Cytogenomic Characterization of a Novel de novo Balanced Reciprocal Translocation t(1;12) by Genome Sequencing Leading to Fusion Gene Formation of EYA3/EFCAB4b

Abstract

Introduction: The accurate detection of breakpoint regions of disease-associated chromosomal rearrangements helps understand the molecular mechanisms and identify the risks involved with disrupted genes.

Methods: In this study, a girl with growth retardation is characterized using positional cloning and genome sequencing. The techniques include fluorescence in situ hybridization (FISH) with paint (WCP) and bacterial-artificial chromosomes (BAC) probes, PCR, real-time PCR, and short and long-read sequencing.

Results: The translocation was identified by GTG banding and confirmed by WCP FISH. Microarray ruled out the involvement of other copy number variations except for 6 homozygous regions which are not disease-causing variants. Fine mapping with FISH showed split signals with BAC clone RP11-312A3. Genome sequencing of short-read with an average 30× depth and long-read sequencing technology with a 3.8× coverage identified both breakpoints, confirmed by Sanger sequencing, that showed microhomology. The breakpoint at 1p and 12p regions disrupted EYA3 and EFCAB4B genes. Expression analysis of EYA3 showed a 7-fold increase, suggesting the formation of a fusion gene with EFCAB4B. EYA3 is involved in skeleton development, and EFCAB4B plays a role in calcium metabolism, which may be relevant for the patient's phenotype.

Conclusion: The systematic application of genome techniques to translocations and their advantages is discussed.

Keywords: Balanced reciprocal translocation; FISH; Long-read sequencing; Paired-end sequencing; Short stature.

Fig. 1

a Ideograms of derivative chromosomes 1 and 12 and their normal homologs. b Partial karyogram showing the balanced reciprocal translocation t(1;12)(p36.1;p13.32). c Partial ideogram of the translocated chromosomes 1 and 12 showing normal genes on normal chromosomes and fusion genes on the derivative chromosomes 1 and 12. The breakpoint on the 1p region disrupted intron 1 of EYA3, and the 12p region breakpoint disrupted the 14th intron of EFCAB4B. The small fusion gene EYA3/EFCAB4B on der1 includes exon 1 of EYA3 and exon 15 of EFCAB4B and the large fusion gene EFCAM4B/EYA3 on der12 includes exon 2–18 of EYA3 and 1–14 of EFCAB4B. The blue arrow represents the orientation of the genes.

Fig. 2

a Physical map of the 12p13 region showing the BAC clones and the breakpoint region. b Physical map of the 1p36 region showing the BAC clones. c FISH with WCP 12 on the metaphase of the patient showing signals on chromosome 12, der12, and der1. d FISH with BAC clone RP11-62P15 showing signals on normal chromosome 12 and der1. e FISH with BAC clone RP11-689K16 showing signals on the normal chromosome 12 and der12. f FISH with breakpoint-spanning clone RP11-312A3 showing normal signals on normal chromosome 12 and split signals on both derivative chromosomes 1 and 12.

Fig. 3

a Pipeline showing the ID² oxford nanopore sequencing. b Overview of the read data of the BRT. c Pipeline for the short-read paired-end sequencing. d Overview of the read data of the BRT.

Fig. 4

a Sanger sequencing electropherogram of der1 showing the sequences of chromosome 1 (intron 1 of EYA3) in green, 3 bp microhomology (CCA) in yellow, and the sequences of chromosome 12 (intron 14 of EFCAB4B) in red of the junction sequence. b Sanger sequencing electropherogram of der12 showing the sequences of chromosome 12 (intron 14 of EFCAB4B) in red, 4 bp microhomology (CAGA) in yellow, and the sequences of chromosome 1 (intron 1 of EYA3) in green of the junction sequence. c Gel picture showing the junction fragment of 200 bp. d RTqPCR of EYA3 expression. e Gel picture showing the junction fragment of 300 bp of the 2nd breakpoint. f RTqPCR of EFCAB4B expression.