PMC6173455_01
Summarized karyotype, WG-MPS, Sanger sequencing, RT-PCR and X-inactivation results from Case 1.. A) Partial ideograms showing the normal and derivative (der) chromosomes (chr) X (purple) and 1 (orange) (not to scale) in Case 1. Xq13 and 1p31 breakpoints are indicated by arrows. B) Derivative translocation junction sequences (middle line) and matching reference sequences (top and bottom lines) as identified by Sanger sequencing. Microhomology is highlighted in yellow, deleted sequences are underlined, and duplicated sequences are in bold letters. C) Schematic illustration of the Topologically Associated Domain (TAD) structure at the POU3F4 locus (indicated with a dashed line and a blue bar) as created by the 3D Genome Browser (http://promoter.bx.psu.edu/hi-c/). Each dot within individual TADs reflects the interaction between two DNA positions. The corresponding region from the UCSC Genome Browser was aligned underneath the heat map. This illustrates among other genes POU3F4 (red rectangle), the der(X) breakpoint identified in the current study mapping ~87kb upstream POU3F4 (vertical red arrow), as well as specific enhancers identified by Naranjo et al., 2010 upstream POU3F4 (red vertical bars). D) RT-PCR results showing absence of POU3F4 expression in the patient as compared with a normal control. cDNA = RNA reverse transcribed; RNA = RNA not reverse transcribed; H2O = no template sample. E) X-inactivation analysis results showing a skewed X-inactivation pattern (100:0). A PCR product is obtained only from the inactive (methylated) chromosomes. After HpaII digestion, only one peak was shown in the female patient, whereas, no peak was observed in the male control.
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Case 1 is a female patient with hearing loss and a de novo t(X;1)(q13;p31) translocation (Fig 1A) as detected by previous karyotype and FISH analyses. Table 1). The der(1) breakpoint disrupted the non-pathogenic leucine rich repeats and IQ motif containing 3 (LRRIQ3) gene (intron 7/7) (NM_001105659.1), while the der(X) breakpoint mapped ~87kb upstream the POU class 3 homeobox 4 (POU3F4) gene (OMIM-300039) (NM_000307.4) (Fig 1C; Table 1). In addition, RT-PCR analysis revealed absence of POU3F4 expression in the patient as compared with a normal control (Fig 1D). Finally, a highly skewed X-inactivation pattern (100:0) was observed in Case 1 after pre-digestion with the methylation-sensitive restriction enzyme HpaII (Fig 1E).
PMC6173455_02
Summarized WG-MPS and Sanger sequencing results in Case 2.. A) Circos plot illustrating partial chr6, chr7, chr8, and chr12 ideograms (outer circle), deletions (red) and duplications (blue) based on the depth of coverage analysis (inner circle) as well as inter-translocation (green lines) and intra-translocation (purple lines) junctions identified by WG-MPS in Case 2. SOX5 disruption by a heterozygous deletion and two inter-translocation breakpoints is highlighted. B) Derivative chromosome reconstruction of the chromothripsis rearrangement. Fragments from chromosomes 6, 7, 8, and 12 are in red, green, blue, and yellow, respectively. Arrows: fragment orientations; black asterisks: translocation junctions sequenced to the base-pair level; red asterisks: translocation junctions amplified but not sequenced. C) Derivative translocation junction sequences (middle line) and matching reference sequences (top and bottom lines) as identified by Sanger sequencing in Case 2. Microhomology is highlighted, and inserted sequences not aligning to either chromosome are in lower-case letters.
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This chr8 deletion was also identified from the depth of coverage analysis in this study, which additionally detected a patient-specific ~108kb heterozygous deletion on 12p12.1 overlapping exons 1-3 of the SRY-box 5 (SOX5) gene (OMIM-604975) (NM_006940.4) (Fig 2A). Interestingly, by using WG-MPS, cryptic complexity and a chromothripsis rearrangement was revealed including thirty-eight interchromosomal and intrachromosomal translocation junctions in total (Fig 2A) (Table 1; S2 Table). After accurate derivative chromosome reconstruction of the complex rearrangement (Fig 2B), a number of translocation breakpoints (marked with a black asterisk in Fig 2B) were validated by Sanger sequencing (Fig 2C; Table 1). In some of these cases, a PCR product was obtained by long-range PCR; however, breakpoint mapping to the base-pair level with Sanger sequencing was not feasible (marked with a red asterisk in Fig 2B).
PMC6173455_03
Summarized WG-MPS and Sanger sequencing results in Case 3.. A) Partial ideograms showing the normal and derivative (der) chromosomes (chr) 4 (dark purple) and 9 (brown) (not to scale) in Case 3. 4q26 and 9p24 breakpoints are indicated by arrows. B) Derivative translocation junction sequences (middle line) and matching reference sequences (top and bottom lines) as identified by Sanger sequencing. Microhomology is highlighted in yellow, deleted sequences are underlined, duplicated sequences are in bold letters, and inserted sequences not aligning to either chromosome are in lower-case letters. C) IGV screenshot illustrating the der(9) breakpoint disrupting the NFIB gene within intron 10/10 (NM_001190737.1).
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Initial karyotype analysis detected a de novo apparently balanced t(4;9)(q26;p24) translocation (Fig 3A). Table 1). The der(4) breakpoint did not disrupt any genes, whereas the der(9) breakpoint directly disrupted intron 10/10 of the Nuclear Factor I B (NFIB) gene (OMIM-600728) (NM_001190737.1) (Fig 3C).