Aneuploidy as a mechanism for stress-induced liver adaptation

Abstract

Over half of the mature hepatocytes in mice and humans are aneuploid and yet retain full ability to undergo mitosis. This observation has raised the question of whether this unusual somatic genetic variation evolved as an adaptive mechanism in response to hepatic injury. According to this model, hepatotoxic insults select for hepatocytes with specific numerical chromosome abnormalities, rendering them differentially resistant to injury. To test this hypothesis, we utilized a strain of mice heterozygous for a mutation in the homogentisic acid dioxygenase (Hgd) gene located on chromosome 16. Loss of the remaining Hgd allele protects from fumarylacetoacetate hydrolase (Fah) deficiency, a genetic liver disease model. When adult mice heterozygous for Hgd and lacking Fah were exposed to chronic liver damage, injury-resistant nodules consisting of Hgd-null hepatocytes rapidly emerged. To determine whether aneuploidy played a role in this phenomenon, array comparative genomic hybridization (aCGH) and metaphase karyotyping were performed. Strikingly, loss of chromosome 16 was dramatically enriched in all mice that became completely resistant to tyrosinemia-induced hepatic injury. The frequency of chromosome 16-specific aneuploidy was approximately 50%. This result indicates that selection of a specific aneuploid karyotype can result in the adaptation of hepatocytes to chronic liver injury. The extent to which aneuploidy promotes hepatic adaptation in humans remains under investigation.

Figure 1. Spontaneous liver repopulation in response to liver injury.

(A) Diagram of the tyrosine catabolic pathway. Critical components are highlighted in red. Fah deficiency leads to accumulation of fumarylacetoacetate and toxic metabolites. Fah deficiency is ameliorated by NTBC therapy or loss of Hgd. (B) Hgd^+/–Fah^–/– mice were subjected to selection by NTBC withdrawal and regenerating livers analyzed following partial and approximately complete repopulation. (C) During NTBC selection, Hgd^+/–Fah^–/– mice (black lines; n = 3) lost approximately 20% of their starting weight over 1 month and then achieved preselection weight in the subsequent 1 to 2 months. Hgd^–/–Fah^–/– mice (red lines; n = 3) maintained a constant weight. (D and E) Following 3 to 4 weeks of selection, hepatic nodules were dispersed randomly throughout livers from Hgd^+/–Fah^–/– mice. Nodules maintained a healthy appearance (D, arrows, inset is enlarged 250%) (n = 18) and were positive for the proliferation marker Ki67 (E, asterisks) (n = 6). Scale bar: 1 mm. (F) Following approximately complete repopulation, livers had normal coloration (left panel) and were confluent with large nodules (n = 5), which are best observed macroscopically after exsanguination (right panel).

Figure 2. Whole chromosome aneuploidy in hepatocytes.

(A) Hepatocytes isolated from healthy WT mice were karyotyped. The percentages of hepatocytes with chromosome-specific gains (gray bars) and losses (black bars) are indicated for young mice (ages 20 to 21 days; n = 3), adult mice (ages 4 to 5 months; n = 5), and senior mice (ages 10 to 15 months; n = 6). Percentages were derived from pooled data within each group. (B) Hepatocytes from fully repopulated Hgd^+/–Fah^–/– mice were karyotyped (n = 4); a representative profile is shown. (C) Karyotypes were also determined for hepatocytes from Fah^–/– mice that were off NTBC. Percentages were derived from pooled data (n = 4). (D) Chromosome 16 aneuploidy is indicated for all mice karyotyped. Data are shown as mean ± SEM. *P <0.005; **P = 0.03.

Figure 3. Copy number variation in pools of hepatocytes.

Alterations in chromosome copy number were assessed by aCGH analysis using populations enriched for hepatocytes isolated from WT mice (n = 2), Hgd^–/–Fah^–/– mice off NTBC (n = 2) and highly repopulated Hgd^+/–Fah^–/– mice off NTBC (n = 4). Hybridization intensity for hepatic chromosomes is plotted as log₂ ratio versus sex-mismatched diploid chromosomes (derived from splenocytes). The log₂ of –1 indicates chromosome loss (e.g., loss of the X chromosome in mouse 1), whereas log₂ of 1 indicates chromosome gain (e.g., gain of the X chromosome in mouse 2). Copy number changes in the Y chromosome due to gender mismatch were detected but not shown in the plots.

Figure 4. Loss of chromosome 16 in injury-resistant livers from Hgd^+/–Fah^–/– mice.

(A) Copy number variation from aCGH analysis is shown specifically for chromosome 16. Hybridization intensity for hepatic chromosomes is plotted as log₂ ratio versus sex-mismatched diploid chromosomes derived from splenocytes. Green dots indicate log₂ ratio of less than 0, and red dots indicate log₂ ratio of more than 0. The extent of chromosome loss is indicated along each plot (shaded purple). (B) Illustration of mosaicism for copy number loss in a heterogeneous population of cells as detected by aCGH. Whole chromosome loss and terminal deletion events are indicated. (C) Summary showing the maximum extent of chromosome 16 loss, which includes the Hgd locus.

Figure 5. Models of aneuploidy-mediated adaptation and hepatocyte expansion.

(A) Mechanism for adaptation to tryosinemia in Hgd^+/–Fah^–/– mice. Hgd heterozygotes have KO and WT alleles. Loss of WT Hgd promotes resistance to Fah deficiency and occurs by mutation of the WT allele, aneuploidy (i.e., loss chromosome 16 with the WT gene), or a combination of mutation and aneuploidy. Drawings represent diploid Fah^–/– hepatocytes and illustrate chromosome 16 copy number and Hgd status. (B) Model showing hepatocyte adaptation in response to chronic liver injury. Early in life, livers are primarily diploid and have the expected numbers of chromosomes (single black nuclei). During aging, hepatocytes polyploidize (binucleated cells) and become aneuploid (red, blue, green nuclei). Random aneuploidy affects nearly half of hepatocytes in mice and humans. Chronic liver injury can have multiple effects leading to either hepatocellular carcinoma (cells with white nuclei) or expansion injury-resistant hepatocytes (cells with red nuclei).