Journal: medRxiv
Article Title: Gene-Pseudogene Inversions as a Hidden Source of Missing Heritability
doi: 10.1101/2025.10.01.25336578
Figure Lengend Snippet: Identification of SORD/SORD2P inversion as a common pathogenic allele in CMT-SORD. a) IGV visualization of the inversion breakpoint (intron 5 SORD2P and intron 4 SORD). Split and soft-clipped long-reads (red arrows) identify insertions/deletions of Alu elements specific to SORD or SORD2P, thus delineating the genomic interval of the breakpoints of the inversion between SORD and SORD2P (black boxes). Short-read sequencing fails to resolve the inversion due to high sequence homology. b) Schematic representation of the inversion’s genomic effect: the first four exons of SORD are replaced by the first five exons of SORD2P, which harbor multiple nonsense variants, leading to non-sense mediated decay and loss of SORD function. c) Optical Genome Mapping (OGM) of the inversion. The first breakpoint is located within the SORD2P pseudogene, while the second is within SORD. d) Zoom-in of the Hi-C ∼1 Mb region surrounding the SORD-SORD2P locus (skin fibroblasts; hg19; 5 kb resolution; raw counts) in a control (panel A; CTR) and the patient (panel B; proband N, SORD-CM). Statistically significant chromatin contacts identified by FitHiC2 are shown for both the control and patient samples. A patient-specific interaction (dark red curve, panel B) between chromosome 15 positions 45,132,499 and 45,352,501 was detected, consistent with the SORD/SORD2P inversion identified in the patient. Panel C displays a heatmap obtained with the subtraction method (SB map; patient - control), in which the inversion is visualized as a characteristic bow-tie pattern located distant from the matrix (arrows). e) Schematic representation of the breakpoint-specific PCR assay (top) and corresponding gel electrophoresis of PCR products from SORD-CMT patients and controls. Primers target divergent Alu elements in SORD2P intron 6 and SORD intron 5. In the reference genome, primers F1 and F2 are oriented in the same direction and ∼226 kb apart, precluding amplification. Upon inversion, F1 now lies ∼3.2 kb from F2 and in opposite direction, thus enabling amplification of a PCR product. Gel electrophoresis of breakpoint PCR assay shows the presence of a 3.2 Kb PCR product in probands P1 and P2 (heterozygous for SORD c.757delG and the inversion), but not in control (C1) or in probands that do not carry the inversion (S1–S3). f) Pie chart depicting the prevalence of SORD variants in a cohort of 156 CMT-SORD patients. SORD/SORD2P inversions represent the third most common pathogenic allele.
Article Snippet: Sequencing adaptors and indexes were added to DNA fragments using NEBNext Multiplex Oligos for Illumina kit (E7335 and E7500, New England BioLabs) and NEBNext Ultra II Q5 Master Mix (M0544, New England BioLabs).
Techniques: Sequencing, Hi-C, Control, Nucleic Acid Electrophoresis, Amplification
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