A massively parallel reporter assay of MECP2 cis-regulatory elements reveals genetic candidates for male-biased autism

Autism affects male individuals four times more often than female individuals, yet the basis of this sex bias remains unclear. One hypothesis is that this sex bias is due to genetic variants in X chromosome genes where haploinsufficiency causes neurodevelopmental disorders (NDDs). In heterozygous XX female probands, a complete loss-of-function variant in one of these genes often causes syndromic NDD, but the same variant is too deleterious for survival in hemizygous XY males. A mild hypomorphic variant, however, may be tolerated in a female carrier but may produce milder neurological phenotypes in a male proband. We tested this by investigating cis-regulatory elements (CREs) of MECP2, a dosage-sensitive gene on the X chromosome where total loss-of-function variants cause Rett syndrome in female children and severe, usually fatal neonatal encephalopathies in male infants. Using a massively parallel reporter assay in human neurons, we mapped transcription factor binding sites within MECP2 CREs and tested autism-associated variants for functional impact. We identified two noncoding variants that change CRE activity and are associated with male-biased autism phenotypes in the proband pedigrees. One of these, a promoter variant, disrupts NFY binding and reduces MECP2 expression by ∼30%, a magnitude that produces social deficits, hyperactivity, and anxiety-like phenotypes in mice. These findings suggest noncoding MECP2 variants can cause idiopathic, male-biased autism and provide a framework for uncovering regulatory variants in other X chromosome neurodevelopmental genes that may contribute to autism's missing heritability.