Using coding and non-coding rare variants to target candidate genes in patients with severe tinnitus

Tinnitus is the phantom percept of an internal non-verbal set of noises and tones. It is reported by 15% of the population and it is usually associated with hearing and/or brain disorders. The role of structural variants (SVs) in coding and non-coding regions has not been investigated in patients with severe tinnitus. In this study, we performed whole-genome sequencing in 97 unrelated Swedish individuals with chronic tinnitus (TIGER cohort). Rare single nucleotide variants (SNV), large structural variants (LSV), and copy number variations (CNV) were retrieved to perform a gene enrichment analysis in TIGER and in a subgroup of patients with severe tinnitus (SEVTIN, n = 34), according to the tinnitus handicap inventory (THI) scores. An independent exome sequencing dataset of 147 Swedish tinnitus patients was used as a replication cohort (JAGUAR cohort) and population-specific datasets from Sweden (SweGen) and Non-Finish Europeans (NFE) from gnomAD were used as control groups. SEVTIN patients showed a higher prevalence of hyperacusis, hearing loss, and anxiety when they were compared to individuals in the TIGER cohort. We found an enrichment of rare missense variants in 6 and 8 high-constraint genes in SEVTIN and TIGER cohorts, respectively. Of note, an enrichment of missense variants was found in the CACNA1E gene in both SEVTIN and TIGER. We replicated the burden of missense variants in 9 high-constrained genes in the JAGUAR cohort, including the gene NAV2, when data were compared with NFE. Moreover, LSVs in constrained regions overlapping CACNA1E, NAV2, and TMEM132D genes were observed in TIGER and SEVTIN.publishedVersio

The statistical analysis plan for the unification of treatments and interventions for tinnitus patients randomized clinical trial (UNITI-RCT)

Background Tinnitus is a leading cause of disease burden globally. Several therapeutic strategies are recommended in guidelines for the reduction of tinnitus distress; however, little is known about the potentially increased effectiveness of a combination of treatments and personalized treatments for each tinnitus patient. Methods Within the Unification of Treatments and Interventions for Tinnitus Patients project, a multicenter, randomized clinical trial is conducted with the aim to compare the effectiveness of single treatments and combined treatments on tinnitus distress (UNITI-RCT). Five different tinnitus centers across Europe aim to treat chronic tinnitus patients with either cognitive behavioral therapy, sound therapy, structured counseling, or hearing aids alone, or with a combination of two of these treatments, resulting in four treatment arms with single treatment and six treatment arms with combinational treatment. This statistical analysis plan describes the statistical methods to be deployed in the UNITI-RCT. Discussion The UNITI-RCT trial will provide important evidence about whether a combination of treatments is superior to a single treatment alone in the management of chronic tinnitus patients. This pre-specified statistical analysis plan details the methodology for the analysis of the UNITI trial results. Trial registration ClinicalTrials.gov NCT04663828. The trial is ongoing. Date of registration: December 11, 2020. All patients that finished their treatment before 19 December 2022 are included in the main RCT analysis

A novel nonsense variant in the CENPP gene segregates in a Swiss family with autosomal dominant low-frequency sensorineural hearing loss

Low-frequency sensorineural hearing loss (SNHL) is a rare hearing impairment affecting frequencies below 1000 Hz, previously associated with DIAPH1, WSF1, MYO7A, TNC, SLC26A4 or CCDC50 genes. By exome sequencing, we report a novel nonsense variant in CENPP gene, segregating low-frequency SNHL in five affected members in a Swiss family with autosomal dominant inheritance pattern. Audiological evaluation showed up-sloping audiometric configuration with mild-to-moderate losses below 1000 Hz, that progresses to high-frequencies over time. Protein modeling shows that the variant truncates five amino acids at the end, losing electrostatic interactions that alter protein stability. CENPP gene is expressed in the supporting cells of the organ of Corti and takes part as a subunit of the Constitutive Centromere Associated Network in the kinetochore, that fixes the centromere to the spindle microtubules. We report CENPP as a new candidate gene for low-frequency SNHL. Further functional characterization might enable us to elucidate its molecular role in SNHL

Genome-Wide Identification and Functional Classification of Tomato (<i>Solanum lycopersicum</i>) Aldehyde Dehydrogenase (ALDH) Gene Superfamily

<div><p>Aldehyde dehydrogenases (ALDHs) is a protein superfamily that catalyzes the oxidation of aldehyde molecules into their corresponding non-toxic carboxylic acids, and responding to different environmental stresses, offering promising genetic approaches for improving plant adaptation. The aim of the current study is the functional analysis for systematic identification of <i>S</i>. <i>lycopersicum</i> ALDH gene superfamily. We performed genome-based ALDH genes identification and functional classification, phylogenetic relationship, structure and catalytic domains analysis, and microarray based gene expression. Twenty nine unique tomato ALDH sequences encoding 11 ALDH families were identified, including a unique member of the family 19 ALDH. Phylogenetic analysis revealed 13 groups, with a conserved relationship among ALDH families. Functional structure analysis of ALDH2 showed a catalytic mechanism involving <i>Cys-Glu</i> couple. However, the analysis of ALDH3 showed no functional gene duplication or potential neo-functionalities. Gene expression analysis reveals that particular ALDH genes might respond to wounding stress increasing the expression as ALDH2B7. Overall, this study reveals the complexity of <i>S</i>. <i>lycopersicum</i> ALDH gene superfamily and offers new insights into the structure-functional features and evolution of ALDH gene families in vascular plants. The functional characterization of ALDHs is valuable and promoting molecular breeding in tomato for the improvement of stress tolerance and signaling.</p></div

Tomato ALDH3H1 protein structure, coenzyme and ligand-binding domain analysis.

<p>(A) Three-dimensional structure of tomato SlALDH3H1. Structures were depicted as a cartoon diagram. α-helices, β-sheets and coils are depicted in red, yellow and green, respectively. Two views rotated 180 around the x-axis are provided for SlALDH3H1 superfamily of the electrostatic potential representation on the SlALDH3H1 protein surfaces. The surface colors are clamped at red (-10) or blue (+10). (B) Detailed view of the SlALDH3H1 chain and the active (catalytic) site, and the spatial distribution of the coenzyme the amino acids involved in holding NAD+. Residues are depicted as stick and colored according with atoms. (C) Hydrogen-bonding interactions in the SlALDH3H1 coenzyme domain and its interaction with NAD+. Hydrogen bonds are shown in broken lines. (D) Detailed view of the catalytic-binding domain showing residues configuring this domain, and critical catalytic amino acids (C244 and N114) highlighted in pink and purple color in proper position related to the substrate (red dot).</p

Tomato ALDH3F1 (b and d variants) proteins structure, coenzyme and ligand-binding domain analysis.

<p>Three-dimensional structure of tomato (A) SlALDH3F1b missing the complete oligomerization domain and partially the coenzyme domain; (B) SlALDH3F1d. Structures were depicted as a cartoon diagram. α-helices, β-sheets and coils are depicted in red, yellow and green, respectively. Two views rotated 180 around the x-axis are provided for both SlALDH3F1 protein variants of the electrostatic potential representation on the SlALDH3F1 proteins surfaces. The surface colors are clamped at red (-10) or blue (+10); (C) Superimposition of SlALDH3F1b and SlALDH3F1d showing the three main domains (coenzyme, oligomerization and catalytic), and highlighting with black arrows all the 2D-structural elements missing in SlALDH3F1b; (D) Detailed view of the superimposition showing the co-enzyme/catalytic cleft accommodating the NAD+ and substrate holding amino acids; (E) Detailed view of the catalytic-binding domain showing residues integrating this domain, and critical catalytic amino acids (C132 and N116) highlighted in green and blue colors in proper position related to the substrate (fatty aldehyde).</p

Comparative phylogenetic analysis of tomato ALDHs.

<p>Phylogenetic analysis was made using 97 ALDH proteins from <i>Solanum lycopersicum</i> (Sl), <i>Arabidopsis thaliana</i> (At), Zea mays (Zm), <i>Physcomitrella patens</i> (Pp), and <i>Chlamydomonas reinhardtii</i> (Cr).</p

Tomato ALDH orthologous genes.

<p>Identification number (EO) for potential orthologous functional domains is indicated in the third column with their respective names.</p

Tomato ALDH genes and families.

<p>Tomato ALDH genes and families.</p

Coenzyme and ligand-binding domain analysis of ALDH2 superfamily.

<p>(A) Hydrogen-bonding interactions in the SlALDH2B1 coenzyme and ligand-binding domain and its interaction with NADH. Hydrogen bonds are shown in broken lines. Critical catalytic amino acids (C223 and E189) are highlighted in blue background. (B) Distribution of the NADH cofactor (red color) and the spatial distribution of the residues that integrate the cofactor-substrate binding cleft. Residues are depicted as stick and orange colored and green/blue colors the catalytic residues. (C) Detailed representation of the amino acids interacting with NADH and stabilizing the cofactor in the catalytic domain. Additional table shows the key amino acids involving the coenzyme-substrate binding domain. Catalytic crucial residues as grey color shadowed.</p