Fig. 4: Chromosomal rearrangement at the ASIP locus driving ectopic ASIP expression.
a, Schematic of the wild-type allele and the 183-kbp tandem duplication at the ASIP, AHCY and ITCH locus found in the patient. Locations of the breakpoints are presented according to the Dec. 2013 GRCh38/hg38 assembly. b, ASIP expression in PBLs in the patient, mother and father normalized to β-actin and TBP. c, Quantification of breakpoint-specific ITCH–ASIP fusion genomic DNA (gDNA) copy number in the patient, mother and father normalized to the copy number of β-actin gDNA. d, 5′-RACE–PCR from the patient and n = 2 controls (Ctr). The lower band contained the ITCH–ASIP fusion mRNA sequence depicted below. The upper bands are incompletely spliced mRNA variants. e,f, ASIP (e) and ITCH (f) expression across human tissues normalized to expression of β-actin and TBP. Tissue RNA pooled from several individuals was purchased TakaraBio (three technical replicates). g, Luciferase assays in HEK293 cells transfected with vectors with or without a 2.8-kbp ITCH promoter sequence upstream of the luciferase (Luc) reporter gene. Firefly Luc activity was normalized to Renilla Luc activity. Bar plots represent mean ± s.e.m. of n = 3 independent experiments. Statistical significance was assessed using two-sided Student’s t-test. h, Immunoblot of ASIP in cell lysates and supernatants from HEK293 cells transfected with the Taconic plasmid containing the ITCH–ASIP mRNA sequence under control of the 2.8-kbp ITCH promoter. i, Immunoblot of ASIP in cell lysates and supernatants from HEK293 cells transfected with plasmids containing the physiological ASIP coding sequence or the ITCH–ASIP fusion mRNA sequence downstream of no promoter or the ITCH promoter. Asterisk indicates a cloning artifact as an additional band due to introduction of an additional translation start codon upstream of the mRNA sequence from the restriction enzyme NcoI (C’CATGG). This generated a functional open reading frame, exclusively for the ITCH–ASIP mRNA sequence, leading to an ASIP protein with additional N-terminal amino acids. The results shown in h,i were each confirmed by a second independent experiment.
