Supplemental Material for Heft et al., 2020

Sequences encoding Olduvai protein
domains (formerly DUF1220) show the greatest human lineage-specific increase in
copy number of any coding region in the genome and have been associated, in a
dosage-dependent manner, with brain size, cognitive aptitude, autism, and
schizophrenia. Tandem intragenic duplications of a three-domain block, termed
the Olduvai triplet, in four <i>NBPF</i> genes
in the chromosomal 1q21.1-.2 region are primarily responsible for the striking
human-specific copy number increase. Interestingly, most of the Olduvai
triplets are adjacent to, and transcriptionally co-regulated with, three
human-specific <i>NOTCH2NL</i> genes that have been shown to promote cortical
neurogenesis. Until now, the underlying genomic events that drove the Olduvai
hyper-amplification in humans have remained unexplained. Here, we show that the
presence or absence of an alternative first exon of the Olduvai triplet
perfectly discriminates between amplified (58/58) and unamplified (0/12)
triplets. We provide sequence and breakpoint analyses that suggest the
alternative exon was produced by an NAHR-based mechanism involving the
duplicative transposition of an existing Olduvai exon found in the CON3 domain
that typically occurs at the carboxy end of <i>NBPF</i>
genes. We also provide suggestive <i>in
vitro</i> evidence that the alternative exon may promote instability through a putative
G-quadraplex-based (pG4) mechanism. Lastly, we use single-molecule optical
mapping to characterize the intragenic structural variation observed in <i>NBPF</i> genes in 154 unrelated individuals
and 52 related individuals from 16 families and show that the presence of pG4-containing
Olduvai triplets is strongly correlated with high levels of Olduvai copy number
variation. These results suggest that the same driver of genomic instability
that allowed the evolutionarily recent, rapid, and extreme human-specific Olduvai
expansion remains highly active in the human genome.