Massively parallel sequencing reveals the complex structure of an irradiated human chromosome on a mouse background in the Tc1 model of Down syndrome

Abstract

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype--phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439.

Figure 1. Copy number analysis of the Hsa21 in Tc1 mice.

(a) High resolution oligonucleotide microarray comparative genomic hybridisation of Hsa21 in Tc1 mice against a human male pool reference DNA. (b) High resolution oligonucleotide microarray comparative genomic hybridisation of DNA extracted from the cell line HT1080 from which the Tc1 Hsa21 was originally isolated against a human male pool reference DNA. (c) Read depth data for Tc1 Hsa21 from NGS mapped to human chromosome 21 reference from five read paired-end sequence libraries with all the data binned at 10 bp intervals.

Figure 2. Fluorescence in situ hybridisation characterisation of the structure of the Hsa21 in Tc1 mice.

(a) Hsa21 specific paint (green) c-hybridised with a Hsa13/21 alpha satellite centromere probe (red giving yellow signal). (b) Hsa21 telomere specific probe (green) co-hybridised with an Hsa13/21 alpha satellite centromere probe (red). (c) Human chromosome pan-telomeric probe (red) i.e. hybridises to all human and mouse pan telomere sequences, demonstrating that Hsa21 in the Tc1 is structurally altered and is metacentric.

Figure 3. Schematic of the proposed structure of Tc1-Hsa21.

Reference: Ideogram of human chromosome 21, numbers 1–41 indicate regions of Tc1 Hsa21 delineated according to Table S5. Tc1: rearranged structure of the Hsa21 in Tc1 mice. The order of regions 11, 20, 22, 24, 19, 18, 25, 17, 13, 28, 26, 6, 15, 35, 32, 5, 9, 10, 33, 38, 40, 36, 37, 31, 29, 23, 22, 21, 20, and 11 in this schematic are based on FISH mapping data (Fig. S2). The certainty of the rearrangement is indicated by a red line, solid line more certain, dotted line suggested. Inverted chromosome regions are indicated by the red arrow symbol. Region 12 is triplicated but the position of the other two copies is unknown. Position of region 27 is unknown. The positions of acrocentric regions 1, 2, 3, 7 and 8 are unknown and are placed arbitrarily. Regions 26, 30 and 41 are duplications and their positions are suggested by FISH within the resolution of the technique. Regions 14, 16, 34 and 39 are deleted.