Clin Exp Otorhinolaryngol.  2022 Aug;15(3):220-229. 10.21053/ceo.2022.00038.

Hyaluronan Synthase 1: A Novel Candidate Gene Associated With Late-Onset Non-syndromic Hereditary Hearing Loss

• Umugire A
• Lee S
• Lee CJ
• Choi Y
• Kim T
• Cho HH

• KMID: 2532986
• DOI: http://doi.org/10.21053/ceo.2022.00038

Abstract

Objectives
. Hyaluronan synthase 1 (HAS1) is a membrane-bound protein that is abundant in the epidermis and dermis, and it is important for skin function. However, its association with hearing loss has not yet been studied. Herein, we sought to evaluate the potential contribution of HAS1: c.1082G>A to genetic hearing loss.
Methods
. We used whole-exome sequencing to analyze blood DNA samples of six patients of a family with autosomal dominant familial late-onset progressive hearing loss, which was revealed to be related to a variant of the HAS1 gene. Confirmatory Sanger sequencing was performed with samples from 10 members. A missense variant was detected in HAS1 (c.1082 G>A, p.Cys361Tyr). In silico analyses predicted this variant to result in the functional loss of HAS1. Immunostaining was conducted using wild-type mouse samples to verify HAS1 expression.
Results
. Has1 was detected in an otocyst at E10.5. In the pup, Has1 expression was localized in the stria vascularis (SV), hair cells, supporting cells of the organ of Corti, and some spiral ganglion neurons. SV marginal cells markedly expressed Has1 in the adult stage. The hearing threshold in the Has1-depleted condition was investigated by accessing the International Mouse Phenotyping Consortium’s Auditory Brainstem Response (ABR) data. ABR of Has1 knock-out mice showed threshold elevations at 6, 12, and 18 kHz in young male adults.
Conclusion
. HAS1 may have a close relationship with auditory function and genetic hearing loss. Further investigation is needed to reveal the precise role of HAS1 in the auditory system. HAS1 is a candidate gene for future hereditary hearing loss genetic testing.

Keyword

Hyaluronan Synthases; Mutation; Missense; Whole Exome Sequencing; Nonsyndromic Sensorineural Hearing Loss

Figure

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  Fig. 1. Familial pedigree of non-syndromic hereditary hearing loss showing an autosomal dominant inheritance pattern. Red boxes indicate participants who were studied by whole-exome sequencing (3 with hearing loss [II-2, III-1, and III-3] and 3 with normal hearing [II-1, III-4, and IV-3]).

• 

  Fig. 2. Pure tone audiometry assessment showing a late-onset progressive severe hearing loss pattern. Hearing loss started in both ears as early as age 15 (patients III-1 and IV-2) and gradually deteriorated afterwards. At age 23, patient IV-1 showed moderate-to-severe hearing loss, and profound deafness occurred around age 40 (III-1, III-2, and III-3). TA, age at audiometry test; OA, age at onset of hearing impairment.

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  Fig. 3. Normal inner ear anatomy and good outcomes after cochlear implantation (CI) in deaf family members. Temporal bone computed tomography (CT) (A), temporal magnetic resonance imaging (MRI) (B), and postoperative CI-aided pure tone average (PTA; C) results of patient III-1. CT (D), MRI (E), and postoperative CI-aided PTA (F) results of patient III-3. Both patients had no cochleovestibular anomalies (arrows) and showed good hearing performance after CI.

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  Fig. 4. 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.

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  Fig. 5. HAS1 localization in the otic vesicle during mouse embryonic development. HAS1 was already expressed at E10.5 on the apical part of cells lining the luminal surface of the otic vesicle. HAS1 expression continued at E12.5. At E16.5, HAS1 was expressed in the apical membrane of the greater epithelial ridge (GER) region, lateral wall, and hair cells. DAPI, 4′,6-diamidino-2-phenylindole; LER, lesser epithelial ridge. Scale bar: 50 μm.

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  Fig. 6. 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.

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  Fig. 7. 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.

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  Fig. 8. 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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