vitamin D, related receptor polymorphisms, and autism traits. As a result, adjustment for multiple comparisons was not performed to avoid further increasing the risk of Type II error. However, in the absence of multiple-comparison adjustment, the observed associations may partly reflect Type I errors arising from the number of tests conducted. A larger cohort with longer follow-up, clinically confirmed autism diagnoses, and offspring vitamin D concentrations would ideally be required to further elucidate the current observations. 4. Conclusion Contrary to our a priori hypothesis, the findings at present showed no association between gestational vitamin D, VDR polymorphisms, and total ASASC scores. However, limited evidence suggests that gestational vitamin D and the Taql polymorphism may influence specific autism-related behaviours, such as fact-orientation, corresponding with the DSM-5 criteria for restricted and repetitive patterns of behaviour, interests, or activities. Interpreted within the constraints of study limitations, initial insight suggests that while vitamin D may not affect aggregate autism symptomatology, it may modulate specific behavioural traits, highlighting the potential role of genetic factors in shaping the phenotypic expression of autism. The results of this study should be interpreted as hypothesis generating, with validation required in larger, independent cohorts. Declarations Funding Funding for the initial phases of the VIP study were provided by a project grant from the National Health and Medical Research Council (NHMRC) of Australia. The 11-year follow up of the VIP study was funded by the Bupa Foundation. Declaration of Interests The authors declare no conflicts of interest. Ethical Information This study was approved by Barwon Health Human Research Ethics Committee 01/42_E2 (11/02/2013) and acknowledged by Deakin University 2013 − 116 (17/05/2013). All participants provided full informed written consent on behalf of themselves and their children. At the 11-year follow-up, all child participants provided optional assent. Correspondence Esma Fazlić, HERB Level 3 (Deakin University-Barwon Health) 285–299 Ryrie St, Geelong VIC 3220 Australia; Email: [email protected] Author Contribution All authors were involved in the formulation of the research question. Authors JP, NH, and LW were involved with the inception of the initial cohort and regulated the collection and measurement of biological samples. Authors EF and NH conducted statistical analyses. EF prepared the main manuscript text and figures. All authors provided initial edits to first and subsequent drafts and provided critical feedback on the structure and content. Acknowledgement The authors would like to thank the participants of the VIP study for their ongoing participation. Funding for the initial phases of the VIP study were provided by a project grant from the National Health and Medical Research Council (NHMRC) of Australia. The 11-year follow up of the VIP study was funded by the Bupa Foundation. Data Availability The data that support the findings of this study are available from the corresponding author [E.F] upon reasonable request. References Nair, R., Maseeh, A. & Vitamin, D. The sunshine vitamin. J. Pharmacol. Pharmacother . 3 (2), 118–126 (2012). Jäpelt, R. B. & Jakobsen, J. Vitamin D in plants: a review of occurrence, analysis, and biosynthesis. Front. Plant. Sci. 4 , 136 (2013). van den Heuvel, E. G., Lips, P., Schoonmade, L. J., Lanham-New, S. A. & van Schoor, N. M. Comparison of the Effect of Daily Vitamin D2 and Vitamin D3 Supplementation on Serum 25-Hydroxyvitamin D Concentration (Total 25(OH)D, 25(OH)D2, and 25(OH)D3) and Importance of Body Mass Index: A Systematic Review and Meta-Analysis. Adv. Nutr. 15 (1), 100133 (2024). Bikle Daniel, D., Vitamin, D. & Metabolism Mechanism of Action, and Clinical Applications. Chem. Biol. 21 (3), 319–329 (2014). Sommer, I. et al. Vitamin D deficiency as a risk factor for dementia: a systematic review and meta-analysis. BMC Geriatr. 17 (1), 16 (2017). Huang, Y. et al. Association of vitamin D with risk of dementia: a dose-response meta-analysis of observational studies. Front. Neurol. 16 , 1649841 (2025). Peitl, V. et al. Vitamin D and Neurotrophin Levels and Their Impact on the Symptoms of Schizophrenia. Neuropsychobiology 79 (3), 179–185 (2020). Pansri, P. et al. Brain-derived neurotrophic factor increases cell number of neural progenitor cells derived from human induced pluripotent stem cells. PeerJ 9 , e11388 (2021). Calabresi, P., Picconi, B., Tozzi, A. & Di Filippo, M. Dopamine-mediated regulation of corticostriatal synaptic plasticity. Trends Neurosci. 30 (5), 211–219 (2007). Mansouri, F. et al. Protective effects of vitamin D on learning and memory deficit induced by scopolamine in male rats: the roles of brain-derived neurotrophic factor and oxidative stress. Naunyn Schmiedebergs Arch. Pharmacol. 394 (7), 1451–1466 (2021). Galvez-Contreras, A. Y., Campos-Ordonez, T., Gonzalez-Castaneda, R. E. & Gonzalez-Perez, O. Alterations of Growth Factors in Autism and Attention-Deficit/Hyperactivity Disorder. Front. Psychiatry . 8 , 126 (2017). Arlington, V. & Association, A. P. Diagnostic and statistical manual of mental disorders. Am. Psychiatric Association . 5 , 612–613 (2013). Tirani, S. A., Balali, A., Askari, G. & Saneei, P. Maternal serum 25-hydroxy vitamin D levels and risk of autism spectrum and attention-deficit hyperactivity disorders in offspring: A systematic review and dose-response meta-analysis. Psychiatry Res. 319 , 114977 (2023). Lee, B. K. et al. Developmental vitamin D and autism spectrum disorders: findings from the Stockholm Youth Cohort. Mol. Psychiatry . 26 (5), 1578–1588 (2021). Magnusson, C. et al. Maternal vitamin D deficiency and the risk of autism spectrum disorders: population-based study. BJPsych Open. 2 (2), 170–172 (2016). Madley-Dowd, P. et al. Maternal vitamin D during pregnancy and offspring autism and autism-associated traits: a prospective cohort study. Mol. Autism . 13 (1), 44 (2022). Prüfer, K., Veenstra, T. D., Jirikowski, G. F. & Kumar, R. Distribution of 1,25-dihydroxyvitamin D3 receptor immunoreactivity in the rat brain and spinal cord. J. Chem. Neuroanat. 16 (2), 135–145 (1999). Smolders, J. et al. Expression of Vitamin D Receptor and Metabolizing Enzymes in Multiple Sclerosis—Affected Brain Tissue. J. Neuropathology Experimental Neurol. 72 (2), 91–105 (2013). Song, N. et al. The Impact of Vitamin D Receptor Gene Polymorphisms on the Susceptibility of Diabetic Vascular Complications: A Meta-Analysis. Genet. Test. Mol. Biomarkers . 23 (8), 533–556 (2019). Valdivielso, J. M. & Fernandez, E. Vitamin D receptor polymorphisms and diseases. Clin. Chim. Acta . 371 (1), 1–12 (2006). Uitterlinden, A. G., Fang, Y., van Meurs, J. B. J., Pols, H. A. P. & van Leeuwen, J. P. T. M. Genetics and biology of vitamin D receptor polymorphisms. Gene 338 (2), 143–156 (2004). Kerr Whitfield, G. et al. Functionally relevant polymorphisms in the human nuclear vitamin D receptor gene. Mol. Cell. Endocrinol. 177 (1), 145–159 (2001). Arai, H. et al. A Vitamin D Receptor Gene Polymorphism in the Translation Initiation Codon: Effect on Protein Activity and Relation to Bone Mineral Density in Japanese Women. J. Bone Miner. Res. 12 (6), 915–921 (1997). Ingles, S. A. et al. Strength of linkage disequilibrium between two vitamin D receptor markers in five ethnic groups: implications for association studies. Cancer Epidemiol. Biomarkers Prev. 6 (2), 93–98 (1997). Usategui-Martín, R. et al. Receptor (VDR) Gene Polymorphisms Modify the Response to Vitamin D Supplementation: A Systematic Review and Meta-Analysis. Nutrients 14 (2), 360 (2022). Karras, S. N. et al. Vitamin D receptor Fokl polymorphism is a determinant of both maternal and neonatal vitamin D concentrations at birth. J. Steroid Biochem. Mol. Biol. 199 , 105568 (2020). Guerini, F. R. et al. Vitamin D Receptor Polymorphisms Associated with Autism Spectrum Disorder. Autism Res. 13 (5), 680–690 (2020). Saechua, C. et al. Impact of gene polymorphisms involved in the vitamin D metabolic pathway on the susceptibility to and severity of autism spectrum disorder. Sci. Rep. 14 (1), 28333 (2024). Cieślińska, A. et al. Vitamin D Receptor Gene Polymorphisms Associated with Childhood Autism. Brain Sci. ; 7 (9). (2017). Morley, R., Carlin, J. B., Pasco, J. A., Wark, J. D. & Ponsonby, A. L. Maternal 25-hydroxyvitamin D concentration and offspring birth size: effect modification by infant VDR genotype. Eur. J. Clin. Nutr. 63 (6), 802–804 (2009). Hyde, N. K. et al. The Vitamin D in Pregnancy Study: a prospective prebirth cohort in southern Australia. BMJ Open. 10 (8), e036003 (2020). Garnett, M. S., Attwood, T., Peterson, C. & Kelly, A. B. Autism spectrum conditions among children and adolescents: A new profiling tool. Australian J. Psychol. 65 (4), 206–213 (2013). World Health, O. ICD-11: international statistical classification of diseases and related health problems : eleventh revision 2nd edn (World Health Organization, 2004). Graybiel, A. M. & Grafton, S. T. The striatum: where skills and habits meet. Cold Spring Harb Perspect. Biol. 7 (8), a021691 (2015). Lopez, B. R., Lincoln, A. J., Ozonoff, S. & Lai, Z. Examining the Relationship between Executive Functions and Restricted, Repetitive Symptoms of Autistic Disorder. J. Autism Dev. Disord. 35 (4), 445–460 (2005). Rojas, D. C. et al. Regional gray matter volumetric changes in autism associated with social and repetitive behavior symptoms. BMC Psychiatry . 6 , 56 (2006). Wang, Z., Ding, R. & Wang, J. The Association between Vitamin D Status and Autism Spectrum Disorder (ASD): A Systematic Review and Meta-Analysis. Nutrients ; 13 (1). (2020). Tamang, M. K. et al. Developmental vitamin D-deficiency produces autism-relevant behaviours and gut-health associated alterations in a rat model. Translational Psychiatry . 13 (1), 204 (2023). Cui, J. et al. Induction of autism-related behavior in male mice by early-life vitamin D deficiency: association with disruption of the gut microbial composition and homeostasis. Food Funct. 15 (8), 4338–4353 (2024). Moradi, H., Sohrabi, M., Taheri, H., Khodashenas, E. & Movahedi, A. Comparison of the effects of perceptual-motor exercises, vitamin D supplementation and the combination of these interventions on decreasing stereotypical behavior in children with autism disorder. Int. J. Dev. Disabil. 66 (2), 122–132 (2018). Zhang, M. et al. Effects of Vitamin D Supplementation on Children with Autism Spectrum Disorder: A Systematic Review and Meta-analysis. Clin. Psychopharmacol. Neurosci. 21 (2), 240–251 (2023). Kittana, M., Ahmadani, A., Stojanovska, L. & Attlee, A. The Role of Vitamin D Supplementation in Children with Autism Spectrum Disorder: A Narrative Review. Nutrients ; 14 (1). (2021). Beck, C. et al. Maternal vitamin D status, fetal growth patterns, and adverse pregnancy outcomes in a multisite prospective pregnancy cohort. Am. J. Clin. Nutr. 121 (2), 376–384 (2025). Lee, S. B. et al. Maternal vitamin D deficiency in early pregnancy and perinatal and long-term outcomes. Heliyon 9 (9), e19367 (2023). Loomes, R., Hull, L. & Mandy, W. P. L. What Is the Male-to-Female Ratio in Autism Spectrum Disorder? A Systematic Review and Meta-Analysis. J. Am. Acad. Child. Adolesc. Psychiatry . 56 (6), 466–474 (2017). Halladay, A. K. et al. Sex and gender differences in autism spectrum disorder: summarizing evidence gaps and identifying emerging areas of priority. Mol. Autism . 6 (1), 36 (2015). McFayden, T. C., Albright, J., Muskett, A. E. & Scarpa, A. Brief Report: Sex Differences in ASD Diagnosis-A Brief Report on Restricted Interests and Repetitive Behaviors. J. Autism Dev. Disord . 49 (4), 1693–1699 (2019). Harrop, C. et al. Restricted and Repetitive Behaviors in Autism Spectrum Disorders and Typical Development: Cross-Sectional and Longitudinal Comparisons. J. Autism Dev. Disord. 44 (5), 1207–1219 (2014). Jasim, S. & Perry, A. Repetitive and restricted behaviors and interests in autism spectrum disorder: relation to individual characteristics and mental health problems. BMC Psychiatry . 23 (1), 356 (2023). Coşkun, S., Şimşek, Ş., Camkurt, M. A., Çim, A. & Çelik, S. B. Association of polymorphisms in the vitamin D receptor gene and serum 25-hydroxyvitamin D levels in children with autism spectrum disorder. Gene 588 (2), 109–114 (2016). Łaczmański, Ł. et al. Vitamin D receptor gene polymorphisms in Alzheimer's disease patients. Exp. Gerontol. 69 , 142–147 (2015). Tarkesh Esfahani, N., Rahgozar, M., Biglarian, A. & Khorram Khorshid, H. R. Identification of genetic polymorphism interactions in sporadic alzheimer’s disease using logic regression. Iran. Rehabilitation J. 9 (2), 45–50 (2011). Lehmann, D. J. et al. The vitamin D receptor gene is associated with Alzheimer's disease. Neurosci. Lett. 504 (2), 79–82 (2011). Dimitrakis, E. et al. Association of vitamin D receptor gene TaqI polymorphism with Alzheimer's disease in a Southeastern European Caucasian population. Exp. Ther. Med. 23 (5), 341 (2022). Moradkhani, A. et al. Association of vitamin D receptor genetic polymorphisms with the risk of infertility: a systematic review and meta-analysis. BMC Pregnancy Childbirth . 24 (1), 398 (2024). Eyles, D. W. & Vitamin, D. Brain and Behavior. JBMR Plus . 5 (1), e10419 (2021). Meza-Meza, M. R. et al. Vitamin D Receptor (VDR) Genetic Variants: Relationship of FokI Genotypes with VDR Expression and Clinical Disease Activity in Systemic Lupus Erythematosus Patients. Genes (Basel) ; 13 (11). (2022). Thakkinstian, A., D'Este, C. & Attia, J. Haplotype analysis of VDR gene polymorphisms: a meta-analysis. Osteoporos. Int. 15 (9), 729–734 (2004). Glaser, B. et al. Identification of a potential Bipolar risk haplotype in the gene encoding the winged-helix transcription factor RFX4. Mol. Psychiatry . 10 (10), 920–927 (2005). Welch, J. M. et al. Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice. Nature 448 (7156), 894–900 (2007). Peça, J. et al. Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. Nature 472 (7344), 437–442 (2011). Matson, J. L., Dempsey, T. & Fodstad, J. C. Stereotypies and repetitive/restrictive behaviours in infants with autism and pervasive developmental disorder. Dev. Neurorehabilitation . 12 (3), 122–127 (2009). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 14 May, 2026 Reviews received at journal 13 May, 2026 Reviews received at journal 08 May, 2026 Reviewers agreed at journal 21 Apr, 2026 Reviewers agreed at journal 21 Apr, 2026 Reviewers agreed at journal 20 Apr, 2026 Reviewers invited by journal 19 Apr, 2026 Editor assigned by journal 17 Apr, 2026 Submission checks completed at journal 08 Apr, 2026 First submitted to journal 08 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9216765","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":628248876,"identity":"13f0047d-3a48-4c4e-8314-27193eb853a7","order_by":0,"name":"Fazlić Esma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDklEQVRIie3QMUsDMRTA8RcCcXnqenC2h98gEFAOQb9KjoNO9wEOFAwcZKtzB7EfQnBOObgudT9wKoKT4IlQHER951piO3bIfwohP/ISgFBoF9urnNPyZyhoEQFo7Dejfwk2metKrg6w0VuSqFCzyYJnd1EhewIbSWK0rPetyCwU7213NTo6BT57QjjPjIdIegkRVBYe79NJU2BqRH6GkPsJM45INLBs/BCjKFE6PIkRuJckFTNEJLMcX+Kv754crohcewk0HOj5+tgKFDGzRX+LIFL7B1sIoE92ioRKxzcjlDUtbuVceQebvn10WrphMn1etp+r/ELOq2X7Wl4OvIOtx/9u3/58KBQKhdb7BeRVWbUWlpKXAAAAAElFTkSuQmCC","orcid":"","institution":"Barwon Health, Deakin University","correspondingAuthor":true,"prefix":"","firstName":"Fazlić","middleName":"","lastName":"Esma","suffix":""},{"id":628248877,"identity":"ca8a1471-ff1e-40aa-bb93-cda093bb75e6","order_by":1,"name":"Pasco Julie","email":"","orcid":"","institution":"Barwon Health, Deakin University","correspondingAuthor":false,"prefix":"","firstName":"Pasco","middleName":"","lastName":"Julie","suffix":""},{"id":628248878,"identity":"26e30244-2c66-4394-a13f-fd27ad2de825","order_by":2,"name":"Williams Lana","email":"","orcid":"","institution":"Barwon Health, Deakin University","correspondingAuthor":false,"prefix":"","firstName":"Williams","middleName":"","lastName":"Lana","suffix":""},{"id":628248879,"identity":"fa0d7104-07f3-47dd-8976-f640cc57b23d","order_by":3,"name":"Hyde Natalie","email":"","orcid":"","institution":"Barwon Health, Deakin University","correspondingAuthor":false,"prefix":"","firstName":"Hyde","middleName":"","lastName":"Natalie","suffix":""}],"badges":[],"createdAt":"2026-03-25 01:08:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9216765/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9216765/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108181527,"identity":"94ea1e02-20eb-49a1-94fa-5cf4b2963b90","added_by":"auto","created_at":"2026-04-30 08:58:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":399758,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9216765/v1/0b21334b-0550-4a4a-a36b-4630ef900dae.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Association Between Gestational Vitamin D Levels and Offspring Vitamin D Receptor Polymorphisms in Autism-Associated Traits","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eVitamin D is a fat-soluble vitamin recognised for its role in musculoskeletal function, maintaining bone through calcium regulation (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Vitamin D occurs in two forms: D2 and D3. Vitamin D2 is plant-derived, produced primarily by fungi and yeast (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Vitamin D3 is synthesised in the skin by UV irradiation, further transported to the liver where both D2 and D3 are metabolised into the final circulating form, 25-hydroxyvitamin D (25(OH)D) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). While critical in calcium metabolism, additional physiological processes are regulated by vitamin D, notably, brain function (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Vitamin D function in the brain is associated with neuroprotective and neurodevelopmental processes, where lower concentrations are associated with elevated risk of dementia in adults (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Vitamin D is evidenced in the modulation of neurodevelopmental processes, regulating several neurotropic and growth factors to influence neuronal growth, differentiation, maturation and survival (\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Alterations to such processes are implied in the pathogenesis of neurodevelopmental conditions, namely autism (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Autism is a neurodevelopmental condition characterised by differences in social interaction and communication, combined with the presence of repetitive behaviours, activities, or interests (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Potential associations with vitamin D are evident in several studies, where deficient maternal vitamin D status is associated with increased offspring autism risk (\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVitamin D function is contingent on receptor binding. The vitamin D receptor (VDR) is expressed in the mammalian brain, including the hypothalamus, hippocampal formation, amygdala, stria terminalis and neocortex, and is located on chromosome 12q13.1 (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Crucial in the regulation of cellular response to vitamin D, changes to VDR structure and function influence response quality. Such changes include genetic variability in the form of polymorphisms (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Most reported single nucleotide polymorphisms include: Fok1 (rs2228570, adenine\u0026gt;guanine), Bsml (rs1544410 adenine\u0026gt;guanine), Apal (rs7975232 adenine\u0026gt;cytosine), and Taql (rs731236 cytosine\u0026gt;thymine), all resulting in altered functionality (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The Taql polymorphism can modify VDR protein structure and vitamin D binding specificity (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Fok1 is evidenced to alter VDR structure, delivering a shortened receptor associated with elevated transcriptional activity, in comparison with the longer protein (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Bsml and Apal potentially influence VDR expression through strong linkage disequilibrium with a microsatellite repeat in the 3\u0026acute;untranslated region (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The outlined polymorphisms are additionally implied in attenuated vitamin D response. One systematic review reported that variants of the Taql and Fok1 polymorphisms resulted in a better response to vitamin D supplementation (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Another study observed lowered risk of maternal and neonatal vitamin D deficiency associated with the Fok1 polymorphism (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In relation to autism, the transcriptionally less active Fok1 allele was associated with increased autism risk in one study, while another study observed reduced risk (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Alleles of Taql and Apal were further associated with decreased autism incidence in a third study (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Interestingly, VDR polymorphisms may attune the effect of deficient vitamin D in utero, as suggested by Morley et al., 2009, indicating altered birth weight in association with deficient gestational vitamin D was modulated by offspring VDR genotype (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). However, the effect of VDR polymorphisms in relation to maternal vitamin D status and correlation with autism is unknown.\u003c/p\u003e \u003cp\u003eIn this exploratory study, we investigated the association between vitamin D levels in gestation and four different VDR polymorphisms (Apal, Bsml, Taql, Fok1) in relation to offspring autism traits. We hypothesise that vitamin D in pregnancy will be associated with ASASC scores and that associations may be modified by child VDR genotype.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Vitamin D in Pregnancy Study\u003c/h2\u003e \u003cp\u003eParticipants were selected as part of the vitamin D in pregnancy (VIP) study, which aimed to investigate maternal health during gestation and offspring developmental outcomes (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Conducted in south-eastern Australia, 475 pregnant women were recruited from 2002 to 2004 from the antenatal clinic at Barwon Health, Geelong, Australia. Data for 192 mother-child pairs were available at 11 years follow-up. Participants were excluded if they had multiple pregnancies, diabetes, renal disease, sarcoidosis, substance use, or had significant maternal illness or disability. Participant inclusion and exclusion criteria are outlined in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Maternal venous blood samples were collected at two points; during recruitment (\u0026lt;\u0026thinsp;16 weeks' gestation), and at 28\u0026ndash;32 weeks' gestation. Serum samples were centrifuged within two hours and stored at \u0026minus;\u0026thinsp;70\u0026deg;C. 25(OH)D levels were detected in plasma via radioimmunoassay (Immunodiagnostic Systems, Tyne and Wear, UK). This study was approved by Barwon Health Human Research Ethics Committee 01/42_E2 (11/02/2013) and acknowledged by Deakin University 2013\u0026thinsp;\u0026minus;\u0026thinsp;116 (17/05/2013). All methods were performed in accordance with the Australian National Statement on Ethical Conduct in Human Research (2005) and Declaration of Helsinki. All participants provided full informed written consent on behalf of themselves and their children. At the 11-year follow-up, all child participants provided optional assent. All data were de-identified presented as aggregate data to ensure participant anonymity\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVitamin D in Pregnancy Study Participant Inclusion and Exclusion Criteria\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInclusion criteria\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExclusion criteria\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSingleton pregnancy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEvidence of insulin-dependent diabetes, anticonvulsant therapy, renal disease\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAged over 18 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSarcoidosis or familial hypocalciuric hypercalcemia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGestational age of less than 16 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubstance use/abuse\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnlikely to leave the area within the foreseeable future\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaintenance systematic or high-dose glucocorticoid therapy\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\u003eSignificant maternal illness and/or disability\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\u003eDark skinned and/or veiled (as of known elevated risk for deficiency and usually screened and treated)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eInclusion and exclusion criteria were designed to ensure a homogeneous study population and minimize confounding variables related to vitamin D metabolism and absorption.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Offspring Data\u003c/h2\u003e \u003cp\u003eOffspring DNA was extracted from bloodspot samples on Guthrie cards obtained shortly after birth using Chelex reagent (BioRad, Hercules, CA, USA). Genotyping was performed on 10 ng by standard restriction fragment length polymorphism analysis (overnight digestion) to identify the following VDR polymorphisms: Apal, Bsml, Fok1 and Taql. At 11 years follow-up, the Australian Scale for Asperger's Syndrome (ASASC) was administered to identify behaviours and capabilities characteristic of autism during childhood (age 5\u0026ndash;19 years). With established test\u0026ndash;retest reliability and internal consistency, the ASASC consists of 44 items within five dimensions: understand and express emotion, fact orientation, sensory sensitivity, social communication, and rigidity (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Each dimension contains several questions or statements accompanied by a rating scale from \u0026ldquo;1\u0026rdquo; to \u0026ldquo;5\u0026rdquo;. Individual dimension and total scores were compared to groups including neurotypical, autism, and those with a diagnosis other than autism such as anxiety disorder or depression. The internal reliability of the scale was good (α\u0026thinsp;=\u0026thinsp;.87). Parents provided responses to the ASASC questionnaire on behalf of their child. Clinical measures and demographic information were obtained by an independent researcher.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Statistical Analysis\u003c/h2\u003e \u003cp\u003eSummary cohort characteristics were presented as median (IQR) if nonparametric or mean (\u0026plusmn;\u0026thinsp;SD) if parametric. The Shapiro-wilk test was employed to assess normality. Binary data such as sex and total ASASC score were analysed using the Kruskal-Wallis Test. Where data were not distributed, normally appropriate transformations were made. Several confounding variables suggested to potentially influence maternal vitamin D levels and offspring autism susceptibility were selected. Included covariates included maternal age, body mass index, smoking status, season of serum collection and offspring weight, all of which were adjusted for in a backwards stepwise elimination approach. Potential effect modification by offspring VDR genotype and gestational vitamin D was tested in the models predicting ASASC scores as described above. Significant interactions were stratified by genotype and sex to explore potential effect modification. All analyses were conducted using STATA 18.0 (StataCorp LLC, College Station, TX).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eThis study included 192 mother-child pairs. Mean (\u0026plusmn;\u0026thinsp;SD) maternal age during gestation was 29.7 (\u0026plusmn;\u0026thinsp;4.72) years. The median (IQR) maternal 25(OH)D level was 56 (42\u0026ndash;73) nmol/L and 57 (44\u0026ndash;74) nmol/L at the early (\u0026lt;\u0026thinsp;16 weeks' gestation) and late (28\u0026ndash;32 weeks' gestation) timepoints. At birth, 53.7% of offspring were males. Participant characteristics are outlined in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\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 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParticipant Characteristics and Genotype Frequencies\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAll\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMales\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFemales\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMaternal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (yr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.7 (\u0026plusmn;\u0026thinsp;4.72)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeight (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e166.1 (\u0026plusmn;\u0026thinsp;6.74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeight (kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73.12 (\u0026plusmn;\u0026thinsp;16.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.4 (\u0026plusmn;\u0026thinsp;5.54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25(OH)D Recruitment (nmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56 (42\u0026ndash;73)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25(OH)D 28\u0026ndash;32 weeks (nmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57 (44\u0026ndash;74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmoking during pregnancy (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOffspring\u003c/b\u003e, \u003cb\u003en\u003c/b\u003e \u003cb\u003e(%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;103 (53.65%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;89 (46.35%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBirth weight (kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.52 (\u0026plusmn;\u0026thinsp;0.53)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.58 (\u0026plusmn;\u0026thinsp;0.56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.47 (\u0026plusmn;\u0026thinsp;0.50)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFollow up height (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e148.69 (\u0026plusmn;\u0026thinsp;7.62)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e148.51 (\u0026plusmn;\u0026thinsp;7.89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e148.87 (\u0026plusmn;\u0026thinsp;7.37)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFollow up weight (kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.41 (\u0026plusmn;\u0026thinsp;10.19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.02 (\u0026plusmn;\u0026thinsp;10.55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42.79 (\u0026plusmn;\u0026thinsp;9.85)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVDR genotypes (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBsml\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e56.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ebb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eApal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e56.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eaa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTaql\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ett\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFok1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eff\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eData are presented as median (IQR) or mean (\u0026plusmn;\u0026thinsp;SD). 25(OH)D represents serum 25-hydroxyvitamin D (measured in nmol/L).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eNeither early nor late pregnancy 25(OH)D levels were associated with total offspring ASASC scores (early β: -0.07, 95% CI: -0.19\u0026ndash;0.04, p\u0026thinsp;=\u0026thinsp;0.20; late β: -0.02, 95% CI: -0.13\u0026ndash;0.09, p\u0026thinsp;=\u0026thinsp;0.70). Total ASASC scores were additionally unaffected by offspring VDR genotype (Bsml β: -0.30, 95% CI: -4.63\u0026ndash;4.05, p\u0026thinsp;=\u0026thinsp;0.90; Fok1 β: -0.41, 95% CI: -4.50\u0026ndash;3.68, p\u0026thinsp;=\u0026thinsp;0.84; Apal β: 0.81, 95% CI: -3.51\u0026ndash;5.13; p\u0026thinsp;=\u0026thinsp;0.71; Taql β: -1.13, 95% CI: -5.53\u0026ndash;3.27, p\u0026thinsp;=\u0026thinsp;0.61).\u003c/p\u003e \u003cp\u003eIn secondary analyses of ASASC subscales (understanding emotion, fact orientation, sensory sensitivity, social communication, and rigidity), the Taql polymorphism was associated with elevated fact orientation scores (β: 1.07, 95% CI: 0.02\u0026ndash;2.13, p\u0026thinsp;=\u0026thinsp;0.05). Stratification by genotype identified the homozygous recessive genotype was associated with a 2.5-unit score increase over the homozygous dominant genotype (β: 2.52, 95% CI 0.36\u0026ndash;4.69, p\u0026thinsp;=\u0026thinsp;0.02). However, this finding was not significant in the adjusted model (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e displays a full distribution of ASASC results by genotype status. No other ASASC subscale was affected in either sex (all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). No interaction was observed with gestational 25(OH)D with any genotype (all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBeta Coefficients (95% CI) for Associations Between Gestational 25(OH)D, Offspring Genotype, and ASASC Total and Subscale Scores.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eEarly pregnancy 25(OH)D\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUnderstanding Emotion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFact Orientation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSensory Sensitivity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSocial Communication\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRigidity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTotal ASASC Score\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eβ: -0.01, 95% CI: -0.04\u0026ndash;0.02, p\u0026thinsp;=\u0026thinsp;0.51\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eβ: -0.02, 95% CI: -0.05\u0026ndash;0.00, p\u0026thinsp;=\u0026thinsp;0.11\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eβ: -0.02, 95% CI: -0.05\u0026ndash;0.01,\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.22\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ: 0.00, 95% CI: -0.03\u0026ndash;0.03,\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.95\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eβ: -0.02, 95% CI: -0.05\u0026ndash;0.01\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.24\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eβ: -0.07, 95% CI: -0.19\u0026ndash;0.04, p\u0026thinsp;=\u0026thinsp;0.20\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLate pregnancy\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e25(OH)D\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eβ: -0.02, 95% CI: -0.05\u0026ndash;0.01, p\u0026thinsp;=\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eβ: 0.01, 95% CI: -0.02 \u0026ndash; 0.03, p\u0026thinsp;=\u0026thinsp;0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eβ: 0.00, 95% CI: -0.03\u0026ndash;0.03,\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.90\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ: -0.02, 95% CI: -0.05\u0026ndash;0.01,\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eβ: 0.00. 95% CI: -0.03\u0026ndash;0.03\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eβ: -0.02, 95% CI: -0.13\u0026ndash;0.09, p\u0026thinsp;=\u0026thinsp;0.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBsml\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eβ: -0.20, 95% CI: -1.33\u0026ndash;0.93, p\u0026thinsp;=\u0026thinsp;0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eβ: -0.71, 95% CI: -1.77 \u0026ndash; 0.34, p\u0026thinsp;=\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eβ: 0.12, 95% CI: -0.98\u0026ndash;1.22,\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ: 0.29, 95% CI: -0.93\u0026ndash;1.50,\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eβ: 0.27, 95% CI: -0.90\u0026ndash;1.44\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eβ: -0.30, 95% CI: -4.63 \u0026ndash; 4.05, p\u0026thinsp;=\u0026thinsp;0.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFok1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eβ: 0.27, 95% CI: -0.79\u0026ndash;1.33, p\u0026thinsp;=\u0026thinsp;0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eβ: -0.05, 95% CI: -1.04 \u0026ndash; 0.95, p\u0026thinsp;=\u0026thinsp;0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eβ: -0.21, 95% CI: -1.25\u0026ndash;0.82,\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ: -0.19, 95% CI: -1.35\u0026ndash;0.95,\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eβ: -0.14, 95% CI: -1.24\u0026ndash;0.96\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFok1 β: -0.41, 95% CI: -4.50\u0026ndash;3.68, p\u0026thinsp;=\u0026thinsp;0.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eApal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eβ: -0.23, 95% CI: -1.33\u0026ndash;0.88, p\u0026thinsp;=\u0026thinsp;0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eβ: -0.10, 95% CI: -1.14 \u0026ndash; 0.94, p\u0026thinsp;=\u0026thinsp;0.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eβ: 0.51, 95% CI: -0.56\u0026ndash;1.58,\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ: 0.68, 95% CI: -0.51\u0026ndash;1.87,\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eβ: 0.34, 95% CI: -0.81\u0026ndash;1.49,\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eApal β: 0.81, 95% CI: -3.51\u0026ndash;5.13, p\u0026thinsp;=\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTaql\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eβ: -0.00, 95% CI: -1.14\u0026ndash;1.14, p\u0026thinsp;=\u0026thinsp;1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eβ: 1.07, 95% CI: 0.02\u0026ndash;2.13\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.05*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eβ: -0.59, 95% CI: -1.70\u0026ndash;0.51,\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eβ: -1.03, 95% CI: -2.26\u0026ndash;0.19,\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eβ: -0.88,95% CI: -2.06\u0026ndash;0.23\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTaql β: -1.13, 95% CI: -5.53\u0026ndash;3.27, p\u0026thinsp;=\u0026thinsp;0.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eBeta coefficients (β), 95% confidence intervals (CI), and p-values represent the associations between gestational 25-hydroxyvitamin D (25(OH)D) concentrations, offspring vitamin D receptor (VDR) genotypes (Bsml, Fok1, Apal, Taql), and Australian Scale for Autism Spectrum Conditions (ASASC) total and subscale scores.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWhile overall early and late pregnancy 25(OH)D levels were not associated with any subscales (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e), interaction analyses identified higher early 25(OH)D levels were associated with decreased fact orientation scores in males in both adjusted and crude models (Crude β: -0.05, 95% CI -0.08 \u0026ndash; -0.01, p\u0026thinsp;=\u0026thinsp;0.02, Adjusted β: -0.05, 95% CI -0.09 \u0026ndash; -0.01, p\u0026thinsp;=\u0026thinsp;0.02). No significant association was observed in females (p\u0026thinsp;=\u0026thinsp;0.88). No further interaction was observed in both early and late 25(OH)D and ASASC total scores or subscale scores in either sex (all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis longitudinal study did not detect an association between gestational vitamin D concentrations at any timepoint and offspring ASASC scores. This is in contrast with studies which have reported associations in cases of diagnosed autism (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). VDR genotype additionally did not affect total ASASC scores in offspring. A combination of factors may account for the null relationship between total ASASC scores, maternal vitamin D concentration, and offspring VDR genotype. The total ASASC score represents a composite of diverse behavioural domains, where trait-specific effects may have been diluted as suggested by the subscale-specific findings at present. The lack of a direct comparison with diagnosed cases of autism in the broader literature may complicate findings when evaluating separate outcome measures. Further, our relatively small sample size may not reflect changes in vitamin D concentrations large enough to influence ASASC scores across the cohort. Low variability in vitamin D concentration was also observed across our cohort, where concentrations were generally sufficient. This in turn may obscure the true effect of VDR polymorphisms which are possibly contingent on low/deficient vitamin D exposure, as main-effect modelling may fail to capture these conditional associations.\u003c/p\u003e \u003cp\u003eWhile secondary analyses of ASASC subscales, polymorphisms and maternal vitamin D concentrations revealed null associations, limited evidence presented an association between maternal vitamin D, the Taql VDR polymorphism and autism traits in offspring. Elevated early, 25(OH)D levels were associated with decreased fact orientation scores in males in both adjusted and crude models, leaning towards neurotypical scores in accordance with ASASC guidelines. Formal autism diagnosis is based on key core characteristics in accordance with the Diagnostic and Statistical Manual of Mental Disorders 5th Edition (DSM-5), or the International Classification of Diseases 11th Revision (ICD-11) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Core characteristics include communication and social interaction differences, and the presence of restrictive and repetitive behaviours and interests (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). While the ASASC scale does not constitute an autism diagnosis, items within the fact-orienting dimension can be mapped onto the DSM-5 criterion of restricted, repetitive patterns of behaviour, interests or activities (RRBs) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Behaviours characteristic of this criterion include highly restricted, fixated interests that are atypical in intensity or focus, further broken down into an insistence on sameness, inflexible adherence to routines, or ritualized patterns of verbal or nonverbal behaviour (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral neural processes and mechanisms are suggested to influence RRBs, including executive function, and upstream neural dysregulation in the cortico-striatal-thalamo-cortical circuit and cerebellum (\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). While vitamin D deficiency is associated with structural and functional brain abnormalities, its effect on mechanistic drivers of RRBs remains largely unexplored (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). However, animal studies have linked behaviours characteristic of RRBs to deficient vitamin D in utero (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Increased self-grooming in vitamin D deficient rat pups exposed to low vitamin D in utero was observed in one study, potentially overlapping with RRBs observed in autism (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). In a subsequent study, male mice exposed to vitamin D deficiency in utero exhibited increased repetitive behaviours and disrupted social interaction (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). In human studies of vitamin D supplementation, a 13% reduction in stereotypical behaviours (categorised as RRBs) was observed in children diagnosed with autism in comparison to control participants (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). A meta-analysis of children with autism additionally reported significant improvement in stereotypical behaviours following vitamin D supplementation (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). However, this effect has not been researched in relation to maternal vitamin D status. The potential of vitamin D supplementation to ameliorate autism symptoms is additionally not consistently reported, likely owing to varying ages, populations and questionnaires utilised across studies (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eReduced fact orienting scores were further only observed in early pregnancy, a critical period in neurodevelopment particularly sensitive to changes in gestational vitamin D (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Reduced fact-orientation scores were additionally only observed in male offspring. The male to female ratio of autism prevalence is close to 3:1 and is influenced by societal and biological factors (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Autism criteria were initially developed on symptoms presented primarily in males, resulting in missed or inapplicable diagnostic and symptomatic measures in females (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Sex differences in symptom presentation are further observed, namely across RRBs which are less predictive of autism in females (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Additionally, presentation of autism symptoms, including RRBs vary based on sex, age, cognitive level and co-occurring mental health conditions (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe homozygous recessive (tt) genotype of the Taql polymorphism was associated with elevated fact orientation scores in the crude model, suggesting the presence of traits consistent with autism. Interestingly, Coşkun et al., 2016 reported the frequency of the same genotype was higher in children with autism (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Cieślińska et al., 2017 additionally reported decreased autism incidence in association with the dominant allele of the Taql VDR polymorphism (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Taken together, this pattern may be consistent with the dominant allele\u0026rsquo;s exertion of a neuroprotective effect when compared with the recessive variant (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). While research relating to the influence of Taql and neurodevelopment is sparse, the Taql variant is evidenced to confer both elevated or decreased risk of neurodegenerative disease, suggesting the role of Taql both as a risk and protective factor in neurological processes (\u003cspan additionalcitationids=\"CR52 CR53\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Variants of the Taql polymorphism are associated with increased mRNA stability, potentially enhancing vitamin D signalling (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Activation of VDR by its ligand is necessary for mediation of neurogenic and synaptic activity of vitamin D in the brain (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). The efficacy of this response may be dictated by VDR genotype, where Taql heterozygous and recessive genotypes were associated with a better response to vitamin D supplementation in one study (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). However, the effect observed in this study occurred irrespective of maternal vitamin D status, similar to two studies associating Taql with autism where offspring vitamin D concentration was controlled (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). As such, the exact Taql genotypes and variants broadly associated with increased autism risk or symptomatology remain unclear. However, known Taql associations may be the result of linkage disequilibrium with other VDR polymorphisms known to influence VDR expression and activity (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Our finding was once again only observed in the fact orientation ASASC subscale, providing limited evidence of singular genetic factors in the influence phenotypic expression of autism, as has been previously observed in RRBs (\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). Further research into the molecular pathways exerted by the Taql polymorphism is warranted to underpin the genotypes and variants associated with autism risk/symptomatology.\u003c/p\u003e \u003cp\u003eSeveral limitations present in the current study. While the ASASC does not provide an autism diagnosis, its subscales provide insight into autism traits, symptom variability, and their relationship to vitamin D and VDR polymorphisms. The continuous nature of the scale reflects characteristics also present in the general population, such as RRBs which present in other neurodevelopmental conditions (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). As such, ASASC outcomes may be applied beyond the autism population. However, the utility of the present findings in relation to autism risk should be interpreted with caution in the absence of diagnostic information. Parental report in the assessment of autism traits relies on subjective interpretation of behavioural traits, introducing variability in the precision of reported behaviours. Further, the use of the ASASC as a measure of autism symptomatology carries unique outcomes and measures based on the Australian population (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Variations in results across studies may be attributed to differences in utilised scales and questionnaires, given that use of the ASASC in the literature is limited. The limited sample size in the current study may restrict our ability to detect associations between maternal vitamin D, related receptor polymorphisms, and autism traits. As a result, adjustment for multiple comparisons was not performed to avoid further increasing the risk of Type II error. However, in the absence of multiple-comparison adjustment, the observed associations may partly reflect Type I errors arising from the number of tests conducted. A larger cohort with longer follow-up, clinically confirmed autism diagnoses, and offspring vitamin D concentrations would ideally be required to further elucidate the current observations.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eContrary to our \u003cem\u003ea priori\u003c/em\u003e hypothesis, the findings at present showed no association between gestational vitamin D, VDR polymorphisms, and total ASASC scores. However, limited evidence suggests that gestational vitamin D and the Taql polymorphism may influence specific autism-related behaviours, such as fact-orientation, corresponding with the DSM-5 criteria for restricted and repetitive patterns of behaviour, interests, or activities. Interpreted within the constraints of study limitations, initial insight suggests that while vitamin D may not affect aggregate autism symptomatology, it may modulate specific behavioural traits, highlighting the potential role of genetic factors in shaping the phenotypic expression of autism. The results of this study should be interpreted as hypothesis generating, with validation required in larger, independent cohorts.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eFunding for the initial phases of the VIP study were provided by a project grant from the National Health and Medical Research Council (NHMRC) of Australia. The 11-year follow up of the VIP study was funded by the Bupa Foundation.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eDeclaration of Interests\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthical Information\u003c/h2\u003e \u003cp\u003eThis study was approved by Barwon Health Human Research Ethics Committee 01/42_E2 (11/02/2013) and acknowledged by Deakin University 2013\u0026thinsp;\u0026minus;\u0026thinsp;116 (17/05/2013). All participants provided full informed written consent on behalf of themselves and their children. At the 11-year follow-up, all child participants provided optional assent.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCorrespondence\u003c/strong\u003e \u003cp\u003eEsma Fazlić, HERB Level 3 (Deakin University-Barwon Health) 285\u0026ndash;299 Ryrie St, Geelong VIC 3220 Australia; Email: [email protected]\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors were involved in the formulation of the research question. Authors JP, NH, and LW were involved with the inception of the initial cohort and regulated the collection and measurement of biological samples. Authors EF and NH conducted statistical analyses. EF prepared the main manuscript text and figures. All authors provided initial edits to first and subsequent drafts and provided critical feedback on the structure and content.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to thank the participants of the VIP study for their ongoing participation. Funding for the initial phases of the VIP study were provided by a project grant from the National Health and Medical Research Council (NHMRC) of Australia. The 11-year follow up of the VIP study was funded by the Bupa Foundation.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author [E.F] upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNair, R., Maseeh, A. \u0026amp; Vitamin, D. The sunshine vitamin. \u003cem\u003eJ. Pharmacol. Pharmacother\u003c/em\u003e. \u003cb\u003e3\u003c/b\u003e (2), 118\u0026ndash;126 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ\u0026auml;pelt, R. B. \u0026amp; Jakobsen, J. Vitamin D in plants: a review of occurrence, analysis, and biosynthesis. \u003cem\u003eFront. Plant. Sci.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, 136 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan den Heuvel, E. G., Lips, P., Schoonmade, L. J., Lanham-New, S. A. \u0026amp; van Schoor, N. M. Comparison of the Effect of Daily Vitamin D2 and Vitamin D3 Supplementation on Serum 25-Hydroxyvitamin D Concentration (Total 25(OH)D, 25(OH)D2, and 25(OH)D3) and Importance of Body Mass Index: A Systematic Review and Meta-Analysis. \u003cem\u003eAdv. Nutr.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (1), 100133 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBikle Daniel, D., Vitamin, D. \u0026amp; Metabolism Mechanism of Action, and Clinical Applications. \u003cem\u003eChem. Biol.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e (3), 319\u0026ndash;329 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSommer, I. et al. Vitamin D deficiency as a risk factor for dementia: a systematic review and meta-analysis. \u003cem\u003eBMC Geriatr.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e (1), 16 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang, Y. et al. Association of vitamin D with risk of dementia: a dose-response meta-analysis of observational studies. \u003cem\u003eFront. Neurol.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e, 1649841 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeitl, V. et al. Vitamin D and Neurotrophin Levels and Their Impact on the Symptoms of Schizophrenia. \u003cem\u003eNeuropsychobiology\u003c/em\u003e \u003cb\u003e79\u003c/b\u003e (3), 179\u0026ndash;185 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePansri, P. et al. Brain-derived neurotrophic factor increases cell number of neural progenitor cells derived from human induced pluripotent stem cells. \u003cem\u003ePeerJ\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, e11388 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalabresi, P., Picconi, B., Tozzi, A. \u0026amp; Di Filippo, M. Dopamine-mediated regulation of corticostriatal synaptic plasticity. \u003cem\u003eTrends Neurosci.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e (5), 211\u0026ndash;219 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMansouri, F. et al. Protective effects of vitamin D on learning and memory deficit induced by scopolamine in male rats: the roles of brain-derived neurotrophic factor and oxidative stress. \u003cem\u003eNaunyn Schmiedebergs Arch. Pharmacol.\u003c/em\u003e \u003cb\u003e394\u003c/b\u003e (7), 1451\u0026ndash;1466 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalvez-Contreras, A. Y., Campos-Ordonez, T., Gonzalez-Castaneda, R. E. \u0026amp; Gonzalez-Perez, O. Alterations of Growth Factors in Autism and Attention-Deficit/Hyperactivity Disorder. \u003cem\u003eFront. Psychiatry\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e, 126 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArlington, V. \u0026amp; Association, A. P. Diagnostic and statistical manual of mental disorders. \u003cem\u003eAm. Psychiatric Association\u003c/em\u003e. \u003cb\u003e5\u003c/b\u003e, 612\u0026ndash;613 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTirani, S. A., Balali, A., Askari, G. \u0026amp; Saneei, P. Maternal serum 25-hydroxy vitamin D levels and risk of autism spectrum and attention-deficit hyperactivity disorders in offspring: A systematic review and dose-response meta-analysis. \u003cem\u003ePsychiatry Res.\u003c/em\u003e \u003cb\u003e319\u003c/b\u003e, 114977 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, B. K. et al. Developmental vitamin D and autism spectrum disorders: findings from the Stockholm Youth Cohort. \u003cem\u003eMol. Psychiatry\u003c/em\u003e. \u003cb\u003e26\u003c/b\u003e (5), 1578\u0026ndash;1588 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagnusson, C. et al. Maternal vitamin D deficiency and the risk of autism spectrum disorders: population-based study. \u003cem\u003eBJPsych Open.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e (2), 170\u0026ndash;172 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadley-Dowd, P. et al. Maternal vitamin D during pregnancy and offspring autism and autism-associated traits: a prospective cohort study. \u003cem\u003eMol. Autism\u003c/em\u003e. \u003cb\u003e13\u003c/b\u003e (1), 44 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePr\u0026uuml;fer, K., Veenstra, T. D., Jirikowski, G. F. \u0026amp; Kumar, R. Distribution of 1,25-dihydroxyvitamin D3 receptor immunoreactivity in the rat brain and spinal cord. \u003cem\u003eJ. Chem. Neuroanat.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e (2), 135\u0026ndash;145 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmolders, J. et al. Expression of Vitamin D Receptor and Metabolizing Enzymes in Multiple Sclerosis\u0026mdash;Affected Brain Tissue. \u003cem\u003eJ. Neuropathology Experimental Neurol.\u003c/em\u003e \u003cb\u003e72\u003c/b\u003e (2), 91\u0026ndash;105 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong, N. et al. The Impact of Vitamin D Receptor Gene Polymorphisms on the Susceptibility of Diabetic Vascular Complications: A Meta-Analysis. \u003cem\u003eGenet. Test. Mol. Biomarkers\u003c/em\u003e. \u003cb\u003e23\u003c/b\u003e (8), 533\u0026ndash;556 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValdivielso, J. M. \u0026amp; Fernandez, E. Vitamin D receptor polymorphisms and diseases. \u003cem\u003eClin. Chim. Acta\u003c/em\u003e. \u003cb\u003e371\u003c/b\u003e (1), 1\u0026ndash;12 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUitterlinden, A. G., Fang, Y., van Meurs, J. B. J., Pols, H. A. P. \u0026amp; van Leeuwen, J. P. T. M. Genetics and biology of vitamin D receptor polymorphisms. \u003cem\u003eGene\u003c/em\u003e \u003cb\u003e338\u003c/b\u003e (2), 143\u0026ndash;156 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKerr Whitfield, G. et al. Functionally relevant polymorphisms in the human nuclear vitamin D receptor gene. \u003cem\u003eMol. Cell. Endocrinol.\u003c/em\u003e \u003cb\u003e177\u003c/b\u003e (1), 145\u0026ndash;159 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArai, H. et al. A Vitamin D Receptor Gene Polymorphism in the Translation Initiation Codon: Effect on Protein Activity and Relation to Bone Mineral Density in Japanese Women. \u003cem\u003eJ. Bone Miner. Res.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e (6), 915\u0026ndash;921 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIngles, S. A. et al. Strength of linkage disequilibrium between two vitamin D receptor markers in five ethnic groups: implications for association studies. \u003cem\u003eCancer Epidemiol. Biomarkers Prev.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e (2), 93\u0026ndash;98 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUsategui-Mart\u0026iacute;n, R. et al. Receptor (VDR) Gene Polymorphisms Modify the Response to Vitamin D Supplementation: A Systematic Review and Meta-Analysis. \u003cem\u003eNutrients\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (2), 360 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarras, S. N. et al. Vitamin D receptor Fokl polymorphism is a determinant of both maternal and neonatal vitamin D concentrations at birth. \u003cem\u003eJ. Steroid Biochem. Mol. Biol.\u003c/em\u003e \u003cb\u003e199\u003c/b\u003e, 105568 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuerini, F. R. et al. Vitamin D Receptor Polymorphisms Associated with Autism Spectrum Disorder. \u003cem\u003eAutism Res.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e (5), 680\u0026ndash;690 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaechua, C. et al. Impact of gene polymorphisms involved in the vitamin D metabolic pathway on the susceptibility to and severity of autism spectrum disorder. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (1), 28333 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCieślińska, A. et al. Vitamin D Receptor Gene Polymorphisms Associated with Childhood Autism. \u003cem\u003eBrain Sci.\u003c/em\u003e ;\u003cb\u003e7\u003c/b\u003e(9). (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorley, R., Carlin, J. B., Pasco, J. A., Wark, J. D. \u0026amp; Ponsonby, A. L. Maternal 25-hydroxyvitamin D concentration and offspring birth size: effect modification by infant VDR genotype. \u003cem\u003eEur. J. Clin. Nutr.\u003c/em\u003e \u003cb\u003e63\u003c/b\u003e (6), 802\u0026ndash;804 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHyde, N. K. et al. The Vitamin D in Pregnancy Study: a prospective prebirth cohort in southern Australia. \u003cem\u003eBMJ Open.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e (8), e036003 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarnett, M. S., Attwood, T., Peterson, C. \u0026amp; Kelly, A. B. Autism spectrum conditions among children and adolescents: A new profiling tool. \u003cem\u003eAustralian J. Psychol.\u003c/em\u003e \u003cb\u003e65\u003c/b\u003e (4), 206\u0026ndash;213 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Health, O. \u003cem\u003eICD-11: international statistical classification of diseases and related health problems : eleventh revision\u003c/em\u003e 2nd edn (World Health Organization, 2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGraybiel, A. M. \u0026amp; Grafton, S. T. The striatum: where skills and habits meet. \u003cem\u003eCold Spring Harb Perspect. Biol.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e (8), a021691 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLopez, B. R., Lincoln, A. J., Ozonoff, S. \u0026amp; Lai, Z. Examining the Relationship between Executive Functions and Restricted, Repetitive Symptoms of Autistic Disorder. \u003cem\u003eJ. Autism Dev. Disord.\u003c/em\u003e \u003cb\u003e35\u003c/b\u003e (4), 445\u0026ndash;460 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRojas, D. C. et al. Regional gray matter volumetric changes in autism associated with social and repetitive behavior symptoms. \u003cem\u003eBMC Psychiatry\u003c/em\u003e. \u003cb\u003e6\u003c/b\u003e, 56 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Z., Ding, R. \u0026amp; Wang, J. The Association between Vitamin D Status and Autism Spectrum Disorder (ASD): A Systematic Review and Meta-Analysis. \u003cem\u003eNutrients\u003c/em\u003e ;\u003cb\u003e13\u003c/b\u003e(1). (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamang, M. K. et al. Developmental vitamin D-deficiency produces autism-relevant behaviours and gut-health associated alterations in a rat model. \u003cem\u003eTranslational Psychiatry\u003c/em\u003e. \u003cb\u003e13\u003c/b\u003e (1), 204 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui, J. et al. Induction of autism-related behavior in male mice by early-life vitamin D deficiency: association with disruption of the gut microbial composition and homeostasis. \u003cem\u003eFood Funct.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (8), 4338\u0026ndash;4353 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoradi, H., Sohrabi, M., Taheri, H., Khodashenas, E. \u0026amp; Movahedi, A. Comparison of the effects of perceptual-motor exercises, vitamin D supplementation and the combination of these interventions on decreasing stereotypical behavior in children with autism disorder. \u003cem\u003eInt. J. Dev. Disabil.\u003c/em\u003e \u003cb\u003e66\u003c/b\u003e (2), 122\u0026ndash;132 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, M. et al. Effects of Vitamin D Supplementation on Children with Autism Spectrum Disorder: A Systematic Review and Meta-analysis. \u003cem\u003eClin. Psychopharmacol. Neurosci.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e (2), 240\u0026ndash;251 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKittana, M., Ahmadani, A., Stojanovska, L. \u0026amp; Attlee, A. The Role of Vitamin D Supplementation in Children with Autism Spectrum Disorder: A Narrative Review. \u003cem\u003eNutrients\u003c/em\u003e ;\u003cb\u003e14\u003c/b\u003e(1). (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeck, C. et al. Maternal vitamin D status, fetal growth patterns, and adverse pregnancy outcomes in a multisite prospective pregnancy cohort. \u003cem\u003eAm. J. Clin. Nutr.\u003c/em\u003e \u003cb\u003e121\u003c/b\u003e (2), 376\u0026ndash;384 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, S. B. et al. Maternal vitamin D deficiency in early pregnancy and perinatal and long-term outcomes. \u003cem\u003eHeliyon\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e (9), e19367 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoomes, R., Hull, L. \u0026amp; Mandy, W. P. L. What Is the Male-to-Female Ratio in Autism Spectrum Disorder? A Systematic Review and Meta-Analysis. \u003cem\u003eJ. Am. Acad. Child. Adolesc. Psychiatry\u003c/em\u003e. \u003cb\u003e56\u003c/b\u003e (6), 466\u0026ndash;474 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHalladay, A. K. et al. Sex and gender differences in autism spectrum disorder: summarizing evidence gaps and identifying emerging areas of priority. \u003cem\u003eMol. Autism\u003c/em\u003e. \u003cb\u003e6\u003c/b\u003e (1), 36 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcFayden, T. C., Albright, J., Muskett, A. E. \u0026amp; Scarpa, A. Brief Report: Sex Differences in ASD Diagnosis-A Brief Report on Restricted Interests and Repetitive Behaviors. \u003cem\u003eJ. Autism Dev. Disord\u003c/em\u003e. \u003cb\u003e49\u003c/b\u003e (4), 1693\u0026ndash;1699 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarrop, C. et al. Restricted and Repetitive Behaviors in Autism Spectrum Disorders and Typical Development: Cross-Sectional and Longitudinal Comparisons. \u003cem\u003eJ. Autism Dev. Disord.\u003c/em\u003e \u003cb\u003e44\u003c/b\u003e (5), 1207\u0026ndash;1219 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJasim, S. \u0026amp; Perry, A. Repetitive and restricted behaviors and interests in autism spectrum disorder: relation to individual characteristics and mental health problems. \u003cem\u003eBMC Psychiatry\u003c/em\u003e. \u003cb\u003e23\u003c/b\u003e (1), 356 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoşkun, S., Şimşek, Ş., Camkurt, M. A., \u0026Ccedil;im, A. \u0026amp; \u0026Ccedil;elik, S. B. Association of polymorphisms in the vitamin D receptor gene and serum 25-hydroxyvitamin D levels in children with autism spectrum disorder. \u003cem\u003eGene\u003c/em\u003e \u003cb\u003e588\u003c/b\u003e (2), 109\u0026ndash;114 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eŁaczmański, Ł. et al. Vitamin D receptor gene polymorphisms in Alzheimer's disease patients. \u003cem\u003eExp. Gerontol.\u003c/em\u003e \u003cb\u003e69\u003c/b\u003e, 142\u0026ndash;147 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTarkesh Esfahani, N., Rahgozar, M., Biglarian, A. \u0026amp; Khorram Khorshid, H. R. Identification of genetic polymorphism interactions in sporadic alzheimer\u0026rsquo;s disease using logic regression. \u003cem\u003eIran. Rehabilitation J.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e (2), 45\u0026ndash;50 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLehmann, D. J. et al. The vitamin D receptor gene is associated with Alzheimer's disease. \u003cem\u003eNeurosci. Lett.\u003c/em\u003e \u003cb\u003e504\u003c/b\u003e (2), 79\u0026ndash;82 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDimitrakis, E. et al. Association of vitamin D receptor gene TaqI polymorphism with Alzheimer's disease in a Southeastern European Caucasian population. \u003cem\u003eExp. Ther. Med.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e (5), 341 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoradkhani, A. et al. Association of vitamin D receptor genetic polymorphisms with the risk of infertility: a systematic review and meta-analysis. \u003cem\u003eBMC Pregnancy Childbirth\u003c/em\u003e. \u003cb\u003e24\u003c/b\u003e (1), 398 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEyles, D. W. \u0026amp; Vitamin, D. Brain and Behavior. \u003cem\u003eJBMR Plus\u003c/em\u003e. \u003cb\u003e5\u003c/b\u003e (1), e10419 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeza-Meza, M. R. et al. Vitamin D Receptor (VDR) Genetic Variants: Relationship of FokI Genotypes with VDR Expression and Clinical Disease Activity in Systemic Lupus Erythematosus Patients. \u003cem\u003eGenes (Basel)\u003c/em\u003e ;\u003cb\u003e13\u003c/b\u003e(11). (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThakkinstian, A., D'Este, C. \u0026amp; Attia, J. Haplotype analysis of VDR gene polymorphisms: a meta-analysis. \u003cem\u003eOsteoporos. Int.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (9), 729\u0026ndash;734 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlaser, B. et al. Identification of a potential Bipolar risk haplotype in the gene encoding the winged-helix transcription factor RFX4. \u003cem\u003eMol. Psychiatry\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e (10), 920\u0026ndash;927 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWelch, J. M. et al. Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e448\u003c/b\u003e (7156), 894\u0026ndash;900 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePe\u0026ccedil;a, J. et al. Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e472\u003c/b\u003e (7344), 437\u0026ndash;442 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatson, J. L., Dempsey, T. \u0026amp; Fodstad, J. C. Stereotypies and repetitive/restrictive behaviours in infants with autism and pervasive developmental disorder. \u003cem\u003eDev. Neurorehabilitation\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e (3), 122\u0026ndash;127 (2009).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Autism, Vitamin D, Polymorphisms, Gestation, Neurodevelopment","lastPublishedDoi":"10.21203/rs.3.rs-9216765/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9216765/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe relationship between gestational vitamin D status and offspring autism remains inconsistent, potentially due to genetic differences in vitamin D receptor (VDR) interactions. This exploratory study examined associations between maternal serum vitamin D concentrations, offspring VDR genotypes (BsmI, Fok1, ApaI, TaqI), and autism symptomatology using the Australian Scale for Autism Spectrum Conditions (ASASC) in 192 mother\u0026ndash;child pairs at 11-years follow-up. No association was found between gestational vitamin D levels and total ASASC scores, nor between VDR genotype and total ASASC scores. However, secondary subscale analyses revealed that higher early vitamin D concentrations were associated with decreased fact-orienting scores in males (β = \u0026minus;\u0026thinsp;0.05, 95% CI \u0026minus;\u0026thinsp;0.08, \u0026minus;\u0026thinsp;0.01, p\u0026thinsp;=\u0026thinsp;0.02). Additionally, the homozygous recessive TaqI genotype was associated with a 2.5-unit increase in fact-orienting scores compared to the homozygous dominant genotype (β\u0026thinsp;=\u0026thinsp;2.52, 95% CI 0.36, 4.69, p\u0026thinsp;=\u0026thinsp;0.02), though no interaction with gestational vitamin D was observed. While overall autism traits were unrelated to gestational vitamin D or VDR genotype, these exploratory findings suggest that specific autism-related traits may be influenced by maternal vitamin D levels and offspring genetic variation. This highlights the potential role of vitamin D gene-environment interactions in autism.\u003c/p\u003e","manuscriptTitle":"The Association Between Gestational Vitamin D Levels and Offspring Vitamin D Receptor Polymorphisms in Autism-Associated Traits","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-28 20:39:12","doi":"10.21203/rs.3.rs-9216765/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-14T07:40:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-13T14:24:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T16:10:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"264494391293802836621282161808230328386","date":"2026-04-21T13:32:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"73099528450212914746140535098877085268","date":"2026-04-21T05:38:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167037120166366388590222714315924448747","date":"2026-04-20T06:38:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-20T00:21:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-17T16:37:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-08T10:33:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-08T09:20:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"74fa9664-946f-42b3-94b8-20cd4703664d","owner":[],"postedDate":"April 28th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-14T07:40:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-13T14:24:00+00:00","index":67,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T16:10:52+00:00","index":66,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":66867802,"name":"Health sciences/Diseases"},{"id":66867803,"name":"Biological sciences/Genetics"},{"id":66867804,"name":"Health sciences/Medical research"},{"id":66867805,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-05-14T07:55:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-28 20:39:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9216765","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9216765","identity":"rs-9216765","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}