Complete chromosome 21 centromere sequencing of families with Down syndrome reveals centromere size asymmetry

ABSTRACT Down syndrome, the most common form of human intellectual disability, is caused by nondisjunction and chromosome 21 trisomy (T21). Small centromeres have been hypothesized to contribute to its aetiology and studies on mammals suggest that larger centromeres are more efficiently transmitted, yet complete sequencing of chromosome 21 (chr21) centromeres has been particularly challenging. Using long-read sequencing, we sequenced and assembled the centromeres from eight families that include a child with free T21 (1 trio, 6 child–mother duos, and 1 singleton) all resulting from maternal meiosis I errors. Two of these families carry the smallest chr21 centromeres (143 and 181 kbp) observed in female individuals to date, exhibiting a ∼10.7- and ∼19.4-fold centromeric α-satellite higher-order repeat array size difference between the maternally inherited homologs, respectively. In both cases, the longer centromere harbors a poorly defined centromere dip region, marked by DNA hypomethylation, in the proband but not in the mother. A comparison of all proband chr21 centromeres (n=24) to those of controls (n=261) shows that small centromeres are not enriched in families with T21 (p-value=0.73); contrarily, chr21 extreme centromere size asymmetry (>10-fold) is unique of T21 (p-value=0.003), suggesting that this feature may represent a genetic risk factor for a subset of families with free T21. Additionally, phylogenetic reconstruction reveals that human chr21 has been particularly prone to such variation with some of the biggest size differences occurring over the last ∼17 thousand years of human evolution. Competing Interest Statement E.E.E. is a scientific advisory board (SAB) member of Variant Bio, Inc. Footnotes This version adds the sequencing data of 7 additional children with Trisomy 21 and 6 of their parents. It also adds several analysis, including IF-FISH experiments, a larger centromere characterization analysis in population samples, and a comparison of methylation data in blood and cell lines. Data from ChIP-seq analysis were removed from this updated version of the manuscript. LIST OF ABBREVIATIONS - CDR - centromere dip region - chr21 - chromosome 21 - FISH - fluorescence in situ hybridization - Gbp - Gigabase pairs - IF - immunofluorescence - kbp - kilobase pairs - kya - thousand years ago - H1/2/3/4 - haplotype 1/2/3/4 - HiFi - high fidelity - HGSVC - Human Genome Structural Variation Consortium - HPRC - Human Pangenome Reference Consortium - HOR - higher-order repeat - MMI/IIE - maternal meiosis I/II error - Mbp - Megabase pairs - PacBio - Pacific Biosciences - T21 - Trisomy 21 - T2T - telomere-to-telomere - UL-ONT - ultra-long Oxford Nanopore Technologies sequencing