Fig 1.
Isogenic trisomic-disomic mosaic Down syndrome study design.
An individual with mosaicism for trisomy 21 has both trisomic and disomic cells that originated from a single zygote as a constitutional finding. Thus, these trisomic and disomic cells have identical genetic backgrounds (except for the trisomy 21 imbalance) and identical environmental exposures. By measuring trisomic compared to disomic cellular attributes, individual genetic “background” variation is eliminated to allow for direct assessments of trisomy 21-specific influences.
Fig 2.
Cytokinesis-blocked micronucleus assay to quantify somatic cell instability frequencies in the trisomic compared to disomic cells from people with mosaicism for a trisomy 21 imbalance.
This diagram illustrates one mechanism (chromosome or chromatid lagging) whereby micronuclei can form. (A) During the metaphase of a mitotic division one chromatid from chromosome 21 fails to attach to the spindle fibers. As a result, this chromatid lags behind during the anaphase migration and fails to segregate to the spindle poles (left diagram). Following karyokinesis, the laggard chromosome could be excluded from the daughter cell nuclei and become enclosed in a micronucleus. In the right photomicrograph of a trisomic cell, at least a portion of chromosome 21 was excluded into a micronucleus (white arrow). Only 2 signals for the chromosome 21 probe are present in the right daughter nucleus (loss of a chromosome 21 signal) compared to 3 signals that are present in the left daughter nucleus (RUNX1 probe [21q22; green]; RUNX1T1 probe (8q22; red)]. In panel (B) trisomic binucleates (3 signals for the chromosome 21 probe) are shown (illustration on left; photomicrograph on right) that had loss of one replicated chromosome 8 (both sister chromatids) into a micronucleus, resulting in daughter cells that each had a monosomic imbalance for chromosome 8. (C) A disomic binucleated cell (both primary nuclei have 2 signals for the chromosome 21 and chromosome 8 probes) has a single micronucleus that does not contain chromatin for either the RUNX1 (21q22) or the RUNX1T1 (8q22) loci.
Fig 3.
Assessment of imbalances for the RUNX1 (21q22) and RUNX1T1 (8q22) loci in daughter binucleates from micronucleated cells.
This trisomic cell (total of 6 signals for chromosome 21; 4 signals for chromosome 8 [post-replication]) was categorized as “atypical” since the “daughter” binucleate pattern showed an imbalance that: (a) does not appear to result from the micronucleus formation (the micronucleus is 8q22-; 21q22-); and (b) arose from malsegregation events involving both chromosome 21 (pattern of 4 signals [right] and 2 signals [left]) and chromosome 8 (4 signals [right] and zero signals [left]).
Table 1.
Comparison of micronucleus frequencies in isogenic trisomic and disomic cells from 69 study participants with mosaicism.
Fig 4.
Relative proportions of trisomic and disomic cells containing micronuclei in the cross-sectional study.
The data from the isogenic cells of the 69 participants who have mosaicism are ordered by age (youngest on left; oldest on right). The relative proportion of trisomic cells having micronuclei is shown with blue histograms, while the relative proportion of disomic cells is shown with orange histograms. The trisomic cells showed significantly higher levels of micronuclei than the disomic cells (P = <0.0001).
Table 2.
Mixed effects Poisson regression models to assess factors associated with micronuclei in people with mosaicism for a trisomic imbalance of chromosome 21.
Fig 5.
Relative proportions of trisomic and disomic interphase nuclei showing atypical abnormal RUNX1 and/or RUNX1T1 probe signal patterns.
When compared to patterns in the disomic cells (orange histograms), the trisomic nuclei (blue histograms) showed significantly higher levels of atypical patterns (abnormal values involving more than a single chromosomal malsegregation event that were not clearly derived from the micronucleus formation) (p<0.0001).
Table 3.
Longitudinal assessment of micronuclei frequencies in participants with mosaic Down syndrome.
Fig 6.
Hypothesized biological cascade related to increased micronuclei frequencies in cells with a trisomy 21 imbalance.
Micronuclei can arise from a variety of mechanisms, including (but not limited to) factors that arise during mitosis (top of figure) (Guo et al., 2019). Due to nuclear envelope (NE) pores/gaps or rupture, micronuclei can be perceived as “cytoplasmic” DNA, triggering the cGAS-cGAMP-STING pathway, which in turn leads to the production of interferons. This “mark” targets the cells for senescence, the latter of which also contribute to inflammation (via SASP), thereby perpetuating the CIN-inflammation cycle. The micronuclei, either directly via genetic imbalance, or indirectly via inflammation and/or senescence, can contribute to alterations in gene expression. In turn, these alterations could contribute to the acquisition of age-related health conditions in people with Down syndrome or mosaic Down syndrome, but this conjectured relationship is not yet proven (as indicated by the?). SAC = Spindle Assembly Checkpoint; NE = Nuclear Envelope; ROS = reactive oxidative stress; SASP = Senescence Associated Secretory Phenotype.