Alpha Satellite Insertion Close to an Ancestral Centromeric Region

Abstract

Human centromeres are mainly composed of alpha satellite DNA hierarchically organized as higher-order repeats (HORs). Alpha satellite dynamics is shown by sequence homogenization in centromeric arrays and by its transfer to other centromeric locations, for example, during the maturation of new centromeres. We identified during prenatal aneuploidy diagnosis by fluorescent in situ hybridization a de novo insertion of alpha satellite DNA from the centromere of chromosome 18 (D18Z1) into cytoband 15q26. Although bound by CENP-B, this locus did not acquire centromeric functionality as demonstrated by the lack of constriction and the absence of CENP-A binding. The insertion was associated with a 2.8-kbp deletion and likely occurred in the paternal germline. The site was enriched in long terminal repeats and located ∼10 Mbp from the location where a centromere was ancestrally seeded and became inactive in the common ancestor of humans and apes 20-25 million years ago. Long-read mapping to the T2T-CHM13 human genome assembly revealed that the insertion derives from a specific region of chromosome 18 centromeric 12-mer HOR array in which the monomer size follows a regular pattern. The rearrangement did not directly disrupt any gene or predicted regulatory element and did not alter the methylation status of the surrounding region, consistent with the absence of phenotypic consequences in the carrier. This case demonstrates a likely rare but new class of structural variation that we name "alpha satellite insertion." It also expands our knowledge on alphoid DNA dynamics and conveys the possibility that alphoid arrays can relocate near vestigial centromeric sites.

Keywords: alpha satellite; ancestral centromere; structural variation.

Fig. 1.

D18Z1 alpha satellite de novo insertion. (A) FISH results of cultured amniocytes using alpha satellite DNA probes of chromosomes 15 (D15Z1, Texas-Red), 13/21 (D13/21Z1, green), and 18 (D18Z1, aqua), showing the hybridization of D18Z1 at 15q26. (B) FISH results of cultured amniocytes using the 15q25 BAC probes RP11-635O8 (red) and RP11-752G15 (green) flanking the ancestral centromere and the D18Z1 (aqua) probe. (C) Read length, repeat composition (color code in inset), and mapping location of the four selected HiFi and ONT reads (top). Dot plot (window size 20) of the 10-kb alpha satellite sequence from the centromere of chromosome 18 showing its tandem repetitive structure (bottom). (D) Schematic representation of the CHM13-T2T chromosome 18 centromere with its repeat composition (top). A heatmap representation of sequence identity over the region is presented below. The mapping location of the PacBio HiFi and ONT reads is pinpointed by black arrows.

Fig. 2.

Organization of the alpha satellite array. (A) Heatmaps of identity percentages between the 60 alpha satellite monomers derived from the T2T chr18:17500488–17510699 sequence, with monomers ordered either according to their position in the sequence (left) or as determined by clustering (right). In the latter, the position (1–12) in the 12-mer unit and type (D1 or D2) of the monomers are shown. (B) Boxplots of identity percentages between D1–D1, D2–D2, and D1–D2 monomer pairs. (C) Plot of monomer size with monomers ordered according to their position in the original sequence. (D) Schematic representation of the 12-mer HOR units.

Fig. 3.

Functional profiling of the rearrangement site. (A) UCSC view of the 100-kbp region surrounding the rearrangement at 15q26.1. The deleted region is highlighted in yellow, with deletion extremes corresponding to the satellite insertion positions. The GENCODE v32 and ENCODE regulation (H3K4me1, H3K4me3, and H3K27ac) tracks are shown (hg38). No gene and no enrichment of epigenetic marks found near regulatory elements are annotated in the deleted region. The closest gene, ST8SIA2, is mapped 32 kb distally. (B) Methylation pattern of the insertion site in the family trio obtained from the ONT selective sequencing. Methylated (red) and unmethylated (blue) CpGs are shown. The methylation profiles are similar among the family trio.

Fig. 4.

CENP-A and CENP-B immuno-FISH. Cohybridization of the D18Z1 probe (red) with antibodies against CENP-A (top) and CENP-B (bottom) proteins (green) on chromosome metaphases from the proband. The arrows point at the derivative chromosome 15 that is also shown in larger magnification in the insets.

Fig. 5.

(A) Schematic overview of the rearrangement. An alphoid array ∼50–300 kbp in size from the centromere of chromosome 18 inserted into an LTR-rich region of chromosome 15q26, ∼10 Mbp distally from the site where an ancestral centromere was seeded ∼25 Mya. This insertion was coupled with a 2.8-kbp deletion. Dashed lines pinpoint the boundaries of the synteny between chromosome 15 and the ancestral submetacentric chromosome; the dotted line indicates the position of the ancestral centromere. (B) Possible inferred model of alphoid DNA dynamics relative to centromere repositioning. Following a centromere repositioning event, the new centromere is epigenetically specified by CENP-A binding and subsequently acquires alphoid DNA. It is possible that not only centromeric function but also the presence of alphoid DNA can be resurrected at ancestral centromeric sites.