The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies

Abstract

Despite the clinical significance of balanced chromosomal abnormalities (BCAs), their characterization has largely been restricted to cytogenetic resolution. We explored the landscape of BCAs at nucleotide resolution in 273 subjects with a spectrum of congenital anomalies. Whole-genome sequencing revised 93% of karyotypes and demonstrated complexity that was cryptic to karyotyping in 21% of BCAs, highlighting the limitations of conventional cytogenetic approaches. At least 33.9% of BCAs resulted in gene disruption that likely contributed to the developmental phenotype, 5.2% were associated with pathogenic genomic imbalances, and 7.3% disrupted topologically associated domains (TADs) encompassing known syndromic loci. Remarkably, BCA breakpoints in eight subjects altered a single TAD encompassing MEF2C, a known driver of 5q14.3 microdeletion syndrome, resulting in decreased MEF2C expression. We propose that sequence-level resolution dramatically improves prediction of clinical outcomes for balanced rearrangements and provides insight into new pathogenic mechanisms, such as altered regulation due to changes in chromosome topology.

Figure 1

Characterization of BCAs detected by karyotyping at nucleotide resolution a. Genome-wide map of all BCA breakpoints identified in the cohort by whole-genome sequencing. One color is used per BCA to represent all rearrangement breakpoints in each subject. The scatter plot on the outside ring denotes breakpoint density per 1-Mb bin across the genome, with a blue arrow displaying the largest clustering of breakpoints at 5q14.3; b. Scatter plot summarizing the overall genomic imbalance associated with fully reconstructed BCAs at varying size thresholds. Curves represent the fraction of cases with final genomic imbalances greater than the corresponding size provided. Solid lines denote the final genomic imbalances for all BCAs, and are further delineated by deletions (red) or duplications (blue). The final genomic imbalances among fully mapped BCAs is also split between cases that have been pre-screened by CMA (dashed line) versus cases without CMA data (dotted line); c. Sequence signatures of BCA breakpoints. Histogram representing nucleotide signatures at the junction of 662 Sanger-validated breakpoints: inserted nucleotides, blunt ends, microhomology, or longer stretches of homology.

Figure 2

De novo BCAs associated with congenital anomalies disrupt functionally relevant loci. a. Boxplots illustrate specific gene-set enrichments at BCA breakpoints in subjects with congenital anomalies. Each boxplot represents the expected distribution (median, first and third quartiles) based on total intersections between 100,000 sets of simulated breakpoints and a particular gene-set. Red diamonds indicate the observed intersection values. Empirical Monte-Carlo P-values are indicated; b. Venn diagram showing the detailed overlap of disrupted genes previously associated with three neurodevelopmental phenotypes in amalgamated exome and CNV studies. In black: high-confidence genes (3 or more de novo LoF mutations reported), in grey: low-confidence genes (two de novo LoF mutations). c–e) Diagnostic yields associated with the overall cohort and multiple subgroups of BCAs. c. Diagnostic yield associated with all 248 mapped BCAs from subjects with congenital or developmental anomalies; d. Diagnostic yields partitioned by inheritance status; e. Diagnostic yields associated with BCAs depleted for large pathogenic CNVs thanks to CMA pre-screen compared to BCAs that had not been pre-screened by CMA.

Figure 3

Recurrent disruption of long-range regulatory interactions at the 5q14.3 locus. a. Genome-wide distribution of BCA breakpoints in the cohort across each 1-Mb bin. P-values correspond to observed vs. expected cluster sizes after 100,000 Monte Carlo randomizations. Corrected P-values are reported. One cluster, localized to 5q14.3, achieved genome-wide significance (threshold demarcated by red line); b. Hi-C profile and contact domains at the 5q14.3 locus derived from human LCLs. Overlapping Hi-C data suggests that the topology of the MEF2C-contact domain is altered in subjects carrying BCAs. Brain-expressed enhancers located in the region, loops involving MEF2C (yellow circles) and CTCF binding sites (green: forward, red: reverse) are indicated. Multiple pathogenic mechanisms converge on a similar syndrome: multi-genic deletions that encompass MEF2C along with one or both TAD boundaries (n=68), MEF2C-intragenic deletions (n=12) or LoF mutations, deletions that do not encompass MEF2C but overlap one TAD boundary (n=13), and BCA breakpoints distal to MEF2C (breakpoints from seven subjects reported in this study and three previously reported subjects)^,,.; c. Proposed model of the chromatin folding in the region defining a regulatory unit for MEF2C; d. Significantly decreased expression of MEF2C was observed in subjects harboring BCAs distal to MEF2C compared to controls. MEF2C-expression was measured by qRT-PCR, normalized against three endogenous genes and compared to the average MEF2C-expression from 16 age-matched controls (two-sided Wilcoxon rank-sum test: DGAP131, DGAP191, DGAP222: P=0.0085, DGAP218: P=0.0160). Individual expression values, median, first and third quartiles are indicated.

Figure 4

Correlations between phenotypes and genes disrupted in subjects harboring pathogenic BCAs. For each gene, the phenotypes reported in the corresponding subject were digitalized using HPO. One tile represents the normalized count of HPO terms belonging to each organ category reported in the subject(s). Genes clustered together when sharing similarly affected organs, from which five groups can be delineated: 1- genes associated with multiple nervous system and craniofacial abnormalities (dark blue); 2- genes connected to multiple neurological phenotypes (pink); 3- genes associated with craniofacial abnormalities and a few neurological symptoms (black); 4- genes associated with skeletal and limb abnormalities, and with limited neurological involvement (green); 5- genes without neurological involvement (light blue).