A germline chimeric KANK1-DMRT1 transcript derived from a complex structural variant is associated with a congenital heart defect segregating across five generations

Abstract

Structural variants (SVs) pose a challenge to detect and interpret, but their study provides novel biological insights and molecular diagnosis underlying rare diseases. The aim of this study was to resolve a 9p24 rearrangement segregating in a family through five generations with a congenital heart defect (congenital pulmonary and aortic valvular stenosis, and pulmonary artery stenosis), by applying a combined genomic analysis. The analysis involved multiple techniques, including karyotype, chromosomal microarray analysis (CMA), FISH, whole-genome sequencing (WGS), RNA-seq and optical genome mapping (OGM). A complex 9p24 SV was hinted at by CMA results, showing three interspersed duplicated segments. Combined WGS and OGM analyses revealed that the 9p24 duplications constitute a complex SV, on which a set of breakpoints match the boundaries of the CMA duplicated sequences. The proposed structure for this complex rearrangement implies three duplications associated with an inversion of ~ 2Mb region on chromosome 9 with a SINE element insertion at the more distal breakpoint. Interestingly, this hypothesized genomic structure of rearrangement forms a chimeric transcript of the KANK1/DMRT1 loci, which was confirmed by RNA-seq on blood from 9p24 rearrangement carriers. Altogether with breakpoint amplification and FISH analysis, this combined approach allowed a deep characterization of this complex rearrangement. Although the genotype-phenotype correlation remains elusive from the molecular mechanism point of view, this study identified a large genomic rearrangement at 9p segregating with a familial congenital clinical trait, revealing a genetic biomarker that was successfully applied for embryo selection, changing the reproductive perspective of affected individuals.

Keywords: KANK1; congenital pulmonary and aortic valvular stenosis; germline quimeric transcripts; pulmonary artery stenosis; structural variation.

Figure 1. Chromosomal microarray analysis (CMA) revealed a complex pattern of 9p24 duplications

The plot shows the copy number profile (log₂ ratios, Y axis) of the distal region of the short arm of chromosome 9 (9p24), with probes (black dots) depicted according to their genomic coordinates (from pter to the centromere, X axis). To further refine the duplications breakpoints, we applied a CMA (array-CGH) based on a custom 44K (Agilent) platform covering at higher resolution the 9p sequences, which confirmed the presence of two adjacent 9p24.3 duplications (dup1 and dup2) and disclosed a third (dup3) one at 9p24.3 (blue shadows and dark blue horizontal lines in the 9p24 ideogram). Above the CNV plot, regions with polymorphic CNVs are presented (pink horizontal lines), as well as genes mapped to the segment (black lines) with respective exons. Image extracted from Nexus Copy Number software (Bionano).

Figure 2. Family pedigree showing five generations of individuals affected by an isolated congenital heart disease

The arrow indicates the proband (black symbols denoted affected individuals with pulmonary artery and aortic stenosis; orange is a female patient who was born only with heart murmur). All affected individuals evaluated by CMA were carriers of the 9p24 rearrangement (red asterisk), while evaluated normal family members were non-carriers (black minus symbol). The two alive affected individuals of the last generation were not tested. The two unaffected children of the proband were conceived by in vitro fertilization followed by embryo selection, based on the results of the genomic analysis described here.

Figure 3. Optical genome mapping reveals the structure of the 9p24 complex rearrangement

Single molecules view of proband DNA sample mapping to reference chromosome 9 with breakpoints mapped to DOCK8, KANK1 and DMRT1. GRCh38 reference chromosomes with OGM label patterns are shown in green, and assembled maps of hybrid molecules with label patterns are shown in light blue. Alignments between reference maps and hybrid molecules are shown as gray strings. Overlapping genes are depicted as orange bars. Image extracted from Acess software.

Figure 4. Assembling the complex 9p24 chromosome rearrangement

a. IGV screenshot showing WGS read coverage, genes, and breakpoint coordinates in 9p regions. The reference sequence and proposed rearrangement structure are shown below the screenshot, with duplicated segments depicted as colored arrows (red for dup1, green for dup2, and blue for dup3). Schematic representation of the breakpoints (represented by *) identified using WGS (b) and OGM (c) data.

Figure 5. Transcriptome data analysis in blood suggesting the formation of KANK1-DMRT1chimeric transcripts

The top track shows genomic coverage for two duplicated segments, dup1 and dup2. SA and SD tracks show probabilities of splice-acceptor (SA) and splice-donor (SD) sites inferred using the GENA tool (Fishman et al. 2023). The highlighted region (pale green) shows RNA-seq reads detected only in cases, and not in controls. The pairs of arrows above and below indicate, respectively, examples of reads supporting exon junctions of DMRT1 and KANK1, and only DMRT1exons.