PMC10434623_01
Presumed breakpoints in APC
(A) and TP63
(B) by integrative genomics viewer (IGV). Top box of IGV indicates chromosomal positions; middle, mapped read depth; bottom, mapped read sequences. Vertical red arrows indicate 3 (BP1-3) and 1 (BP4) breakpoints in APC and TP63, respectively, which were presumed by soft-clipped reads and read-depth gaps. Horizontal grey arrows indicate the positions and directions of primers for Sanger sequencing (
Supplementary Table S2
).
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f1
Figure 1 ). Following a tentative definition of DNA fragments APC-1 to -4 and TP63-1 and -2 based on the IGV review, we presumed the occurrence of a rearrangement between APC and TP63 and designed PCR primers for S2 ). Sanger sequencing was conducted using the BigDye Terminator v3.1 or v1.1 Cycle Sequencing Kit with an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). The SEQUENCHER v.5.4.6 (GENE CODES Corp., Ann Arbor, MI) was used to analyze and visualize the electropherograms. Sanger sequencing successfully validated each breakpoint and revealed a complex interchromosomal rearrangement between APC and TP63 ( Figures 2A-E  and  Supplementary Figure S1 ).
Sanger sequencing around presumed breakpoints in APC and TP63 and G-banding of chromosomes 3 and 5. (A, B) Electrophoresis of amplicons of target regions for Sanger sequencing. Arrow heads indicate the target size (A: 155bp, B: 474bp) of PCR products. (C-E) Sequencing chromatograms around presumed breakpoints and schematics of rearranged genomic fragments. Fragment labels correspond to those shown in 
Figures 1A and B
. (F, G) Right panels show G-banding images of paired chromosomes 3 (F) and 5 (G). Left panels show G-banding images of paired chromosomes 3 (F) and 5 (G). Right panels show chromosome schematics based on G-banding and spectral karyotyping (SKY) analyses.
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S2 ). Sanger sequencing was conducted using the BigDye Terminator v3.1 or v1.1 Cycle Sequencing Kit with an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). The SEQUENCHER v.5.4.6 (GENE CODES Corp., Ann Arbor, MI) was used to analyze and visualize the electropherograms. Sanger sequencing successfully validated each breakpoint and revealed a complex interchromosomal rearrangement between APC and TP63 ( Figures 2A-E  and  Supplementary Figure S1 ). In addition, G-band and spectral karyotyping (SKY) at a commercial laboratory identified derivative chromosomes 3 (der[3]) and 5 (der[5]), exhibiting presumed interchromosomal rearrangements by MGP testing, as well as a novel insertion of 5q15-22 into 3q12 in der[3] and a rearrangement between 5q15 and 3q28 in der[5] ( Figures 2F, G  and  Supplementary Figure S2 ), which were not identified in MGP testing and subsequent Sanger sequencing.
Breakpoints identified by long-read sequencing. IGV shows mapped reads with soft-clipped sequences around breakpoints on 3q12 (A), 5q15 (B), and 5q21.3 (C). identified by long-read sequencing. Fragments were tentatively defined, based on the breakpoints. Grey arrow heads indicate soft-clipped alternative reads mapped in IGV. The soft-clipped alternative sequences were confirmed by BLAT and schematically displayed by fragment arrows.
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f3
Table S4 ), in addition to the proven breakpoints in APC and TP63 ( Supplementary Figure S3 ). The APC structural variant was assumed to cause protein truncation of APC (p.Ser1340Ter) and to be a likely pathogenic variant.
Chromosomal rearrangements identified by a series of genomic analyses. Upper 2 panels represent the wild-type chromosomes 3 and 5 and fragments tentatively defined by a series of genomic analysis. Bottom 2 panels (der3 and der5) represent the derivative chromosomes 3 and 5. Red vertical bars represent an additional insertion variant which is identified in fragment X (
Supplementary Figure S4
) and an insertion of unknown origin, which is identified within the structural variant of APC.
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Figure S4 ,  Supplementary Table S4 ), located in an intronic region of the Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) gene. Finally, we present a schematic of the whole landscape of the structural variants of chromosomes 3 and 5 in  Figure 4  and  Supplementary Table S4 .