Fig 1: Whole-exome sequencing and Sanger sequencing revealed a missense point variant of HAS1. (A) Sanger sequencing confirmed a point variant in hearing-impaired family members. (B) Missense point variant on chromosome 19 in the hyaluronan synthase 1 (HAS1) gene (c.1082G>A). (C) “Damaging” and “disease-causing” were the predictions of in silico analyses by Sorting Intolerant from Tolerant (SIFT), Polymorphism Phenotyping v2 (PolyPhen2), Mutation Taster, and REVEL. TA, age at audiometry test; OA, age at onset of hearing impairment.
Fig 2: CircHYBID expression and hyaluronan (HA) accumulation were decreased by interleukin (IL)-1ß treatment. IL-6 (A), TNF-a (B), and HA (E) levels in cell culture supernatant were measured using enzyme-linked immunosorbent assay (ELISA). The mRNA expression of circHYBID (hsa_circ_0003893) (C), HA-binding protein involved in hyaluronan depolymerization (HYBID) (D), HA synthase 1 (HAS1), HA synthase 2 (HAS2), and HA synthase 3(HAS3) (F) were analyzed by quantitative real-time polymerase chain reaction in chondrocytes in the presence or absence of IL-1ß treatment. The protein expression of HAS1, HAS2, HAS3, and HYBID (G) was analyzed by Western blotting in chondrocytes in the presence or absence of IL-1ß treatment. GAPDH was used as the internal control. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Fig 3: Has1 expression in adult mouse cochlea. (A) Has1 expression was more confined within the stria vascularis (SV) in 7-week-old mice. (B) Magnified view of the organ of Corti in (A). The expression in hair cells was weaker than in the SV. Hensen’s and Claudius cells showed similarly weak expression of Has1. (C) Magnified view of the SV in (A). Has1 was robustly localized in the apical membrane of SV marginal cells. (D) Magnified image of the Rosenthal canal. Has1 was detected in some spiral ganglion cells. ICAM2, intercellular adhesion molecule 2; DAPI, 4',6-diamidino-2-phenylindole; OHC, outer hair cell; IHC, inner hair cell. Scale bar: 100 µm.
Fig 4: Has1 expression in neonatal mouse cochlea. (A) Postnatal day 3. Has1 was detected in the cochlea. Intercellular adhesion molecule 2 (ICAM2), an endothelial cell marker, indicates cochlear vessels. (B) Magnified view of the organ of Corti in (A) showing Has1 expression in the inner hair cell (IHC) and outer hair cells (OHCs). Has1 was also weakly expressed in supporting cells and inner sulcus cells. (C) Magnified image of the stria vascularis in (A). The apical membrane of marginal cells showed Has1 expression. (D) Magnified image of the spiral ganglion region in (A). Some spiral ganglion cells also expressed Has1. DAPI, 4',6-diamidino-2-phenylindole. Scale bar: 100 µm.
Fig 5: Hearing threshold elevation in Has1 knock-out (KO) mice. Hearing levels of 14-week-old Has1 KO mice. Female Has1 KO hearing levels were comparable to those of wild-type (WT) mice (female WT: n=230, female KO: n=2). Male Has1 KO mice showed significant elevations in their hearing thresholds (male WT: n=228, male KO: n=2, P<0.05) at 6, 12, and 18 kHz compared to the WT control mice. Public data from International Mouse Phenotyping Consortium (www.mousephenotype.org) were analyzed with R version 4.0.2 (using the ilcoxon rank sum test). Hom, homozygote. *Statistically significant, P<0.05.
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