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SUPPLEMENTARY INFORMATION
SUPPLEMENTARY METHODS
Topological Domains and Genes Predicted to Show Haploinsufficiency Bordering
DGAP242 Translocation Breakpoints
Six Mb windows surrounding the chromosomal breakpoints were analyzed for topologically
associating domains (TADs), gene annotation, predicted haploinsufficiency (HI), OMIMassociated phenotypes and inner ear expression. TADs were predicted using human embryonic
stem cell Hi-C domains from the Hi-C project (chromosome.sdsc.edu) and converted to hg19 for
comparison to the breakpoints.1 Gene annotation was obtained from the University of California
Santa Cruz Genome Browser (genome.ucsc.edu).2 If the breakpoint occurred within the open
reading frame of any gene, that gene was considered disrupted. If a gene resided within the TAD,
it was evaluated for potential dysregulation. To assess pathogenicity, HI prediction was
downloaded from DECIPHER (decipher.sanger.ac.uk).3 Only disrupted or dysregulated genes
with HI scores of ≤10% were considered as possibly pathogenic. Inner ear cell-type specific gene
expression was determined using the Shared Harvard Inner Ear Laboratory Database (SHIELD)
(shield.hms.harvard.edu).4,5 Possible effects of disrupted or dysregulated pathogenic genes were
determined by assessment of OMIM phenotypes from the Online Mendelian Inheritance in Man
Catalog of Human Genes and Genetic Disorders (www.omim.org) and mapping of phenotypes to
genes implicated within TAD regions.6
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SUPPLEMENTARY FIGURES
Supplementary Figure 1. Topologically associating domains (TADs) disrupted by DGAP242’s
chromosomal translocation and genes predicted to show haploinsufficiency (HI). The top row is
the chromosome section, containing the banding patterns of the 6 Mb chromosome region
surrounding the breakpoint (red arrowhead). The second row includes genes present in that
section of the chromosome (green rectangles). The third row corresponds to predicted HI genes
(red rectangles). The fourth row includes the topological domain data, with blue rectangles
representing Hi-C domains, and black rectangles representing domain boundary regions, or
regions where no contacts were detected. Units are in bp x 107. Panel A: Chromosome 1 (chr1)
TAD and HI presence. Predicted HI genes are PROX1, USH2A (OMIM #276901), ESRRG and
TGFB2 (OMIM #614816), which are located ~2.7, ~1.1, 0, and ~1.5 Mb from the translocation
breakpoint, respectively. Note that ESRRG is directly disrupted by the translocation. Panel B:
Chromosome 5 (chr5) TAD and HI presence. Predicted HI genes are NR2F1 (OMIM #615722),
FAM172A, KIAA0825, ANKRD32 and MCTP1, which are located ~625, ~108, 0, ~519, and ~483
kb from the translocation breakpoint, respectively. Note that KIAA0825 is directly disrupted by
the translocation. Gene transcript orientation for haploinsufficient genes is denoted by a forward
arrow (encoded on the positive reference strand) or reverse arrow (encoded on the negative
reference strand).
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SUPPLEMENTARY TABLES
Supplementary Table 1. Anchored primers for targeted locus amplification
Sequence (5’-3’)
Name
Target region
TLA1a
chr1_downstream_fw1
AACATTTACAAATCCCCAACAC
TLA1b
chr1_downstream_rev1
GGATCAGTGGTGCAGAAATA
TLA2a
chr1_upstream_fw1
GGACAGATTTGCACTTGTTAC
TLA2b
chr1_upstream_rev1
CTTGAAATCGCCAGCATTTG
TLA3a
chr5_downstream_fw1
TCAAGGTAATGAAATTCAGGGA
TLA3b
chr5_downstream_rev1
GTTCTCACTACTGACACCTTAA
TLA4a
chr5_upstream_fw1
TCAAGGAGTAAGACCCAGAG
TLA4b
chr5_upstream_rev1
AGGGTCTGGGTGCATATATT
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Supplementary Table 2. Expression of candidate genes in mouse inner ear hair cells in
ascending order of false discovery rate (FDR). Genes that both reside within a TAD disrupted by
the breakpoints and have a HI score ≤10% were assessed for enriched expression in hair cells,
using the SHIELD database (shield.hms.harvard.edu).4,5 An asterisk (*) denotes significantly
enriched expression levels in inner ear hair cells (FDR < 0.01).
Gene
Fold Enrichment in Hair Cells
False Discovery Rate (FDR)
USH2A*
24.76
2.91E-05
FAM172A*
2.93
2.18E-03
ESRRG*
4.55
8.20E-03
MCTP1
1.16
5.97E-02
PROX1
5.48
7.11E-02
KIAA0825
1.32
3.14E-01
ANKRD32
2.39
3.33E-01
NR2F1
1.23
6.80E-01
TGFB2
1.85
7.43E-01
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SUPPLEMENTARY DISCUSSION
Assessment of candidate genes in the breakpoint-containing TADs
When we extended our analysis to the identification of possible dysregulated genes by
examining the TADs disrupted by the breakpoints and identifying genes with a HI score ≤10%,
several additional predicted HI genes were identified within the chr1 and chr5 TAD breakpoint
regions: PROX1, USH2A, TGFB2, NR2F1, FAM172A, ANKRD32 and MCTP1 (Supplementary
Figure 1). Of these candidates, only USH2A, TGFB2 and NR2F1 are associated with an OMIM
phenotype (#276901 Usher Syndrome, Type IIA, USH2A; #614816 Loeys-Dietz Syndrome 4,
LDS4; and #615722 Bosch-Boonstra-Schaaf Optic Atrophy Syndrome, BBSOAS; respectively).
Of all three diseases, only Usher Syndrome has a phenotype of SNHL.
When we analyzed the inner ear expression pattern of these genes using data from the
SHIELD database, only USH2A and FAM172A have significantly enriched expression levels in
inner ear hair cells, suggesting a potential role for these genes in HL (Supplementary Table 2).4,5
However, of these genes, FAM172A is not predicted to be associated with pathogenicity and
USH2A is associated with an autosomal recessive disorder, which is inconsistent with the
predicted HI score (%HI = 4.17) and any dysregulation that may have occurred by the
heterozygous chromosomal rearrangement in DGAP242.
Etiology of the t(1;5)
The cause of DGAP242’s de novo translocation is unknown. One possibility is that the AT-rich
nature of the sequences at the chromosome 1 and 5 breakpoints made them more susceptible to
rearrangement. AT-rich sequences have been hypothesized to induce genome instability by
forming vulnerable secondary structures. Lesions in these regions could then be repaired by the
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non-homologous end-joining repair pathway, leading to improper ligation of non-homologous
chromosomes.7 Another possibility is that this translocation occurred by a microhomologymediated repair mechanism, which has been reported to underpin many non-recurrent
translocations.8 It has not escaped our attention that a 16 bp region of microhomology
(TGACCTTGGACAGCTG) resides 55 bp downstream of the chromosome 1 breakpoint and 643
bp downstream of the chromosome 5 breakpoint.
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SUPPLEMENTARY REFERENCES
1.
Dixon JR, Selvaraj S, Yue F et al: Topological domains in mammalian genomes
identified by analysis of chromatin interactions. Nature 2012; 485: 376-380.
2.
Rosenbloom KR, Armstrong J, Barber GP et al: The UCSC Genome Browser database:
2015 update. Nucleic Acids Res 2015; 43: D670-681.
3.
Huang N, Lee I, Marcotte EM, Hurles ME: Characterising and predicting
haploinsufficiency in the human genome. PLoS Genet 2010; 6: e1001154.
4.
Scheffer DI, Shen J, Corey DP, Chen ZY: Gene Expression by Mouse Inner Ear Hair
Cells during Development. J Neurosci 2015; 35: 6366-6380.
5.
Shen J, Scheffer DI, Kwan KY, Corey DP: SHIELD: an integrative gene expression
database for inner ear research. Database (Oxford) 2015; 2015: bav071.
6.
Online Mendelian Inheritance in Man, OMIM®. Johns Hopkins University (Baltimore,
MD): McKusick-Nathans Institute of Genetic Medicine, 2015.
7.
Kato T, Kurahashi H, Emanuel BS: Chromosomal translocations and palindromic ATrich repeats. Curr Opin Genet Dev 2012; 22: 221-228.
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8.
Verdin H, D'Haene B, Beysen D et al: Microhomology-mediated mechanisms underlie
non-recurrent disease-causing microdeletions of the FOXL2 gene or its regulatory
domain. PLoS Genet 2013; 9: e1003358.
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