Grant, R. L. | bioRxiv Science

bioRxiv2026

Linked origins but distinct roles for extreme length and sequence variation at a tandem repeat in CACNA1C

Length variation in tandem repeats is a well-established driver of disease risk and is commonly assumed to arise from persistent genomic instability. Here, we characterize TRACT, a 30-bp variable number tandem repeat (VNTR) intronic to the calcium channel gene CACNA1C. TRACT exhibits extreme length variation (3-30+ kb) and has been previously linked to risk for bipolar disorder and schizophrenia. By examining multiple human cohorts, we find that TRACT alleles are strikingly bimodal in both length and sequence composition. Short alleles (TRACTS, [~]6 kb) and long alleles (TRACTL, [~]24 kb) are enriched for distinct 30-bp variants and are found on separate haplotypes that arose prior to the human migration out of Africa. Our data suggest that these ancient alleles expanded via perfect repeat tracts that were disrupted by accumulated mutations to result in relative length stability in extant humans, where there is no evidence for overt germline or somatic instability. Interestingly, neuropsychiatric disease risk is associated with specific 30-bp variants within TRACTS alleles, but not with overall TRACT length or with 30-bp variants enriched in TRACTL alleles. Instead, TRACTL alleles are associated with decreased gene expression in fibroblasts and testis. Together, these findings motivate joint examination of both sequence composition and length variation to fully understand the effects of VNTRs on evolution, trait variation, and disease risk.

genetics↗

bioRxiv2021

Genetic studies of human-chimpanzee divergence using stem cell fusions

Complete genome sequencing has identified millions of DNA changes that differ between humans and chimpanzees. Although a subset of these changes likely underlies important phenotypic differences between humans and chimpanzees, it is currently difficult to distinguish causal from incidental changes and to map specific phenotypes to particular genome locations. To facilitate further genetic study of human-chimpanzee divergence, we have generated human and chimpanzee auto-tetraploids and allo-tetraploids by fusing induced pluripotent stem cells (iPSCs) of each species. The resulting tetraploid iPSCs can be stably maintained and retain the ability to differentiate along ectoderm, mesoderm, and endoderm lineages. RNA sequencing identifies thousands of genes whose expression differs between humans and chimpanzees when assessed in single-species diploid or auto-tetraploid iPSCs. Analysis of gene expression patterns in inter-specific allo-tetraploid iPSCs shows that human-chimpanzee expression differences arise from substantial contributions of both cis-acting changes linked to the genes themselves, and trans-acting changes elsewhere in the genome. To enable further genetic mapping of species differences, we tested chemical treatments for stimulating genome-wide mitotic recombination between human and chimpanzee chromosomes, and CRISPR methods for inducing species-specific changes on particular chromosomes in allo-tetraploid cells. We successfully generated derivative cells with nested deletions or inter-specific recombination on the X chromosome. These studies identify a long distance cis-regulatory domain of the Fragile X-associated gene (FMR1), confirm an important role for the X chromosome in trans-regulation of other expression differences, and illustrate the potential of this system for more detailed mapping of the molecular basis of human and chimpanzee evolution. Significance StatementComparative studies of humans and chimpanzees have revealed many anatomical, physiological, behavioral, and molecular differences. However, it has been challenging to map these differences to particular chromosome regions. Here, we develop a genetic approach in fused stem cell lines that makes it possible to map human-chimpanzee molecular and cellular differences to specific regions of the genome. We illustrate this approach by mapping chromosome regions responsible for species-specific gene expression differences in fused tetraploid cells. This approach is general, and could be used in the future to map the genomic changes that control many other humanchimpanzee differences in various cell types or organoids in vitro.

evolutionary biology↗