In-depth genetic and molecular characterization of diaphanous related formin 2 (DIAPH2) and its role in the inner ear

Chiara Chiereghin; Michela Robusto; Morag A. Lewis; Susana Caetano; Valentina Massa; Pierangela Castorina; Umberto Ambrosetti; Karen P. Steel; Stefano Duga; Rosanna Asselta; Giulia Soldà

doi:10.1371/journal.pone.0273586

In-depth genetic and molecular characterization of diaphanous related formin 2 (DIAPH2) and its role in the inner ear

Chiara Chiereghin, Michela Robusto, Morag A. Lewis, Susana Caetano, Valentina Massa, Pierangela Castorina, Umberto Ambrosetti, Karen P. Steel, Stefano Duga, Rosanna Asselta, Giulia Soldà^*

^*Corresponding author for this work

Wolfson SPaRC

Research output: Contribution to journal › Article › peer-review

Abstract

Diaphanous related formins are regulatory cytoskeletal protein involved in actin elongation and microtubule stabilization. In humans, defects in two of the three diaphanous genes (DIAPH1 and DIAPH3) have been associated with different types of hearing loss. Here, we investigate the role of the third member of the family, DIAPH2, in nonsyndromic hearing loss, prompted by the identification, by exome sequencing, of a predicted pathogenic missense variant in DIAPH2. This variant occurs at a conserved site and segregated with nonsyndromic X-linked hearing loss in an Italian family. Our immunohistochemical studies indicated that the mouse ortholog protein Diaph2 is expressed during development in the cochlea, specifically in the actin-rich stereocilia of the sensory outer hair cells. In-vitro studies showed a functional impairment of the mutant DIAPH2 protein upon RhoA-dependent activation. Finally, Diaph2 knock-out and knock-in mice were generated by CRISPR/Cas9 technology and auditory brainstem response measurements performed at 4, 8 and 14 weeks. However, no hearing impairment was detected. Our findings indicate that DIAPH2 may play a role in the inner ear; further studies are however needed to clarify the contribution of DIAPH2 to deafness.

Original language	English
Article number	e0273586
Number of pages	22
Journal	PLoS ONE
Volume	18
Issue number	1
DOIs	https://doi.org/10.1371/journal.pone.0273586
Publication status	Published - 23 Jan 2023

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title = "In-depth genetic and molecular characterization of diaphanous related formin 2 (DIAPH2) and its role in the inner ear",

abstract = "Diaphanous related formins are regulatory cytoskeletal protein involved in actin elongation and microtubule stabilization. In humans, defects in two of the three diaphanous genes (DIAPH1 and DIAPH3) have been associated with different types of hearing loss. Here, we investigate the role of the third member of the family, DIAPH2, in nonsyndromic hearing loss, prompted by the identification, by exome sequencing, of a predicted pathogenic missense variant in DIAPH2. This variant occurs at a conserved site and segregated with nonsyndromic X-linked hearing loss in an Italian family. Our immunohistochemical studies indicated that the mouse ortholog protein Diaph2 is expressed during development in the cochlea, specifically in the actin-rich stereocilia of the sensory outer hair cells. In-vitro studies showed a functional impairment of the mutant DIAPH2 protein upon RhoA-dependent activation. Finally, Diaph2 knock-out and knock-in mice were generated by CRISPR/Cas9 technology and auditory brainstem response measurements performed at 4, 8 and 14 weeks. However, no hearing impairment was detected. Our findings indicate that DIAPH2 may play a role in the inner ear; further studies are however needed to clarify the contribution of DIAPH2 to deafness.",

author = "Chiara Chiereghin and Michela Robusto and Lewis, {Morag A.} and Susana Caetano and Valentina Massa and Pierangela Castorina and Umberto Ambrosetti and Steel, {Karen P.} and Stefano Duga and Rosanna Asselta and Giulia Sold{\`a}",

note = "Funding Information: This study was funded by Fondazione Cariplo, grant N. 2013-0825 to RA and the National Institute for Health Research (NIHR) Biomedical Research Centre to KPS. CC received a travel grant from Boehringer Ingelheim Fonds to pursue a short-term research stay in KPS{\textquoteright}s laboratory at King{\textquoteright}s College London. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Elysia James and Dr Neil Ingham for assisting with waveform analysis and Dr Oliver Baker and the King{\textquoteright}s College London Gene Editing and Embryology Core for their design and implementation of targeted genome editing of the mouse Diaph2. We thank the Marine Biological Laboratory (Woods Hole, MA, USA) and the Biology of Inner Ear 2017 course organizers for technical assistance in immunofluorescence colocalization studies on vibratome cochlear sections. We are indebted to Prof. Stefano Duga, co-author, mentor and friend, who has recently passed away (10/11/2021). Finally, we are grateful to all the members of the here described NSHL family, who have been extremely collaborative and interested in the research we have been performing. Publisher Copyright: {\textcopyright} 2023 Chiereghin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.",

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T1 - In-depth genetic and molecular characterization of diaphanous related formin 2 (DIAPH2) and its role in the inner ear

AU - Chiereghin, Chiara

AU - Robusto, Michela

AU - Lewis, Morag A.

AU - Caetano, Susana

AU - Massa, Valentina

AU - Castorina, Pierangela

AU - Ambrosetti, Umberto

AU - Steel, Karen P.

AU - Duga, Stefano

AU - Asselta, Rosanna

AU - Soldà, Giulia

N1 - Funding Information: This study was funded by Fondazione Cariplo, grant N. 2013-0825 to RA and the National Institute for Health Research (NIHR) Biomedical Research Centre to KPS. CC received a travel grant from Boehringer Ingelheim Fonds to pursue a short-term research stay in KPS’s laboratory at King’s College London. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Elysia James and Dr Neil Ingham for assisting with waveform analysis and Dr Oliver Baker and the King’s College London Gene Editing and Embryology Core for their design and implementation of targeted genome editing of the mouse Diaph2. We thank the Marine Biological Laboratory (Woods Hole, MA, USA) and the Biology of Inner Ear 2017 course organizers for technical assistance in immunofluorescence colocalization studies on vibratome cochlear sections. We are indebted to Prof. Stefano Duga, co-author, mentor and friend, who has recently passed away (10/11/2021). Finally, we are grateful to all the members of the here described NSHL family, who have been extremely collaborative and interested in the research we have been performing. Publisher Copyright: © 2023 Chiereghin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

PY - 2023/1/23

Y1 - 2023/1/23

N2 - Diaphanous related formins are regulatory cytoskeletal protein involved in actin elongation and microtubule stabilization. In humans, defects in two of the three diaphanous genes (DIAPH1 and DIAPH3) have been associated with different types of hearing loss. Here, we investigate the role of the third member of the family, DIAPH2, in nonsyndromic hearing loss, prompted by the identification, by exome sequencing, of a predicted pathogenic missense variant in DIAPH2. This variant occurs at a conserved site and segregated with nonsyndromic X-linked hearing loss in an Italian family. Our immunohistochemical studies indicated that the mouse ortholog protein Diaph2 is expressed during development in the cochlea, specifically in the actin-rich stereocilia of the sensory outer hair cells. In-vitro studies showed a functional impairment of the mutant DIAPH2 protein upon RhoA-dependent activation. Finally, Diaph2 knock-out and knock-in mice were generated by CRISPR/Cas9 technology and auditory brainstem response measurements performed at 4, 8 and 14 weeks. However, no hearing impairment was detected. Our findings indicate that DIAPH2 may play a role in the inner ear; further studies are however needed to clarify the contribution of DIAPH2 to deafness.

AB - Diaphanous related formins are regulatory cytoskeletal protein involved in actin elongation and microtubule stabilization. In humans, defects in two of the three diaphanous genes (DIAPH1 and DIAPH3) have been associated with different types of hearing loss. Here, we investigate the role of the third member of the family, DIAPH2, in nonsyndromic hearing loss, prompted by the identification, by exome sequencing, of a predicted pathogenic missense variant in DIAPH2. This variant occurs at a conserved site and segregated with nonsyndromic X-linked hearing loss in an Italian family. Our immunohistochemical studies indicated that the mouse ortholog protein Diaph2 is expressed during development in the cochlea, specifically in the actin-rich stereocilia of the sensory outer hair cells. In-vitro studies showed a functional impairment of the mutant DIAPH2 protein upon RhoA-dependent activation. Finally, Diaph2 knock-out and knock-in mice were generated by CRISPR/Cas9 technology and auditory brainstem response measurements performed at 4, 8 and 14 weeks. However, no hearing impairment was detected. Our findings indicate that DIAPH2 may play a role in the inner ear; further studies are however needed to clarify the contribution of DIAPH2 to deafness.

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In-depth genetic and molecular characterization of diaphanous related formin 2 (DIAPH2) and its role in the inner ear

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