Liquid plasma promotes angiogenesis through upregulation of endothelial nitric oxide synthase-induced extracellular matrix metabolism: potential applications of liquid plasma for vascular injuries.

BACKGROUND: Applications of nonthermal plasma have expanded beyond the biomedical field to include antibacterial, anti-inflammatory, wound healing, and tissue regeneration. Plasma enhances epithelial cell repair; however, the potential damage to deep tissues and vascular structures remains under investigation. RESULT: This study assessed whether liquid plasma (LP) increased nitric oxide (NO) production in human umbilical vein endothelial cells by modulating endothelial NO synthase (eNOS) phosphorylation and potential signaling pathways. First, we developed a liquid plasma product and confirmed the angiogenic effect of LP using the Matrigel plug assay. We found that the NO content increased in plasma-treated water. NO in plasma-treated water promoted cell migration and angiogenesis in scratch and tube formation assays via vascular endothelial growth factor mRNA expression. In addition to endothelial cell proliferation and migration, LP influenced extracellular matrix metabolism and matrix metalloproteinase activity. These effects were abolished by treatment with NG-L-monomethyl arginine, a specific inhibitor of NO synthase. Furthermore, we investigated the signaling pathways mediating the phosphorylation and activation of eNOS in LP-treated cells and the role of LKB1-adenosine monophosphate-activated protein kinase in signaling. Downregulation of adenosine monophosphate-activated protein kinase by siRNA partially inhibited LP-induced eNOS phosphorylation, angiogenesis, and migration. CONCLUSION: The present study suggests that LP treatment may be a novel strategy for promoting angiogenesis in vascular damage. Video Abstract.

A nonsense TMEM43 variant leads to disruption of connexin-linked function and autosomal dominant auditory neuropathy spectrum disorder.

Genes that are primarily expressed in cochlear glia-like supporting cells (GLSs) have not been clearly associated with progressive deafness. Herein, we present a deafness locus mapped to chromosome 3p25.1 and an auditory neuropathy spectrum disorder (ANSD) gene, TMEM43, mainly expressed in GLSs. We identify p.(Arg372Ter) of TMEM43 by linkage analysis and exome sequencing in two large Asian families segregating ANSD, which is characterized by inability to discriminate speech despite preserved sensitivity to sound. The knock-in mouse with the p.(Arg372Ter) variant recapitulates a progressive hearing loss with histological abnormalities in GLSs. Mechanistically, TMEM43 interacts with the Connexin26 and Connexin30 gap junction channels, disrupting the passive conductance current in GLSs in a dominant-negative fashion when the p.(Arg372Ter) variant is introduced. Based on these mechanistic insights, cochlear implant was performed on three subjects, and speech discrimination was successfully restored. Our study highlights a pathological role of cochlear GLSs by identifying a deafness gene and its causal relationship with ANSD.

Rising of LOXHD1 as a signature causative gene of down-sloping hearing loss in people in their teens and 20s.

BACKGROUND: Down-sloping sensorineural hearing loss (SNHL) in people in their teens and 20s hampers efficient learning and communication and in-depth social interactions. Nonetheless, its aetiology remains largely unclear, with the exception of some potential causative genes, none of which stands out especially in people in their teens and 20s. Here, we examined the role and genotype-phenotype correlation of lipoxygenase homology domain 1 (LOXHD1) in down-sloping SNHL through a cohort study. METHODS: Based on the Seoul National University Bundang Hospital (SNUBH) genetic deafness cohort, in which the patients show varying degrees of deafness and different onset ages (n=1055), we have established the 'SNUBH Teenager-Young Adult Down-sloping SNHL' cohort (10-35 years old) (n=47), all of whom underwent exome sequencing. Three-dimensional molecular modelling, minigene splicing assay and short tandem repeat marker genotyping were performed, and medical records were reviewed. RESULTS: LOXHD1 accounted for 33.3% of all genetically diagnosed cases of down-sloping SNHL (n=18) and 12.8% of cases in the whole down-sloping SNHL cohort (n=47) of young adults. We identified a potential common founder allele, as well as an interesting genotype-phenotype correlation. We also showed that transcript 6 is necessary and probably sufficient for normal hearing. CONCLUSIONS: LOXHD1 exceeds other genes in its contribution to down-sloping SNHL in young adults, rising as a signature causative gene, and shows a potential but interesting genotype-phenotype correlation.

Natural Course of Residual Hearing with Reference to GJB2 and SLC26A4 Genotypes: Clinical Implications for Hearing Rehabilitation.

BACKGROUND: Understanding the characteristics of residual hearing at low frequencies and its natural course in relation to molecular genetic etiology may be important in developing rehabilitation strategies. Thus, we aimed to explore the characteristics and natural course of residual hearing at low frequencies associated with the two most frequent deafness genes: GJB2 and SLC26A4. METHODS: Initially, 53 GJB2 and 65 SLC26A4 subjects were enrolled, respectively. Only those whose audiograms exhibited hearing thresholds ≤70 dB at 250 and 500 Hz, and who had at least 1-year follow-up period between the first and last audiograms, were included. Collectively, the clinical characteristics of 14 ears from eight subjects with GJB2 variants, and 31 ears from 22 subjects with SLC26A4 variants fulfilled the strict criteria. In this study, a dropout rate refers to an incidence of dropping out of the cohort by cochlear implant surgery due to severe hearing deterioration. RESULTS: Among the ears with complete serial audiogram data set, significant residual hearing at low frequencies at the time of inclusion was observed in 18.8% of those with GJB2 variants (15 out of 80 ears) and 42.6% of those with SLC26A4 variants (46 out of 108 ears), revealing a difference between two deafness genes. Subsequently, ears with SLC26A4 variants (11 of 46 ears, 23.9%) turned out to have a higher dropout rate for cochlear implantation due to hearing deterioration within the first year than those with GJB2 variants (1 of 15, 6.7%), albeit with no statistical significance. Throughout the follow-up period (mean: 37.2 ± 6.8, range: 12 to 80 months), deterioration of residual hearing at low frequencies at 250 Hz (dB HL/y) and 500 Hz (dB HL/y) of those with GJB2 variants exhibited 3.1 ± 1.3 (range: 0 to 15) and 5.2 ± 1.6 (range: 0 to 20), respectively, suggesting the deterioration of residual hearing in GJB2 variants was rather slow and gradual. Specifically, GJB2 p.Leu79Cysfs*3 show less remarkable residual hearing at low frequencies, but then a relatively stable nature. In contrast, SLC26A4 variants demonstrated a significantly higher dropout rate due to severe hearing deterioration requiring cochlear implantation compared with the GJB2 variants. This trend was observed not only in the first-year follow-up period but also in the follow-up periods thereafter. The p.His723Arg;c.919-2A>G genotype of SLC26A4, in particular, was associated with a high propensity for sudden hearing deterioration, as indicated by the dropout rate, which was as high as 46.2% for cochlear implantation due to hearing deterioration during the first year follow-up period. Furthermore, the dropout rate for cochlear implantation was observed in 7.1% of those with GJB2 variants (one out of 14 ears) and 30.3% of those with SLC26A4 variants (10 out of 33 ears) throughout the entire follow-up period. CONCLUSIONS: Our results suggest that there is a difference with respect to the progressive nature of residual hearing at low frequencies between the two most common genes responsible for hearing loss, which may provide clinical implications of having individualized rehabilitation and timely intervention.

Novel genotype-phenotype correlation of functionally characterized LMX1A variants linked to sensorineural hearing loss.

LMX1A, encoding the LIM homeobox transcription factor, is essential for inner ear development. Despite previous reports of three human LMX1A variants with nonsyndromic hearing loss (NSHL) in the literature, functional characterization of these variants has never been performed. Encouraged by identification of a de novo, heterozygous, missense variant (c.595A > G; p.Arg199Gly) located in the homeodomain of LMX1A in a subject with congenital severe-to-profound deafness through Exome sequencing, we performed luciferase assay to evaluate transcriptional activity of all LMX1A variants reported in the literature including p.Arg199Gly. Resultantly, p.Arg199Gly manifesting the most severe NSHL showed the biggest reduction of transcriptional activity in contrast with moderately reduced activity of p.Cys97Ser and p.Val241Leu associated with less severe progressive NSHL, proposing a genotype-phenotype correlation. Further, our dominant LMX1A variant exerted pathogenic effects via haploinsufficiency rather than dominant-negative effect. Collectively, we provide a potential genotype-phenotype correlation of LMX1A variants as well as the pathogenic mechanism of LMX1A-related NSHL.

POLD1 variants leading to reduced polymerase activity can cause hearing loss without syndromic features.

DNA polymerase δ, whose catalytic subunit is encoded by POLD1, is responsible for synthesizing the lagging strand of DNA. Single heterozygous POLD1 mutations in domains with polymerase and exonuclease activities have been reported to cause syndromic deafness as a part of multisystem metabolic disorder or predisposition to cancer. However, the phenotypes of diverse combinations of POLD1 genotypes have not been elucidated in humans. We found that five members of a multiplex family segregating autosomal recessive nonsyndromic sensorineural hearing loss (NS-SNHL) have revealed novel compound heterozygous POLD1 variants (p.Gly1100Arg and a presumptive null function variant, p.Ser197Hisfs*54). The recombinant p.Gly1100Arg polymerase δ showed a reduced polymerase activity by 30-40%, but exhibited normal exonuclease activity. The polymerase activity in cell extracts from the affected subject carrying the two POLD1 mutant alleles was about 33% of normal controls. We suggest that significantly decreased polymerase δ activity, but not a complete absence, with normal exonuclease activity could lead to NS-SNHL.

Significant Mendelian genetic contribution to pediatric mild-to-moderate hearing loss and its comprehensive diagnostic approach.

PURPOSE: Timely diagnosis and identification of etiology of pediatric mild-to-moderate sensorineural hearing loss (SNHL) are both medically and socioeconomically important. However, the exact etiologic spectrum remains uncertain. We aimed to establish a genetic etiological spectrum, including copy-number variations (CNVs) and efficient genetic testing pipeline, of this defect. METHODS: A cohort of prospectively recruited pediatric patients with mild-to-moderate nonsyndromic SNHL from 2014 through 2018 (n = 110) was established. Exome sequencing, multiplex ligation-dependent probe amplification (MLPA), and nested customized polymerase chain reaction (PCR) for exclusion of a pseudogene, STRCP, from a subset (n = 83) of the cohort, were performed. Semen analysis was also performed to determine infertility (n = 2). RESULTS: Genetic etiology was confirmed in nearly two-thirds (52/83 = 62.7%) of subjects, with STRC-related deafness (n = 29, 34.9%) being the most prevalent, followed by MPZL2-related deafness (n = 9, 10.8%). This strikingly high proportion of Mendelian genetic contribution was due particularly to the frequent detection of CNVs involving STRC in one-third (27/83) of our subjects. We also questioned the association of homozygous continuous gene deletion of STRC and CATSPER2 with deafness-infertility syndrome (MIM61102). CONCLUSION: Approximately two-thirds of sporadic pediatric mild-to-moderate SNHL have a clear Mendelian genetic etiology, and one-third is associated with CNVs involving STRC. Based on this, we propose a new guideline for molecular diagnosis of these children.

Differential disruption of autoinhibition and defect in assembly of cytoskeleton during cell division decide the fate of human DIAPH1-related cytoskeletopathy.

BACKGROUND: Diaphanous-related formin 1 (DIA1), which assembles the unbranched actin microfilament and microtubule cytoskeleton, is encoded by DIAPH1. Constitutive activation by the disruption of autoinhibitory interactions between the N-terminal diaphanous inhibitory domain (DID) and C-terminal diaphanous autoregulatory domain (DAD) dysregulates DIA1, resulting in both hearing loss and blood cell abnormalities. METHODS AND RESULTS: Here, we report the first constitutively active mutant in the DID (p.A265S) of humans with only hearing loss and not blood cell abnormality through whole exome sequencing. The previously reported DAD mutants and our DID mutant (p.A265S) shared the finding of diminished autoinhibitory interaction, abnormally upregulated actin polymerisation activity and increased localisations at the plasma membrane. However, the obvious defect in the DIA1-driven assembly of cytoskeleton 'during cell division' was only from the DAD mutants, not from p.A265S, which did not show any blood cell abnormality. We also evaluated the five DID mutants in the hydrophobic pocket since four of these five additional mutants were predicted to critically disrupt interaction between the DID and DAD. These additional pathogenic DID mutants revealed varying degrees of defect in the DIA1-driven cytoskeleton assembly, including nearly normal phenotype during cell division as well as obvious impaired autoinhibition, again coinciding with our key observation in DIA1 mutant (p.A265S) in the DID. CONCLUSION: Here, we report the first mutant in the DID of humans with only hearing loss. The differential cell biological phenotypes of DIA1 during cell division appear to be potential determinants of the clinical severity of DIAPH1-related cytoskeletopathy in humans.

Clarification of glycosylphosphatidylinositol anchorage of OTOANCORIN and human OTOA variants associated with deafness.

Otoancorin (OTOA), encoded by OTOA, is required for the development of the tectorial membrane in the inner ear. Mutations in this gene cause nonsyndromic hearing loss (DFNB22). The molecular mechanisms underlying most DFNB22 remain poorly understood. Disruption of glycosylphosphatidylinositol (GPI) anchorage has been assumed to be the pathophysiology mandating experimental validation. From a Korean deaf family, we identified two trans OTOA variants (c.1320 + 5 G > C and p.Gln589ArgfsX55 [NM_144672.3]) . The pathogenic potential of c.1320 + 5 G > C was confirmed by a minigene splicing assay. To experimentally determine the GPI anchorage, wild-type (WT) and mutant OTOA harboring p.Gln589ArgfsX55 were expressed in HEK293T cells. The mutant OTOA with p.Gln589ArgfsX55 resulted in an uncontrolled release of OTOA into the medium in contrast with phosphatidylinositol-specific phospholipase C-induced controlled release of WT OTOA from the cell surface. Together, the results of this reverse translational study confirmed GPI-anchorage of OTOA and showed that downstream sequences from the 589th amino acid are critical for GPI-anchorage.

Analyzing Somatic Genome Rearrangements in Human Cancers by Using Whole-Exome Sequencing.

Although exome sequencing data are generated primarily to detect single-nucleotide variants and indels, they can also be used to identify a subset of genomic rearrangements whose breakpoints are located in or near exons. Using >4,600 tumor and normal pairs across 15 cancer types, we identified over 9,000 high confidence somatic rearrangements, including a large number of gene fusions. We find that the 5' fusion partners of functional fusions are often housekeeping genes, whereas the 3' fusion partners are enriched in tyrosine kinases. We establish the oncogenic potential of ROR1-DNAJC6 and CEP85L-ROS1 fusions by showing that they can promote cell proliferation in vitro and tumor formation in vivo. Furthermore, we found that ∼4% of the samples have massively rearranged chromosomes, many of which are associated with upregulation of oncogenes such as ERBB2 and TERT. Although the sensitivity of detecting structural alterations from exomes is considerably lower than that from whole genomes, this approach will be fruitful for the multitude of exomes that have been and will be generated, both in cancer and in other diseases.

Identification of a Potential Founder Effect of a Novel PDZD7 Variant Involved in Moderate-to-Severe Sensorineural Hearing Loss in Koreans.

PDZD7, a PDZ domain-containing scaffold protein, is critical for the organization of Usher syndrome type 2 (USH2) interactome. Recently, biallelic PDZD7 variants have been associated with autosomal-recessive, non-syndromic hearing loss (ARNSHL). Indeed, we identified novel, likely pathogenic PDZD7 variants based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines from Korean families manifesting putative moderate-to-severe prelingual ARNSHL; these were c.490C>T (p.Arg164Trp), c.1669delC (p.Arg557Glyfs*13), and c.1526G>A (p.Gly509Glu), with p.Arg164Trp being a predominantly recurring variant. Given the recurring missense variant (p.Arg164Trp) from our cohort, we compared the genotyping data using six short tandem-repeat (STR) markers within or flanking PDZD7 between four probands carrying p.Arg164Trp and 81 normal-hearing controls. We observed an identical haplotype across three out of six STR genotyping markers exclusively shared by two unrelated hearing impaired probands but not by any of the 81 normal-hearing controls, suggesting a potential founder effect. However, STR genotyping, based on six STR markers, revealed various p.Arg164Trp-linked haplotypes shared by all of the affected subjects. In conclusion, PDZD7 can be an important causative gene for moderate to severe ARNSHL in Koreans. Moreover, at least some, if not all, p.Arg164Trp alleles in Koreans could exert a potential founder effect and arise from diverse haplotypes as a mutational hot spot.

A clinical guidance to DFNA22 drawn from a Korean cohort study with an autosomal dominant deaf population: A retrospective cohort study.

BACKGROUND: The MYO6 gene, if altered, can cause nonsyndromic hearing loss (NSHL) either in an autosomal dominant (AD) (DFNA22) or recessive form. The present study identified MYO6 variants in the cohort of Korean AD NSHL families and investigated the audiological phenotypes of DFNA22 with respect to suggesting clinical guides for the counseling of DFNA22. METHODS: A retrospective cohort study was performed on 81 AD NSHL families in two hospitals. Among them, five families (SH21, SB60, SB247, SB290 and SB305) segregating with MYO6 variant were genetically and clinically assessed. RESULTS: We identified two novel missense variants of MYO6: p.G223R (SB290) and p.T158R (SB305). A known heterozygous truncation variant, p.R205X, reported previously (SH21, SB60), was identified (SB247). The overall frequency of DFNA22 among such cases was 6.2%. Specifically, we found p.R205X from three of five DFNA22 families (60%). Five DFNA22 families demonstrated extremely diverse audiogram configurations and age of onset with even intrafamilial variations, whereas the severity of hearing loss mostly ranged within moderate. CONCLUSIONS: We report a recurring predominant allele and two new missense variants of MYO6, highlighting the significant contribution of MYO6 to AD NSHL in the Korean population. Extremely diverse audiological configurations of DFNA22 suggest that MYO6 should be considered in future genetic studies of patients with AD NSHL. Gradual progression with a good speech audiometry score could provide physicians with clinical insight with respect to advising patients to use hearing aids or consider middle ear implants, whereas, in the case of certain exceptional circumstances, physicians could provide patients with the option to consider a cochlear implant.

Mutational and phenotypic spectrum of OTOF-related auditory neuropathy in Koreans: eliciting reciprocal interaction between bench and clinics.

BACKGROUND: While auditory neuropathy spectrum disorder (ANSD) is a heterogeneous disorder and its management quite varies depending upon the etiology, even including self-resolution, OTOF is an important molecular etiology of prelingual ANSD and has emerged as an attractive target for implementation of precision medicine in terms of timing and prognosis prediction of auditory rehabilitation. However, to date, the literature is lacking in the genotype-phenotype relationship of this gene as well as efficient molecular testing strategy in the clinic in many populations and to make things more complicated in Koreans, the most prevalent variant p.Arg1939Gln among Korean ANSD children frequently evaded detection by next generation sequencing (NGS), resulting in delayed genetic diagnosis and late cochlear implantation (CI). The aims of this study are to document the mutational and phenotypic spectrum of OTOF-related ANSD (DFNB9) in the Korean population, further establishing genotype-phenotype correlation and proposing a set of the most commonly found OTOF variants to be screened first. METHODS: Genetic diagnosis through the NGS-based sequencing was made on patients with ANSD in two tertiary hospitals. Genotype and phenotypes of eleven DFNB9 patients were reviewed. For data analysis, Mann-Whitney test and Fisher's exact test were applied. RESULTS: This study disclosed four prevalent variants in Koreans: p.Arg1939Gln with an allele frequency of 40.9%, p.Glu841Lys (13.6%), p.Leu1011Pro and p.Arg1856Trp (9.1%). Three novel variants (c.4227 + 5G > C, p.Gly1845Glu, and p.Pro1931Thr) were identified. Interestingly, a significant association of p.Arg1939Gln with worse ASSR thresholds was observed despite consistently no ABR response. Ten of 11 DFNB9 patients received CI for auditory rehabilitation, showing favorable outcomes with more rapid improvement on early-CI group (age at CI ≤ 18 mo.) than late-CI group. CONCLUSIONS: This study included the largest Korean DFNB9 cohort to date and proposed a set of the most frequent four OTOF variants, allowing the potential prioritization of exons during Sanger sequencing. Further, a significant association of p.Arg1939Gln homozygotes with poor residual hearing was observed. We may have to suspect p.Arg1939Gln homozygosity in cases of poor auditory thresholds in ANSD children with putative negative OTOF variants solely screened by NGS. Reciprocal feedback between bench and clinics regarding DFNB9 would complement each other.

Expansion of phenotypic spectrum of MYO15A pathogenic variants to include postlingual onset of progressive partial deafness.

BACKGROUND: MYO15A variants, except those in the N-terminal domain, have been shown to be associated with congenital or pre-lingual severe-to-profound hearing loss (DFNB3), which ultimately requires cochlear implantation in early childhood. Recently, such variants have also been shown to possibly cause moderate-to-severe hearing loss. Herein, we also demonstrate that some MYO15A mutant alleles can cause postlingual onset of progressive partial deafness. METHODS: Two multiplex Korean families (SB246 and SB224), manifesting postlingual, progressive, partial deafness in an autosomal recessive fashion, were recruited. Molecular genetics testing was performed in two different pipelines, in a parallel fashion, for the SB246 family: targeted exome sequencing (TES) of 129 known deafness genes from the proband and whole exome sequencing (WES) of all affected subjects. Only the former pipeline was performed for the SB224 family. Rigorous bioinformatics analyses encompassing structural variations were executed to investigate any causative variants. RESULTS: In the SB246 family, two different molecular diagnostic pipelines provided exactly the same candidate variants: c.5504G > A (p.R1835H) in the motor domain and c.10245_10247delCTC (p.S3417del) in the FERM domain of MYO15A. In the SB224 family, c.9790C > T (p.Q3264X) and c.10263C > G (p.I3421M) in the FERM domain were detected as candidate variants. CONCLUSIONS: Some recessive MYO15A variants can cause postlingual onset of progressive partial deafness. The phenotypic spectrum of DFNB3 should be extended to include such partial deafness. The mechanism for a milder phenotype could be due to the milder pathogenic potential from hypomorphic alleles of MYO15A or the presence of modifier genes. This merits further investigation.

Molecular breakdown: a comprehensive view of anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer.

Most anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancers (NSCLCs) show good clinical response to ALK inhibitors. However, some ALK-rearranged NSCLC patients show various primary responses with unknown reasons. Previous studies focused on the clinical aspects of ALK fusions in small cohorts, or were conducted in vitro and/or in vivo to investigate the function of ALK. One of the suggested theories describes how echinoderm microtubule-associated protein-like 4 (EML4)-ALK variants play a role towards different sensitivities in ALK inhibitors. Until now, there has been no integrated comprehensive study that dissects ALK at the molecular level in a large scale. Here, we report the largest extensive molecular analysis of 158 ALK-rearranged NSCLCs and have investigated these findings in a cell line construct experiment. We discovered that NSCLCs with EML4-ALK short forms (variant 3/others) had more advanced stage and frequent metastases than cases with the long forms (variant 1/others) (p = 0.057, p < 0.05). In vitro experiments revealed that EML4-ALK short forms show lower sensitivity to ALK inhibitors than do long forms. Clinical analysis also showed a trend for the short forms showing worse PFS. Interestingly, we found that breakpoints of ALK are evenly distributed mainly in intron 19 and almost all of them undergo a non-homologous end-joining repair to generate ALK fusions. We also discovered four novel somatic ALK mutations in NSCLC (T1151R, R1192P, A1280V, and L1535Q) that confer primary resistance; all of them showed strong resistance to ALK inhibitors, as G1202R does. Through targeted deep sequencing, we discovered three novel ALK fusion partners (GCC2, LMO7, and PHACTR1), and different ALK fusion partners showed different intracellular localization. With our findings that the EML4-ALK variants, new ALK somatic mutations, and novel ALK-fusion partners may affect sensitivity to ALK inhibitors, we stress the importance of targeted therapy to take the ALK molecular profiling into consideration. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Discovery of MYH14 as an important and unique deafness gene causing prelingually severe autosomal dominant nonsyndromic hearing loss.

BACKGROUND: Pathogenic variants of MYH14 are known to be associated (in either a syndromic or nonsyndromic manner) with hearing loss. Interestingly, all reported cases to date of MYH14-related nonsyndromic hearing loss with detailed phenotypes have demonstrated mild-to-moderate progressive hearing loss with postlingual onset. METHODS: In the present study, targeted resequencing (TRS) of known deafness genes was performed to identify the causative variant in two multiplex families segregating autosomal dominant (AD) inherited hearing loss. RESULTS: TRS uncovered two novel variants of MYH14 (c.A572G: p.Asp191Gly in the myosin head domain and c.C73T:p.Gln25* in exon 2) from two multiplex deafness Korean families. Notably, both probands showed phenotypes of congenital or prelingual severe hearing loss. It is remarkably uncommon to encounter such a severe-to-profound, prelingual, AD hearing loss. Given that the first variant, p. Asp191Gly, was the first documented missense allele discovered in the myosin head domain of this gene related to either congenital or prelingual severe nonsyndromic hearing loss, and also that the second variant, p. Gln25*, lead to a null allele, more severe phenotypes from our probands may have been the result of either genotype-phenotype correlation or genetic backgrounds, or both. CONCLUSIONS: In the present study, we report that MYH14 can manifest as nonsyndromic prelingual severe sensorineural hearing loss in an AD fashion in Koreans. The results of the present study suggest that further genetic studies of similar patients should consider MYH14 as a causative gene, and cochlear implantation during infant or early childhood should be indicated for those patients with certain MYH14 pathogenic variants.

Precision medicine approaches to lung adenocarcinoma with concomitant MET and HER2 amplification.

BACKGROUND: Patient-derived xenograft (PDX) models are important tools in precision medicine and for the development of targeted therapies to treat cancer patients. This study aimed to evaluate our precision medicine strategy that integrates genomic profiling and preclinical drug-screening platforms, in order to personalize cancer treatments using PDX models. METHODS: We performed array-comparative genomic hybridization, microarray, and targeted next-generation sequencing analyses, in order to determine the oncogenic driver mutations. PDX cells were obtained from PDXs and subsequently screened in vitro with 17 targeted agents. RESULTS: PDX tumors recapitulated the histopathologic and genetic features of the patient tumors. Among the samples from lung cancer patients that were molecularly-profiled, copy number analysis identified unique focal MET amplification in one sample, 033 T, without RTK/RAS/RAF oncogene mutations. Although HER2 amplification in 033 T was not detected in the cancer panel, the selection of HER2-amplified clones was found in PDXs and PDX cells. Additionally, MET and HER2 overexpression were found in patient tumors, PDXs, and PDX cells. Crizotinib or EGFR tyrosine kinase inhibitor treatments significantly inhibited cell growth and impaired tumor sphere formation in 033 T PDX cells. CONCLUSIONS: We established PDX cell models using surgical samples from lung cancer patients, and investigated their preclinical and clinical implications for personalized targeted therapy. Additionally, we suggest that MET and EGFR inhibitor-based therapy can be used to treat MET and HER2-overexpressing lung cancers, without receptor tyrosine kinase /RAS/RAF pathway alterations.

The Analysis of A Frequent TMPRSS3 Allele Containing P.V116M and P.V291L in A Cis Configuration among Deaf Koreans.

We performed targeted re-sequencing to identify the genetic etiology of early-onset postlingual deafness and encountered a frequent TMPRSS3 allele harboring two variants in a cis configuration. We aimed to evaluate the pathogenicity of the allele. Among 88 cochlear implantees with autosomal recessive non-syndromic hearing loss, subjects with GJB2 and SLC26A4 mutations were excluded. Thirty-one probands manifesting early-onset postlingual deafness were sorted. Through targeted re-sequencing, we detected two families with a TMPRSS3 mutant allele containing p.V116M and p.V291L in a cis configuration, p.[p.V116M; p.V291L]. A minor allele frequency was calculated and proteolytic activity was measured. A p.[p.V116M; p.V291L] allele demonstrated a significantly higher frequency compared to normal controls and merited attention due to its high frequency (4.84%, 3/62). The first family showed a novel deleterious splice site variant-c.783-1G>A-in a trans allele, while the other showed homozygosity. The progression to deafness was noted within the first decade, suggesting DFNB10. The proteolytic activity was significantly reduced, confirming the severe pathogenicity. This frequent mutant allele significantly contributes to early-onset postlingual deafness in Koreans. For clinical implication and proper auditory rehabilitation, it is important to pay attention to this allele with a severe pathogenic potential.

Cancer-Specific Sequences in the Diagnosis and Treatment of NUT Carcinoma.

PURPOSE: NUT carcinoma (NC) is a solid tumor caused by the rearrangement of NUTM1 that usually develops in midline structures, such as the thorax. No standard treatment has been established despite high lethality. Thus, we investigated whether targeting the junction region of NUTM1 fusion breakpoints could serve as a potential treatment option for NC. Materials and Methods: We designed and evaluated a series of small interfering RNAs (siRNAs) targeting the junction region of BRD4-NUTM1 fusion (B4N), the most common form of NUTM1 fusion. Droplet digital polymerase chain reaction using the blood of patients was also tested to evaluate the treatment responses by the junction sequence of the B4N fusion transcripts. RESULTS: As expected, the majority of NC fusion types were B4N (12 of 18, 67%). B4N fusion-specific siRNA treatment on NC cells showed specific inhibitory effects on the B4N fusion transcript and fusion protein without affecting the endogenous expression of the parent genes, resulting in decreased relative cell growth and attenuation of tumor size. In addition, the fusion transcript levels in platelet-rich-plasma samples of the NC patients with systemic metastasis showed a negative correlation with therapeutic effect, suggesting its potential as a measure of treatment responsiveness. CONCLUSION: This study suggests that tumor-specific sequences could be used to treat patients with fusion genes as part of precision medicine for a rare but deadly disease.

Improving genetic diagnosis by disease-specific, ACMG/AMP variant interpretation guidelines for hearing loss.

The 2018 Hearing Loss Expert Panel (HL-EP)-specific guidelines specified from the universal 2015 ACMG/AMP guidelines are proposed to be used in genetic HL, which prompted this study. A genetic HL cohort comprising 135 unrelated probands with available exome sequencing data was established. Overall, 169 variants were prioritized as candidates and interpreted using the 2015 ACMG/AMP and 2018 HL-EP guidelines. Changes in rule application and variant classification between the guidelines were compared. The concordance rate of variant classification of each variant between the guidelines was 71.60%, with significant difference. The proportion of pathogenic variants increased from 13.02% (2015) to 29.59% (2018). Variant classifications of autosomal recessive (AR) variants that previously belonged to VUS or likely pathogenic in the 2015 guidelines were changed toward pathogenic in the 2018 guidelines more frequently than those of autosomal dominant variants (29.17% vs. 6.38%, P = 0.005). Stratification of the PM3 and PP1 rules in the 2018 guidelines led to more substantial escalation than that in the 2015 guidelines. We compared the disease-specific guidelines (2018) with the universal guidelines (2015) using real-world data. Owing to the sophistication of case-level data, the HL-specific guidelines have more explicitly classified AR variants toward "likely pathogenic" or "pathogenic", serving as potential references for other recessive genetic diseases.