Genetic Clue to Autism's Fourfold Difference In Boys and Girls

By Dr. Chinta SidharthanReviewed by Lauren HardakerOct 22 2026

Rare changes in a gene on the X chromosome altered its activity in human neurons, offering new clues to why autism is diagnosed four times more often in boys than girls.

Study: A massively parallel reporter assay of MECP2cis-regulatory elements reveals genetic candidates for male-biased autism. Image credit: MIA Studio/Shutterstock.com

Autism is diagnosed about four times more often in males than in females, yet the reasons behind this gap remain unclear. In a recent study published in The American Journal of Human Genetics, a team of researchers examined the regulatory region of the X-linked gene MECP2, which encodes methyl-CpG-binding protein 2, and tested whether small changes in this control region could help explain why autism appears more frequently in boys and men.

Why X-Linked Genes May Influence Autism Risk

Autism is a heritable condition marked by repetitive or restricted behaviors and altered social interactions. Males are diagnosed four times more often than females. Earlier genetic work found that several genes showing the strongest sex differences in harmful new mutations among people with neurodevelopmental disorders are located on the X chromosome, with these mutations occurring more frequently in affected females. Males with the same gene variants often have more severe neurodevelopmental conditions that are underrepresented in autism cohorts, while females may be partly protected by having a second X chromosome. 

One of these genes, MECP2, is sensitive to dosage. Too little of its protein causes Rett syndrome in girls and severe newborn brain disease in boys, while extra copies cause a separate syndrome. Mouse studies suggest that moderate shifts in protein level produce milder traits resembling autism.

Researchers Screen Thousands of Families for Rare MECP2 Variants

In this study, the research team examined whether subtle changes in the regulatory deoxyribonucleic acid (DNA) of MECP2 contribute to autism. They analyzed whole-genome sequencing data from 30,916 people in autism cohorts of the Centers for Common Disease Genomics, comprising 8,245 families, 9,437 autistic individuals who defined each family (proband), and 4,989 unaffected siblings.

Variants were called with two separate programs, and only those identified by both were retained. The team then searched five regulatory regions of MECP2 for rare variants that appeared in a single family and were transmitted by an unaffected mother to a male proband, filtering for quality and sequencing depth. This yielded 22 variants.

Candidate cis-regulatory elements (CREs) were also selected from open chromatin maps of the developing human prefrontal cortex. They comprised the promoter, two elements described earlier in mice (CRE 2 and CRE 6), and two newly identified ones (CRE A and CRE B). Each was divided into overlapping 270-base-pair tiles. In each tile, we disrupted predicted transcription factor binding sites, the proteins that control gene activity, by shuffling their letters twice. The library also held the 22 variants, 19 control variants seen in at least 50 presumably neurotypical males in a public population database, and control sequences of known activity.

The library was tested with a massively parallel reporter assay (MPRA), in which each sequence controls a reporter gene tagged with a random barcode, allowing activity to be read by comparing ribonucleic acid (RNA) with DNA counts. It was delivered by lentivirus into neurons grown from induced pluripotent stem cells (iPSCs). Statistical tests identified shuffles and variants that changed activity, and motif matching predicted which proteins were involved. Various assays, along with gene silencing and editing methods, were used to validate the results.

Rare MECP2 Variants Alter Gene Regulation in Neurons

The study found that two rare variants in the noncoding, regulatory regions of MECP2, both inherited from unaffected mothers, altered regulatory activity. One variant, inherited by two autistic brothers, weakened the activity of a regulatory region that normally suppresses gene expression. The other, found in an autistic male, reduced MECP2 RNA and protein levels by approximately 30% in laboratory-grown human neurons.

The assay mapped active binding sites in four of the five regions. The CRE 2 enhancer held four activator sites and three repressor sites, while CRE B had none in neurons. Silencing the genes Transcription Factor 3 (TCF3) and Zinc Finger Protein 281 (ZNF281) lowered MECP2 RNA by about 55% and 47%, respectively, after three weeks.

Furthermore, a promoter variant from a male proband with autism and attention-deficit hyperactivity disorder (ADHD) but no features of Rett syndrome lowered reporter activity. His unaffected brother did not carry it.

In neurons engineered to carry the promoter variant, binding of nuclear factor Y (NFY), a protein complex that helps start gene activity, at the MECP2 promoter ranked in the 51st percentile among bound promoters in unedited neurons and the 9th in edited ones. MECP2 RNA and protein both dropped by about 30% in these laboratory-grown neurons. Previous mouse studies found that a similar reduction in Mecp2 expression produced social difficulties, hyperactivity, and anxiety-like behaviors. Expression of the downstream gene EBF3 also fell by about 50%, while SLC2A1 expression reduced by about 10%.

These results suggest that mild changes in gene control may be tolerated by carrier mothers but contribute to autism in sons who have only one X chromosome. The researchers propose that similar variants in other X chromosome genes could help explain some of autism's male predominance and previously unexplained genetic risk. The authors cautioned that reporter assays may not fully capture how regulatory DNA functions in its natural genomic environment. The experiments also focused on one type of laboratory-grown neuron, and additional genetic cases and molecular studies will be needed to confirm the variants' contribution to autism.

X-Linked Regulatory Variants May Help Explain Autism Risk

In summary, the study showed that small changes in the regulatory DNA of MECP2 could alter its activity and were associated with autism in males. The researchers refined the regulatory map of the gene and tied a promoter variant to a 30% reduction in MECP2 levels. The findings provide a framework for searching other X chromosome genes for similar variants that might explain part of the sex difference in autism.

Reference

Meyer-Schuman, R., Cherry, F., Sui, Y., Papastathopoulos-Katsaros, A., Zhong, Y., Li, Y., Wang, T., Hennick, K., Karunakaran, D., Berk-Rauch, H., Liu, Z., Chakravarti, A., Nowakowski, T., Eichler, E. E., & Zoghbi, H. (n.d.). A massively parallel reporter assay of MECP2 cis-regulatory elements reveals genetic candidates for male-biased autism. The American Journal of Human Genetics. DOI:10.1016/j.ajhg.2026.09.011. https://www.cell.com/ajhg/fulltext/S0002-9297(26)00352-6