The Next Generation of Population-Based DFNB16 Carrier Screening and Diagnosis: STRC Copy-Number Variant Analysis from Genome Sequencing Data.

BACKGROUND: Deafness, autosomal recessive 16 (DFNB16) is caused by compound heterozygous or homozygous variants in STRC and is the second most common form of genetic hearing loss. Due to the nearly identical sequences of STRC and the pseudogene STRCP1, analysis of this region is challenging in clinical testing. METHODS: We developed a method that accurately identifies the copy number of STRC and STRCP1 using standard short-read genome sequencing. Then, we used whole genome sequencing (WGS) data to investigate the population distribution of STRC copy number in 6813 neonates and the correlation between STRC and STRCP1 copy number. RESULTS: The comparison of WGS results with multiplex ligation-dependent probe amplification demonstrated high sensitivity (100%; 95% CI, 97.5%-100%) and specificity (98.8%; 95% CI, 97.7%-99.5%) in detecting heterozygous deletion of STRC from short-read genome sequencing data. The population analysis revealed that 5.22% of the general population has STRC copy number changes, almost half of which (2.33%; 95% CI, 1.99%-2.72%) were clinically significant, including heterozygous and homozygous STRC deletions. There was a strong inverse correlation between STRC and STRCP1 copy number. CONCLUSIONS: We developed a novel and reliable method to determine STRC copy number based on standard short-read based WGS data. Incorporating this method into analytic pipelines would improve the clinical utility of WGS in the screening and diagnosis of hearing loss. Finally, we provide population-based evidence of pseudogene-mediated gene conversions between STRC and STRCP1.

Comprehensive interpretation of single-nucleotide substitutions in GJB2 reveals the genetic and phenotypic landscape of GJB2-related hearing loss.

Genetic variants in GJB2 are the most frequent cause of congenital and childhood hearing loss worldwide. The purpose of this study was to delineate the genetic and phenotypic landscape of GJB2 SNV variants. All possible single-nucleotide substitution variants of the coding region of GJB2 (N = 2043) were manually curated following the ACMG/AMP hearing loss guidelines. As a result, 60 (2.9%), 177 (8.7%), 1499 (73.4%), 301 (14.7%) and 6 (0.3%) of the variants were classified as pathogenic, likely pathogenic, variant of uncertain significance, likely benign, and benign, respectively. 53% (84/158) of the pathogenic/likely pathogenic missense variants were not present in ClinVar. The second transmembrane domain and the 310 helix were highly enriched for pathogenic missense variants, while the intracellular loops were tolerant to variation. The N-terminal tail and the extracellular loop showed high clustering of variants that are associated with syndromic or dominant non-syndromic hearing loss. In conclusion, our study interpreted all possible single-nucleotide substitution coding variants, characterized novel clinically significant variants in GJB2, and revealed significant genotype-phenotype correlations at this common hearing loss locus. Our work provides a prototype for other genes with similarly high genetic and phenotypic heterogeneity.

Comprehensive genetic testing improves the clinical diagnosis and medical management of pediatric patients with isolated hearing loss.

PURPOSE: Genetic testing is widely used in diagnosing genetic hearing loss in patients. Other than providing genetic etiology, the benefits of genetic testing in pediatric patients with hearing loss are less investigated. METHODS: From 2018-2020, pediatric patients who initially presented isolated hearing loss were enrolled. Comprehensive genetic testing, including GJB2/SLC26A4 multiplex amplicon sequencing, STRC/OTOA copy number variation analysis, and exome sequencing, were hierarchically offered. Clinical follow-up and examinations were performed. RESULTS: A total of 80 pediatric patients who initially presented isolated hearing loss were considered as nonsyndromic hearing loss and enrolled in this study. The definitive diagnosis yield was 66% (53/80) and the likely diagnosis yield was 8% (6/80) through comprehensive genetic testing. With the aid of genetic testing and further clinical follow-up and examinations, the clinical diagnoses and medical management were altered in eleven patients (19%, 11/59); five were syndromic hearing loss; six were nonsyndromic hearing loss mimics. CONCLUSION: Syndromic hearing loss and nonsyndromic hearing loss mimics are common in pediatric patients who initially present with isolated hearing loss. The comprehensive genetic testing provides not only a high diagnostic yield but also valuable information for clinicians to uncover subclinical or pre-symptomatic phenotypes, which allows early diagnosis of SHL, and leads to precise genetic counseling and changes the medical management.

Utility of Whole Genome Sequencing for Population Screening of Deafness-Related Genetic Variants and Cytomegalovirus Infection in Newborns.

Background: Hearing loss affects approximately two out of every 1,000 newborns. Genetic factors and congenital cytomegalovirus (CMV) infections account for around 90% of the etiology. The purpose of this study was to develop and test a whole genome sequencing (WGS) approach to detect deafness-related genetic variants and CMV infections simultaneously in newborns. Method: Deafness-related genes causing congenital or childhood hearing loss were curated and selected for newborn screening. Nine dried blood spots from newborns with known genetic variants (n = 6) or CMV infections (n = 3) were employed to develop and validate the WGS testing and analytic pipeline. We then pilot tested the WGS analysis on 51 de-identified clinical samples. Results: 92 gene-disease pairs were selected for screening hearing loss in newborns. In the validation test, WGS accurately detected all types of genetic variants, including single nucleotide variations, insertions/deletions, and copy number variations in the nuclear or mitochondrial genome. Sequence reads mapping to the CMV reference genome were discovered in CMV infected samples. In the pilot test, WGS identified nine out of 51 (18%) newborns carrying pathogenic variants associated with deafness. Conclusion: WGS can simultaneously detect genetic variants and CMV infections in dried blood spot specimens from newborns. Our study provides proof of principle that genome sequencing can be a promising alternative for newborn screening of hearing loss.

Magnetically Reusable and Well-dispersed Nanoparticles for Oxygen Detection in Water.

In this study, we aimed to synthesize magnetically well-dispersed nanosensors for detecting dissolved oxygen (DO) in water, and explore their biological applications. Firstly, we synthesized two kinds of magnetic nanoparticle with average sizes of approximately 82 nm by one-step emulsion polymerization: polystyrene magnetic nanoparticles (Fe3O4@Os1-PS) and polymethylmethacrylate magnetic nanoparticles (Fe3O4@Os1-PMMA). Both types of nanoparticle present good dispersibility and fluorescence stability. The nanoparticles could be used as oxygen sensors that exhibited a high DO-sensitivity response in the range 0-39.30 mg/L, with a strong linear relationship. The nanoparticles have good magnetic properties, and so they could be recycled by magnet for further use. Recovered Fe3O4@Os1-PS still presented high stability after continued use in oxygen sensing for one month. Furthermore, Fe3O4@Os1-PS was employed for detecting the bacterial oxygen consumption of Escherichia coli (E-coli) to monitor the metabolism of bacteria. The results show that Fe3O4@Os1-PS provide high biocompatibility and non-toxicity. Polystyrene magnetic nanoparticles therefore present significant potential for application in biological oxygen sensing.

Rational Design of a Polymer-Based Ratiometric K+ Indicator for High-Throughput Monitoring Intracellular K+ Fluctuations.

Highly selective fluorescent K+ sensors are of great importance for monitoring K+ fluctuations in various biological processes. In particular, highly efficient ratiometric K+ sensors that can emit in dual wavelengths and facilitate the quantitative determination of K+ are highly anticipated. Herein, we present the first polymer-based ratiometric fluorescent K+ indicator (PK1) for quantitatively detecting K+ in aqueous solutions and high-throughput monitoring K+ fluctuations in living cells. PK1 was synthesized by conjugating a small molecular K+ probe and a red emission reference dye to a hydrophilic polymer skeleton. The newly synthesized PK1 can form highly stable nanoparticles in aqueous solutions and work in 100% water without the aid of any organic solvents or surfactants. PK1 is sensitive to K+ with a fluorescence enhancement of sevenfold after interactions with K+ at 1000 mM and inert to other metal ions, physiological pH, or dye concentration vibrations. More importantly, the fluorescence intensity ratio at 572 and 638 nm is linearly correlated with log [K+] in the range of 2-500 mM (R2 = 0.998), which will facilitate the quantitative detection of K+. Practical application of PK1 in detecting different K+-rich samples demonstrates its great potential in quantitative detection of K+. PK1 can be quickly internalized by live cells and shows no obvious cytotoxicity. We also demonstrate that PK1 could be used for monitoring K+ fluctuations under different stimulations by using a confocal microscope and especially a microplate reader, which is high throughput and time saving. The rational design of PK1 will broaden the design concept of ratiometric fluorescent K+ sensors and facilitate the quantitative detection of K+.

Close Temporal Relationship between Oscillating Cytosolic K+ and Growth in Root Hairs of Arabidopsis.

Root hair elongation relies on polarized cell expansion at the growing tip. As a major osmotically active ion, potassium is expected to be continuously assimilated to maintain cell turgor during hair tip growth. However, due to the lack of practicable detection methods, the dynamics and physiological role of K+ in hair growth are still unclear. In this report, we apply the small-molecule fluorescent K+ sensor NK3 in Arabidopsis root hairs for the first time. By employing NK3, oscillating cytoplasmic K+ dynamics can be resolved at the tip of growing root hairs, similar to the growth oscillation pattern. Cross-correlation analysis indicates that K+ oscillation leads the growth oscillations by approximately 1.5 s. Artificially increasing cytoplasmic K+ level showed no significant influence on hair growth rate, but led to the formation of swelling structures at the tip, an increase of cytosolic Ca2+ level and microfilament depolymerization, implying the involvement of antagonistic regulatory factors (e.g., Ca2+ signaling) in the causality between cytoplasmic K+ and hair growth. These results suggest that, in each round of oscillating root hair elongation, the oscillatory cell expansion accelerates on the heels of cytosolic K+ increment, and decelerates with the activation of antagonistic regulators, thus forming a negative feedback loop which ensures the normal growth of root hairs.

A mitochondria-targeting NIR fluorescent potassium ion sensor: real-time investigation of the mitochondrial K+ regulation of apoptosis in situ.

The first NIR fluorescent mitochondria-targeting K+ sensor, denoted as TAC-Rh, was developed. The produced sensor consists of a rhodamine analog as the fluorophore and triazacryptand (TAC) as the K+ recognition unit. Compared to the K+ sensors reported previously, TAC-Rh exhibits two unique optical properties: the largest Stokes shifts (120 nm) and the longest emission peak wavelength (720 nm). With the assistance of this novel sensor, real-time changes of K+ concentrations in mitochondria during apoptosis were monitored for the first time. Moreover, it was also the first time that the relationship between mitochondrial K+ flux and apoptosis was investigated in real time using fluorescence imaging.

Tricolor dual sensor for ratiometrically analyzing potassium ions and dissolved oxygen.

A potassium ion­oxygen (K+-O2) dual fluorescent sensing film was developed. The film contains three probes, which are K+ probe (KS), O2 probe (OS), and reference probe (RP) in a polymer film composed of poly(ethylene glycol) methyl ether methacrylate (PEGMA), poly(ethylene glycol) dimethacrylate (PEGDMA) and methacrylic acid (MAA). The RP showed blue emission, the KS exhibited green emission, and the OS showed red emission. The emission peaks of three probes do not interfere with each other, which enable the sensing film to be used for ratiometrically and quantitatively detecting the concentrations of K+ and dissolved oxygen (DO). The sensing films showed high sensitivity and selectivity to potassium ions over other metal ions and also good sensitivity for DO from deoxygenated to oxygenated conditions. The sensing film was demonstrated to be capable of analyzing K+ and DO concentrations with experimental errors smaller than ±8.5% in aqueous solutions, showing the potential applications of the sensing films.

Pyrazinamide and derivatives block ethylene biosynthesis by inhibiting ACC oxidase.

Ethylene is an important phytohormone that promotes the ripening of fruits and senescence of flowers thereby reducing their shelf lives. Specific ethylene biosynthesis inhibitors would help to decrease postharvest loss. Here, we identify pyrazinamide (PZA), a clinical drug used to treat tuberculosis, as an inhibitor of ethylene biosynthesis in Arabidopsis thaliana, using a chemical genetics approach. PZA is converted to pyrazinecarboxylic acid (POA) in plant cells, suppressing the activity of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), the enzyme catalysing the final step of ethylene formation. The crystal structures of Arabidopsis ACO2 in complex with POA or 2-Picolinic Acid (2-PA), a POA-related compound, reveal that POA/2-PA bind at the active site of ACO, preventing the enzyme from interacting with its natural substrates. Our work suggests that PZA and its derivatives may be promising regulators of plant metabolism, in particular ethylene biosynthesis.

Characterization of a Novel Megabirnavirus from Sclerotinia sclerotiorum Reveals Horizontal Gene Transfer from Single-Stranded RNA Virus to Double-Stranded RNA Virus.

UNLABELLED: Mycoviruses have been detected in all major groups of filamentous fungi, and their study represents an important branch of virology. Here, we characterized a novel double-stranded RNA (dsRNA) mycovirus, Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1), in an apparently hypovirulent strain (SX466) of Sclerotinia sclerotiorum. Two similarly sized dsRNA segments (L1- and L2-dsRNA), the genome of SsMBV1, are packaged in rigid spherical particles purified from strain SX466. The full-length cDNA sequence of L1-dsRNA/SsMBV1 comprises two large open reading frames (ORF1 and ORF2), which encode a putative coat protein and an RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis of the RdRp domain clearly indicates that SsMBV1 is related to Rosellinia necatrix megabirnavirus 1 (RnMBV1). L2-dsRNA/SsMBV1 comprises two nonoverlapping ORFs (ORFA and ORFB) encoding two hypothetical proteins with unknown functions. The 5'-terminal regions of L1- and L2-dsRNA/SsMBV1 share strictly conserved sequences and form stable stem-loop structures. Although L2-dsRNA/SsMBV1 is dispensable for replication, genome packaging, and pathogenicity of SsMBV1, it enhances transcript accumulation of L1-dsRNA/SsMBV1 and stability of virus-like particles (VLPs). Interestingly, a conserved papain-like protease domain similar to a multifunctional protein (p29) of Cryphonectria hypovirus 1 was detected in the ORFA-encoded protein of L2-dsRNA/SsMBV1. Phylogenetic analysis based on the protease domain suggests that horizontal gene transfer may have occurred from a single-stranded RNA (ssRNA) virus (hypovirus) to a dsRNA virus, SsMBV1. Our results reveal that SsMBV1 has a slight impact on the fundamental biological characteristics of its host regardless of the presence or absence of L2-dsRNA/SsMBV1. IMPORTANCE: Mycoviruses are widespread in all major fungal groups, and they possess diverse genomes of mostly ssRNA and dsRNA and, recently, circular ssDNA. Here, we have characterized a novel dsRNA virus (Sclerotinia sclerotiorum megabirnavirus 1 [SsMBV1]) that was isolated from an apparently hypovirulent strain, SX466, of Sclerotinia sclerotiorum. Although SsMBV1 is phylogenetically related to RnMBV1, SsMBV1 is markedly distinct from other reported megabirnaviruses with two features of VLPs and conserved domains. Our results convincingly showed that SsMBV1 is viable in the absence of L2-dsRNA/SsMBV1 (a potential large satellite-like RNA or genuine genomic virus component). More interestingly, we detected a conserved papain-like protease domain that commonly exists in ssRNA viruses, including members of the families Potyviridae and Hypoviridae. Phylogenetic analysis based on the protease domain suggests that horizontal gene transfer might have occurred from an ssRNA virus to a dsRNA virus, which may provide new insights into the evolutionary history of dsRNA and ssRNA viruses.