Biochemical and Sociodemographic Correlates of Autism Spectrum Disorder Severity in Pakistani Children: A Mixed-Methods Sibling-Matched Study

Background Autism spectrum disorder (ASD) arises from complex genetic and environmental interactions. Populations in South Asia remain underrepresented in ASD research despite high consanguinity rates, distinct cultural exposures, and widespread micronutrient deficiencies. Methods We employed a mixed-methods design incorporating a systematic literature review, parental interviews, and biochemical assays in a sibling-matched cohort. Serum 25-hydroxy-vitamin D and vitamin B12 were measured in 25 ASD-control sibling pairs. Sociodemographic predictors of ASD severity were evaluated in 48 children with ASD using binary logistic regression, with severity dichotomized as mild versus moderate and severe. Predictor variables included gender, family history of neurodevelopmental disorders, and screen time. Results Binary logistic regression indicated that children with a family history of neurodevelopmental disorders were significantly more likely to have moderate–severe ASD (p = 0.044), suggesting a genetic or familial contribution to symptom severity. Gender and screen time were not associated with severity. Biochemical analyses showed no significant differences in serum vitamin D or vitamin B12 between ASD cases and control siblings. However, within the ASD group, lower vitamin D concentrations correlated with higher clinical severity (ρ = -0.432), supporting a potential link between vitamin D status and symptom burden. Conclusions In this Pakistani cohort, a positive family history of neurodevelopmental conditions was the only sociodemographic factor associated with greater ASD severity, while lower serum vitamin D levels were also related to more severe symptoms. These findings highlight the potential interplay between genetic vulnerability and nutritional status, identifying vitamin D as a modifiable and low-cost target in ASD management. Larger, longitudinal studies are warranted to confirm these associations in South Asian populations.