Genetic and functional characterization of inherited complex chromosomal rearrangements in a family with multisystem anomalies

Abstract

Purpose: Complex chromosomal rearrangements (CCRs) are rare structural variants involving 3 or more chromosomal breakpoints. Most de novo-reported CCRs pose challenges for diagnosis and management. They often require karyotyping, fluorescence in situ hybridization, and chromosomal microarray analysis (CMA) for clinical diagnosis because of the limitations of each method. Here, we report an inherited, exceptionally complex CCR involving 4 chromosomes and 13 breakpoints in a family with multisystem anomalies.

Methods: We evaluated the CCRs using karyotyping, fluorescence in situ hybridization, CMA, and 2 emerging genomic technologies: high-throughput chromosome conformation capture sequencing aka genomic proximity mapping and optical genome mapping. We also performed functional studies using transcriptome and methylome analyses.

Results: The proband, who had intellectual disability and immune deficiency, shared CCRs with her unaffected mother involving chromosomes 1, 7, and 11 by karyotyping. However, CMA revealed a duplication and 3 deletions in the proband, in contrast to her mother's balanced genome. High-throughput chromosome conformation capture sequencing aka genomic proximity mapping and optical genome mapping detected the CCRs and copy-number alterations but also uncovered additional breakpoints at high resolution, including an insertion in 4p and 2 cryptic rearrangements at 7p. Transcriptome and methylome analyses identified likely biological pathways associated with the proband's phenotypes.

Conclusion: Combining cytogenetic and genomic methods provided comprehensive characterization and defined the breakpoints at high resolution in both proband and mother. This underscores the value of novel cytogenetic and genomic techniques in deciphering complex genome rearrangements and the significance of integrative genomic analysis and functional characterization in understanding clinical phenotypes.

Keywords: Complex chromosomal rearrangements; Genomic proximity mapping (GPM); Hi-C; Optical genome mapping (OGM).

Figure 1

Results of G-banded chromosome analysis and single-nucleotide polymorphism (SNP) genomic microarray analysis of the proband and the proband’s mother. A. Karyotype of the proband. B. Four copy-number variants detected in the proband by SNP genomic microarray analysis with genomic coordinates on the x-axis and Log₂R and B-allele frequency (BAF) on the y-axis (see Table 1 for details). C. Karyotype of the proband's mother. The arrows in (A) and (C) point out the abnormal chromosomes 1, 7, and 11 in the proband and the proband’s mother, respectively.

Figure 2

Fluorescence in situ hybridization (FISH) analysis results of the proband’s mother (A) and the proband (B). Dual-color FISH using subtelomeric probes set on metaphase cells identified localizations of 1p (green) and 1q (red), 7p (green) and 7q (red), 11p (green) and 11q (red) on chromosome 1, der(1), chromosome 7, der(7), chromosome 11, and der(11). Schematic diagrams in the lower right corner of (A) and (B) show localization of FISH signals on ideograms of the respective rearranged chromosomes.

Figure 3

Optical genome mapping results in the proband and her mother. Circos plots display the complex chromosomal rearrangements (CCRs) in the proband (A) and the proband’s mother (C). The outer rings represent chromosomes 1-22, X and Y; inner rings show the CCRs detected. Circos tracks include cytoband, structural variant track, copy-number variant track (red deletion, blue duplication) and translocations. (B and D) Detailed views of rearrangements around the breakpoints. In the proband, a total of 5 translocations were shown (B). In the proband’s mother, 8 translocations observed, including 5 same as those in the proband (C) and 3 in the proband’s mother only (D).

Figure 4

GPM (Hi-C) results in the proband (A and B) and her mother (C and D). (A and C) Genome-wide differential Hi-C heatmaps represent the complex chromosomal rearrangements in the proband and her mother when compared with a normal female control. Red color indicates gain of interactions in the sample and blue color indicates loss of interactions in the sample. Chromosome by chromosome view of Hi-C heatmaps in the proband (B) and her mother (D): 4 pairs of inter-chromosomal interactions were shown for the proband (B), including chromosome 1 with 7, chromosome 1 with 11, chromosome 4 with 7, and chromosome 7 with 11; 3 pairs of interchromosomal interactions and one pair of intrachromosomal interactions specifically observed in the proband’s mother (D). Each left panel in (B) and (D) shows the original heatmap, and the right panel shows the representative ideogram of the translocation. PB, proband; PBM, proband’s mother.

Figure 5

Proposed model of complex chromosomal rearrangement (CCR) transmission from proband’s mother to the proband, resulting in unbalanced chromosomal regions. Postulated CCRs in the proband’s mother (A) and the proband (D) include chromosome 1, 4, 7, and 11. Although derivative chromosome 7p in the proband’s mother (A) comprises segments of 1q, 11q, and 7q, recombinant chromosome 7 rec(7) in the proband contains an intact 7p sequence. Hypothesized meiotic recombination event in the proband’s mother, in which 3-way CCR heterozygote forms a quadrivalent structure (B). Crossover occurs between homologous 7p and derivative 7p and followed by 2:2 alternative segregation (C). The 3 arrows in (A) indicate the novel structural variants identified by GPM and OGM.

Figure 6

Impact of the complex chromosomal rearrangements on gene expression and DNA methylation. Differential expression analysis of genes in copy-number variant regions by comparing the proband with normal female controls and proband’s mother (orange and blue dots, respectively). A. RNA sequencing identified pathways affected in the proband. Upregulated genes in immune response and downregulated genes in neuronal development. B. Functional pathways affected identified by top genes enriched by significant CpGs. C. Correlation of the DNA methylation profile with the RNA expression profile. Red color bar indicates the methylation change of downregulated genes and blue color bar indicates the methylation change of upregulated genes. FC, fold change; GO, Gene Ontology; ncRNA, non-coding RNA; tRNA, transfer RNA; UTR, untranslated region.