Constitutional copy number amplifications: rare or under-evaluated? Revisiting a 25-year-old cold case

Abstract

We reanalyzed through a cytogenomics approach a case published 20 years ago, describing a girl with developmental delay and epilepsy. Karyotype and FISH analysis showed a de novo 2.3 Mb terminal inverted-duplication at 8q24.3. The interpretation was inconsistent with the absence of a more distal deletion as expected for distal inverted duplications, and it was inconceivable to highlight rearrangements smaller than 5-10 Mb at that time. Chromosomal microarray (CMA), optical genome mapping (OGM), and short-read whole genome sequencing (srWGS) identified a complex configuration at 8q24.3, which resembles events like chromoanasynthesis or DUP-TRP/INV-DUP (duplication-triplication/inverted-duplication), both characterized by clustered duplications and triplications, some of which are inverted. In the EBV-line genes located in the amplified regions were overexpressed. Despite a more precise definition of the imbalance, we were unable to provide a clear-cut explanation for the proband's clinical features.

Fig. 1. Characterisation and interpretation of the 8q24.3 rearrangement.

A Cytogenetics and cytogenomics data: a Cut-out of chromosome 8 from a 400-banding karyotype; b CMA, RT-PCR and OGM of the 8q24.3 region showing nearly identical profiles. Numbers represent the log2 ratio (ADM2) of CMA and fractional copy numbers of OGM. B Genome sequencing details (a) NGS coverage plot (50x) of the 8q24.3 region (chr8:142,664,805-145,138,635, hg38) in control and the proband showing the duplicated (lighter blue box), triplicated (blue box), and amplified regions (darker blue boc) present in the patient only; quantification of copy number gains was based on the increased coverage in the plot (see Table S1). The three light gray boxes, present both in the control and the proband, indicate portions with poor coverage. b Schematic drawing of the order of the shattered fragments, according to hg38. In white, neutral copy number fragments (A, C, E, f, T); in red, deleted fragments (B, F, G); in light blue, the duplicated fragment D; in blue, the triplicated fragments H, h, I, L, S, and in dark blue, the amplified ones M, N, n, O, P, q, R, s. Left or right-oriented arrows represent discordant reads as they appear when aligned back to the reference genome (hg38). Note that hN junction one breakpoint lies within a clusted of segmental duplication (see Table S3) c Partial reconstruction of rearranged blocks; numbers within circles correspond to the breakpoint junctions verified by Sanger sequencing analysis (see Fig. S4). C The plausible replication-based mechanism of the 8q24.4 rearrangement. The schematic drawing indicates the 8q normal chromosome segmented according to the breakpoints (see panels B and Table S1). In the proposed replication pathway, arrows in the normal orientation (from left to right) or inverted (from right to left) denote the 8q24.3 regions shuffled on the patient’s chromosome 8q. The dashed lines indicate the movement from one replication fork to another, either in the normal or reverse orientation. At the bottom is a diagram of the final new rearranged chromosome 8 [rea(8q)], where the distal portion 8q24.3 presents inversions, deletions, duplication, triplication, and amplification. It is not certain where the block of fragments CfDO and DEnMNqLMNnOPQqRrST are inserted.

Fig. 2. Genome view of the rearranged genomic region 8q24.3 spanning the distal 2.5 Mb of chromosome 8q (GRCh38/hg38).

a The colored horizontal lines represent the rearranged segments of our patient’s identified by genome sequencing. Each color indicates the copy number status: gray means normal copy numbers; red denotes deletions; light blue indicates duplication; blue represents triplication, and dark blue amplification. b Magnified view of the 8q24.3 DUP/TRIP/AMP. From top to bottom: UCSC genes (GRCh38/hg38); triplosensitivity genes (pTriplo) map: in red circles those having a probability of triplosensitivity (pTriplo ≥0.94) [17]; segmental duplications >1000 bps.