Genotype-phenotype spectrum and correlation of PHARC Syndrome due to pathogenic ABHD12 variants.

BACKGROUND: A comprehensive understanding of the genetic basis of rare diseases and their regulatory mechanisms is essential for human molecular genetics. However, the genetic mutant spectrum of pathogenic genes within the Chinese population remains underrepresented. Here, we reported previously unreported functional ABHD12 variants in two Chinese families and explored the correlation between genetic polymorphisms and phenotypes linked to PHARC syndrome. METHODS: Participants with biallelic pathogenic ABHD12 variants were recruited from the Chinese Deafness Genetics Cohort. These participants underwent whole-genome sequencing. Subsequently, a comprehensive literature review was conducted. RESULTS: Two Han Chinese families were identified, one with a compound heterozygous variant and the other with a novel homozygous variant in ABHD12. Among 65 PHARC patients, including 62 from the literature and 3 from this study, approximately 90% (57 out of 63) exhibited hearing loss, 82% (50 out of 61) had cataracts, 82% (46 out of 56) presented with retinitis pigmentosa, 79% (42 out of 53) experienced polyneuropathy, and 63% (36 out of 57) displayed ataxia. Seventeen different patterns were observed in the five main phenotypes of PHARC syndrome. A total of 33 pathogenic variants were identified in the ABHD12. Compared with other genotypes, individuals with biallelic truncating variants showed a higher incidence of polyneuropathy (p = 0.006), but no statistically significant differences were observed in the incidence of hearing loss, ataxia, retinitis pigmentosa and cataracts. CONCLUSIONS: The diagnosis of PHARC syndrome is challenging because of its genetic heterogeneity. Therefore, exploring novel variants and establishing genotype-phenotype correlations can significantly enhance gene diagnosis and genetic counseling for this complex disease.

Extracellular Vesicle ASC: A Novel Mediator for Lung-Brain Axis in Preterm Brain Injury.

Bronchopulmonary dysplasia (BPD) and neurodevelopmental impairment (NDI) are among the most common morbidities affecting preterm infants. Although BPD is a predictor of poor NDI, it is currently uncertain how BPD contributes to brain injury in preterm infants. Extracellular vesicles (EVs) are involved in inter-organ communication in diverse pathological processes. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly and activation of inflammatory response. We assessed expression profiles of alveolar macrophage (AM) markers, CD11b, CD11c, and CD206, and ASC in EVs isolated from the plasma of preterm infants at risk for BPD at 1 week of age. We found that infants on higher fraction inspired oxygen (FiO2) therapy (HO2, ≥30%) had increased levels of AM-derived EV-ASC compared with infants on lower FiO2 (LO2, <30%). To assess the function of these EVs, we performed adoptive transfer experiments by injecting them into the circulation of newborn mice. We discovered that mice that received EVs from infants on HO2 had increased lung inflammation, decreased alveolarization, and disrupted vascular development, the hallmarks of BPD. Importantly, these EVs crossed the blood-brain barrier and the EVs from infants on HO2 caused inflammation, reduced cell survival, and increased cell death with features of pyroptosis and necroptosis in the hippocampus. These results highlight a novel role for AM-derived EV-ASC in mediating the lung-to-brain crosstalk that is critical in the pathogenesis of BPD and brain injury and identify potential novel targets for preventing and treating BPD and brain injury in preterm infants.

Forensic investigative genetic genealogy: expanding pedigree tracing and genetic inquiry in the genomic era.

Genetic genealogy provides crucial insights into the complex biological relationships within contemporary and ancient human populations by analyzing shared alleles and chromosomal segments that are identical by descent to understand kinship, migration patterns, and population dynamics. Within forensic science, forensic investigative genetic genealogy (FIGG) has gained prominence by leveraging next-generation sequencing technologies and population-specific genomic resources, opening new investigative avenues. In this review, we synthesize current knowledge, underscore recent advancements, and discuss the growing role of FIGG in forensic genomics. FIGG has been pivotal in revitalizing dormant inquiries and offering new genetic leads in numerous cold cases. Its effectiveness relies on the extensive single-nucleotide polymorphism profiles contributed by individuals from diverse populations to specialized genomic databases. Advances in computational genomics and the growth of human genomic databases have spurred a profound shift in the application of genetic genealogy across forensics, anthropology, and ancient DNA studies. As the field progresses, FIGG is evolving from a nascent practice into a more sophisticated and specialized discipline, shaping the future of forensic investigations.

IC100, a humanized therapeutic monoclonal anti-ASC antibody alleviates oxygen-induced retinopathy in mice.

BACKGROUND: Retinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation. METHODS: We first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR. RESULTS: ASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2. CONCLUSION: These data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.

[Prediction of quality markers of Angong Niuhuang Pills based on LC-MS and pull-down with SPR chips].

Elucidating the quality markers(Q-markers) of traditional Chinese medicines is essential for understanding the mechanisms of action and promoting the rational use of traditional Chinese medicines as well as for developing traditional Chinese medicine-derived drugs. Studies have shown that surface plasmon resonance(SPR) is promising in this field. This study proposed a method based on pull-down with SPR chips to predict the Q-markers of Angong Niuhuang pills(AGNHP). Firstly, 71 main chemical components of AGNHP were analyzed by UPLC-Q-TOF-MS, and then network pharmacology was employed to predict the potential targets of AGNHP against stroke. Secondly, the STAT3 protein chip was constructed, and the extract of AGNHP was recovered by pull-down of the SPR system for STAT3 ligand. The potential active ingredients were collected, enriched, and identified as coptisine, palmatine, epiberberine, berberine, worenine, demethyleneberberine, jatrorrhizine, tetrahydrocoptisine, baicalein, and baicalin methyl ester. Next, the affinity constants of the 10 active ingredients were determined as 44.7, 44, 58.1, 51.3, 39.7, 32.1, 49.2, 69.1, 19.7, and 24.9 µmol·L~(-1), respectively. The molecular docking results showed that the 10 compounds could compete for binding with STAT3. This is the first report that SPR combined with UPLC-Q-TOF-MS is reliable and feasible for determining the active ingredients of AGNHP at the molecular level from complex systems. STAT3 could be used as a potential target for the biological quality evaluation of AGNHP.

Cellulose Nanofibrils of High Immunoaffinity for Efficient Enrichment of Small Extracellular Vesicles.

Extracellular vesicles (EVs), crucial in facilitating the transport of diverse molecular cargoes for intercellular communication, have shown great potential in diagnostics, therapeutics, and drug delivery. The challenge of developing effective preparation methods for EVs is heightened by their intrinsic heterogeneity and complexity. Here, a novel strategy for high EV enrichment is developed by utilizing EV-affinitive-modified cellulose nanofibrils. Specifically, modified cellulose with rich carboxyl groups has outstanding dispersing properties, able to be dispersed into cellulose nanofibrils in solution. These cellulose nanofibrils are utilized as scaffolds for the immobilization of EV-affinitive antibody of CD63 by chemical conjugation. The CD63-modified nanofibrils demonstrate a superior EV capture efficiency of 86.4% compared with other reported methods. The high performance of this system is further validated by the efficient capture of EVs from biological blood plasma, allowing the detection of bioactive markers from EV-derived miRNAs and proteins. The authors envision that these modified cellulose nanofibrils of enhanced capability on EV enrichment will open new avenues in various biomedical applications.

Exploring noncoding variants in genetic diseases: from detection to functional insights.

Previous studies on genetic diseases predominantly focused on protein-coding variations, overlooking the vast noncoding regions in the human genome. The development of high-throughput sequencing technologies and functional genomics tools has enabled the systematic identification of functional noncoding variants. These variants can impact gene expression, regulation, and chromatin conformation, thereby contributing to disease pathogenesis. Understanding the mechanisms that underlie the impact of noncoding variants on genetic diseases is indispensable for the development of precisely targeted therapies and the implementation of personalized medicine strategies. The intricacies of noncoding regions introduce a multitude of challenges and research opportunities. In this review, we introduce a spectrum of noncoding variants involved in genetic diseases, along with research strategies and advanced technologies for their precise identification and in-depth understanding of the complexity of the noncoding genome. We will delve into the research challenges and propose potential solutions for unraveling the genetic basis of rare and complex diseases.

Molecular diagnosis, clinical evaluation and phenotypic spectrum of Townes-Brocks syndrome: insights from a large Chinese hearing loss cohort.

BACKGROUND: Townes-Brocks syndrome (TBS) is a rare genetic disorder characterised by multiple malformations. Due to its phenotypic heterogeneity and rarity, diagnosis and recognition of TBS can be challenging and there has been a lack of investigation of patients with atypical TBS in large cohorts and delineation of their phenotypic characteristics. METHODS: We screened SALL1 and DACT1 variants using next-generation sequencing in the China Deafness Genetics Consortium (CDGC) cohort enrolling 20 666 unrelated hearing loss (HL) cases. Comprehensive clinical evaluations were conducted on seven members from a three-generation TBS family. Combining data from previously reported cases, we also provided a landscape of phenotypes and genotypes of patients with TBS. RESULTS: We identified five novel and two reported pathogenic/likely pathogenic (P/LP) SALL1 variants from seven families. Audiological features in patients differed in severity and binaural asymmetry. Moreover, previously undocumented malformations in the middle and inner ear were detected in one patient. By comprehensive clinical evaluations, we further provide evidence for the causal relationship between SALL1 variation and certain endocrine abnormalities. Penetrance analysis within familial contexts revealed incomplete penetrance among first-generation patients with TBS and a higher disease burden among their affected offspring. CONCLUSION: This study presents the first insight of genetic screening for patients with TBS in a large HL cohort. We broadened the phenotypic-genotypic spectrum of TBS and our results supported an underestimated prevalence of TBS. Due to the rarity and phenotypic heterogeneity of rare diseases, broader spectrum molecular tests, especially whole genome sequencing, can improve the situation of underdiagnosis and provide effective recommendations for clinical management.

NCAD v1.0: a database for non-coding variant annotation and interpretation.

The application of whole genome sequencing is expanding in clinical diagnostics across various genetic disorders, and the significance of non-coding variants in penetrant diseases is increasingly being demonstrated. Therefore, it is urgent to improve the diagnostic yield by exploring the pathogenic mechanisms of variants in non-coding regions. However, the interpretation of non-coding variants remains a significant challenge, due to the complex functional regulatory mechanisms of non-coding regions and the current limitations of available databases and tools. Hence, we develop the non-coding variant annotation database (NCAD, http://www.ncawdb.net/), encompassing comprehensive insights into 665,679,194 variants, regulatory elements, and element interaction details. Integrating data from 96 sources, spanning both GRCh37 and GRCh38 versions, NCAD v1.0 provides vital information to support the genetic diagnosis of non-coding variants, including allele frequencies of 12 diverse populations, with a particular focus on the population frequency information for 230,235,698 variants in 20,964 Chinese individuals. Moreover, it offers prediction scores for variant functionality, five categories of regulatory elements, and four types of non-coding RNAs. With its rich data and comprehensive coverage, NCAD serves as a valuable platform, empowering researchers and clinicians with profound insights into non-coding regulatory mechanisms while facilitating the interpretation of non-coding variants.

Quantitative thresholds for variant enrichment in 13,845 cases: improving pathogenicity classification in genetic hearing loss.

BACKGROUND: The American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines recommend using variant enrichment among cases as "strong" evidence for pathogenicity per the PS4 criterion. However, quantitative support for PS4 thresholds from real-world Mendelian case-control cohorts is lacking. METHODS: To address this gap, we evaluated and established PS4 thresholds using data from the Chinese Deafness Genetics Consortium. A total of 9,050 variants from 13,845 patients with hearing loss (HL) and 6,570 ancestry-matched controls were analyzed. Positive likelihood ratio and local positive likelihood ratio values were calculated to determine the thresholds corresponding to each strength of evidence across three variant subsets. RESULTS: In subset 1, consisting of variants present in both cases and controls with an allele frequency (AF) in cases ≥ 0.0005, an odds ratio (OR) ≥ 6 achieved strong evidence, while OR ≥ 3 represented moderate evidence. For subset 2, which encompassed variants present in both cases and controls with a case AF < 0.0005, and subset 3, comprising variants found only in cases and absent from controls, we defined the PS4_Supporting threshold (OR > 2.27 or allele count ≥ 3) and the PS4_Moderate threshold (allele count ≥ 6), respectively. Reanalysis applying the adjusted PS4 criteria changed the classification of 15 variants and enabled diagnosis of an additional four patients. CONCLUSIONS: Our study quantified evidence strength thresholds for variant enrichment in genetic HL cases, highlighting the importance of defining disease/gene-specific thresholds to improve the precision and accuracy of clinical genetic testing.

GSDMD gene knockout alleviates hyperoxia-induced hippocampal brain injury in neonatal mice.

BACKGROUND: Neonatal hyperoxia exposure is associated with brain injury and poor neurodevelopment outcomes in preterm infants. Our previous studies in neonatal rodent models have shown that hyperoxia stimulates the brain's inflammasome pathway, leading to the activation of gasdermin D (GSDMD), a key executor of pyroptotic inflammatory cell death. Moreover, we found pharmacological inhibition of caspase-1, which blocks GSDMD activation, attenuates hyperoxia-induced brain injury in neonatal mice. We hypothesized that GSDMD plays a pathogenic role in hyperoxia-induced neonatal brain injury and that GSDMD gene knockout (KO) will alleviate hyperoxia-induced brain injury. METHODS: Newborn GSDMD knockout mice and their wildtype (WT) littermates were randomized within 24 h after birth to be exposed to room air or hyperoxia (85% O2) from postnatal days 1 to 14. Hippocampal brain inflammatory injury was assessed in brain sections by immunohistology for allograft inflammatory factor 1 (AIF1) and CD68, markers of microglial activation. Cell proliferation was evaluated by Ki-67 staining, and cell death was determined by TUNEL assay. RNA sequencing of the hippocampus was performed to identify the transcriptional effects of hyperoxia and GSDMD-KO, and qRT-PCR was performed to confirm some of the significantly regulated genes. RESULTS: Hyperoxia-exposed WT mice had increased microglia consistent with activation, which was associated with decreased cell proliferation and increased cell death in the hippocampal area. Conversely, hyperoxia-exposed GSDMD-KO mice exhibited considerable resistance to hyperoxia as O2 exposure did not increase AIF1 + , CD68 + , or TUNEL + cell numbers or decrease cell proliferation. Hyperoxia exposure differentially regulated 258 genes in WT and only 16 in GSDMD-KO mice compared to room air-exposed WT and GSDMD-KO, respectively. Gene set enrichment analysis showed that in the WT brain, hyperoxia differentially regulated genes associated with neuronal and vascular development and differentiation, axonogenesis, glial cell differentiation, hypoxia-induced factor 1 pathway, and neuronal growth factor pathways. These changes were prevented by GSDMD-KO. CONCLUSIONS: GSDMD-KO alleviates hyperoxia-induced inflammatory injury, cell survival and death, and alterations of transcriptional gene expression of pathways involved in neuronal growth, development, and differentiation in the hippocampus of neonatal mice. This suggests that GSDMD plays a pathogenic role in preterm brain injury, and targeting GSDMD may be beneficial in preventing and treating brain injury and poor neurodevelopmental outcomes in preterm infants.

GSDMD deficiency ameliorates hyperoxia-induced BPD and ROP in neonatal mice.

Bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) are among the most common morbidities affecting extremely premature infants who receive oxygen therapy. Many clinical studies indicate that BPD is associated with advanced ROP. However, the mechanistic link between hyperoxia, BPD, and ROP remains to be explored. Gasdermin D (GSDMD) is a key executor of inflammasome-induced pyroptosis and inflammation. Inhibition of GSDMD has been shown to attenuate hyperoxia-induced BPD and brain injury in neonatal mice. The objective of this study was to further define the mechanistic roles of GSDMD in the pathogenesis of hyperoxia-induced BPD and ROP in mouse models. Here we show that global GSDMD knockout (GSDMD-KO) protects against hyperoxia-induced BPD by reducing macrophage infiltration, improving alveolarization and vascular development, and decreasing cell death. In addition, GSDMD deficiency prevented hyperoxia-induced ROP by reducing vasoobliteration and neovascularization, improving thinning of multiple retinal tissue layers, and decreasing microglial activation. RNA sequencing analyses of lungs and retinas showed that similar genes, including those from inflammatory, cell death, tissue remodeling, and tissue and vascular developmental signaling pathways, were induced by hyperoxia and impacted by GSDMD-KO in both models. These data highlight the importance of GSDMD in the pathogenesis of BPD and ROP and suggest that targeting GSDMD may be beneficial in preventing and treating BPD and ROP in premature infants.

Spike protein mediated membrane fusion during SARS-CoV-2 infection.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a serious threat to public health and has quickly become a global concern. The infection of SARS-CoV-2 begins with the binding of its spike protein to the receptor-angiotensin-converting enzyme 2 (ACE2), which, after a series of conformation changes, results in the fusion of viral-cell membranes and the release of the viral RNA genome into the cytoplasm. In addition, infected host cells can express spike protein on their cell surface, which will interact with ACE2 on neighboring cells, leading to cell membrane fusion and the formation of multinucleated cells or syncytia. Both viral entry and syncytia formation are mediated by spike-ACE2 interaction and share some common mechanisms of membrane fusion. Here in this review, we will summarize our current understanding of spike-mediated membrane fusion, which may shed light on future broad-spectrum antiviral development.

Proactive functional classification of all possible missense single-nucleotide variants in KCNQ4.

Clinical exome sequencing has yielded extensive disease-related missense single-nucleotide variants (SNVs) of uncertain significance, leading to diagnostic uncertainty. KCNQ4 is one of the most commonly responsible genes for autosomal dominant nonsyndromic hearing loss. According to the gnomAD cohort, approximately one in 100 people harbors missense variants in KCNQ4 (missense variants with minor allele frequency > 0.1% were excluded), but most are of unknown consequence. To prospectively characterize the function of all 4085 possible missense SNVs of human KCNQ4, we recorded the whole-cell currents using the patch-clamp technique and categorized 1068 missense SNVs as loss of function, as well as 728 loss-of-function SNVs located in the transmembrane domains. Further, to mimic the heterozygous condition in Deafness nonsyndromic autosomal dominant 2 (DFNA2) patients caused by KCNQ4 variants, we coexpressed loss-of-function variants with wild-type KCNQ4 and found 516 variants showed impaired or only partially rescued heterogeneous channel function. Overall, our functional classification is highly concordant with the auditory phenotypes in Kcnq4 mutant mice and the assessments of pathogenicity in clinical variant interpretations. Taken together, our results provide strong functional evidence to support the pathogenicity classification of newly discovered KCNQ4 missense variants in clinical genetic testing.

Identification of a novel DFNA5 mutation, IVS7-2 a > G, in a Chinese family with non-syndromic sensorineural hearing loss.

BACKGROUND: To date, seven DFNA5 mutations have been reported in families with autosomal dominant non-syndromic hearing loss worldwide. All the mutations cause exon 8 skipping at the mRNA level, that led to the protein truncated and the protein could exert a gain of ototoxic function. OBJECTIVE: In this study, we found an autosomal-dominant non-syndromic hearing loss Chinese pedigree which spanned four generations and comprised 43 members. We want to identify the causative gene and mutation. METHODS: Application of microsatellite markers on DFNA 23 loci preliminary screening of 25 genes, data were analyzed by linkage analysis. RESULTS: We mapped the locus to the region between D7S629 and D7S516 (two-point lod-score of 5.39) with the application of 8 microsatellite markers. By direct sequencing of candidate genes in mapping region, we identified a novel missense mutation ivs7-2 A > G in DFNA5 gene, which was faithfully cosegregated with hearing loss in the family. CONCLUSION AND SIGNIFICANCE: The missense mutation in intron 7 of DFNA5 causes skipping of exon 8, resulting in premature termination of the open reading frame. This type of mutation has repeatedly confirmed that it provides more evidence for the previous view and provides a more solid foundation for future research.

Cytoplasmic domain and enzymatic activity of ACE2 are not required for PI4KB dependent endocytosis entry of SARS-CoV-2 into host cells.

The recent COVID-19 pandemic poses a global health emergency. Cellular entry of the causative agent SARS-CoV-2 is mediated by its spike protein interacting with cellular receptor-human angiotensin converting enzyme 2 (ACE2). Here, by using lentivirus based pseudotypes bearing spike protein, we demonstrated that entry of SARS-CoV-2 into host cells was dependent on clathrin-mediated endocytosis, and phosphoinositides played essential roles during this process. In addition, we showed that the intracellular domain and the catalytic activity of ACE2 were not required for efficient virus entry. Finally, we showed that the current predominant Delta variant, although with high infectivity and high syncytium formation, also entered cells through clathrin-mediated endocytosis. These results provide new insights into SARS-CoV-2 cellular entry and present proof of principle that targeting viral entry could be an effective way to treat different variant infections.

N-Acetyltransferase 8 Promotes Viral Replication by Increasing the Stability of Enterovirus 71 Nonstructural Proteins.

Enterovirus 71 (EV71) is deemed a reemergent pathogen, with recent outbreaks worldwide. EV71 infection causes hand, foot, and mouth disease (HFMD) and has been associated with severe cardiac and central nervous system complications and even death. Viruses need host factors to complete their life cycle; therefore, the identification of the host factors for EV71 infection is pivotal to new antiviral research. Emerging evidence has highlighted the importance of protein acetylation during infection by various human viruses. The endoplasmic reticulum (ER), as the prominent organelle of EV71 replication, also has a unique acetylation regulation mechanism. However, the pathogenesis of EV71 and its relationship with the ER-based acetylation machinery are not fully understood. In this study, we demonstrated for the first time that the ER-resident acetyltransferase N-acetyltransferase 8 (NAT8) is a host factor for EV71 infection. Inhibiting NAT8 with CRISPR or a small compound significantly suppressed EV71 infection in SK-N-SH cells. NAT8 promoted EV71 replication in an acetyltransferase-activity-dependent manner. Additionally, we found that NAT8 facilitates EV71 infection by interacting with EV71 2B, 3AB, and 3C proteins and increasing the stability of these proteins. These results uncovered a novel function of NAT8 and elucidated a new mechanism underlying the regulation of EV71 replication. IMPORTANCE EV71 is one of the most common pathogens causing HFMD in young children, and some patients experience severe or fatal neurological consequences. To ensure efficient replication, the virus must hijack multiple host factors for its own benefit. Here, we show that the ER-resident acetyltransferase NAT8 is a host factor for EV71 infection. EV71 fails to complete its infection in various cells in the absence of NAT8. We further show that NAT8 benefits EV71 replication in an acetyltransferase-activity-dependent manner. Finally, we show that NAT8 facilitates EV71 infection by interacting with EV71 2B, 3AB, and 3C proteins and increasing the stability of these proteins. These results uncovered a novel function of NAT8 in EV71 infection and elucidated a new mechanism underlying the regulation of EV71 replication.

DVPred: a disease-specific prediction tool for variant pathogenicity classification for hearing loss.

Numerous computational prediction tools have been introduced to estimate the functional impact of variants in the human genome based on evolutionary constraints and biochemical metrics. However, their implementation in diagnostic settings to classify variants faced challenges with accuracy and validity. Most existing tools are pan-genome and pan-diseases, which neglected gene- and disease-specific properties and limited the accessibility of curated data. As a proof-of-concept, we developed a disease-specific prediction tool named Deafness Variant deleteriousness Prediction tool (DVPred) that focused on the 157 genes reportedly causing genetic hearing loss (HL). DVPred applied the gradient boosting decision tree (GBDT) algorithm to the dataset consisting of expert-curated pathogenic and benign variants from a large in-house HL patient cohort and public databases. With the incorporation of variant-level and gene-level features, DVPred outperformed the existing universal tools. It boasts an area under the curve (AUC) of 0.98, and showed consistent performance (AUC = 0.985) in an independent assessment dataset. We further demonstrated that multiple gene-level metrics, including low complexity genomic regions and substitution intolerance scores, were the top features of the model. A comprehensive analysis of missense variants showed a gene-specific ratio of predicted deleterious and neutral variants, implying varied tolerance or intolerance to variation in different genes. DVPred explored the utility of disease-specific strategy in improving the deafness variant prediction tool. It can improve the prioritization of pathogenic variants among massive variants identified by high-throughput sequencing on HL genes. It also shed light on the development of variant prediction tools for other genetic disorders.

The natural history of OTOF-related auditory neuropathy spectrum disorders: a multicenter study.

Pathogenic variations in the OTOF gene are a common cause of hearing loss. To refine the natural history and genotype-phenotype correlations of OTOF-related auditory neuropathy spectrum disorders (ANSD), audiograms and distortion product otoacoustic emissions (DPOAEs) were collected from a diverse cohort of individuals diagnosed with OTOF-related ANSD by comprehensive genetic testing and also reported in the literature. Comparative analysis was undertaken to define genotype-phenotype relationships using a Monte Carlo algorithm. 67 audiograms and 25 DPOAEs from 49 unique individuals positive for OTOF-related ANSD were collected. 51 unique OTOF pathogenic variants were identified of which 21 were missense and 30 were loss of function (LoF; nonsense, splice-site, copy number variants, and indels). There was a statistically significant difference in low, middle, and high frequency hearing thresholds between missense/missense and LoF/missense genotypes as compared to LoF/LoF genotypes (average hearing threshold for low, middle and high frequencies 70.9, 76.0, and 73.4 dB vs 88.5, 95.6, and 94.7 dB) via Tukey's test with age as a co-variate (P = 0.0180, 0.0327, and 0.0347, respectively). Hearing declined during adolescence with missense/missense and LoF/missense genotypes, with an annual mid-frequency threshold deterioration of 0.87 dB/year and 1.87 dB/year, respectively. 8.5% of frequencies measured via DPOAE were lost per year in individuals with serial tests. Audioprofiling of OTOF-related ANSD suggests significantly worse hearing with LoF/LoF genotypes. The unique pattern of variably progressive OTOF-related autosomal recessive ANSD may be amenable to gene therapy in selected clinical scenarios.