Identifying Contact Time Required for Secondary Transmission of Clostridioides difficile Infections by Using Real-Time Locating System.

Considering patient room shortages and prevalence of other communicable diseases, reassessing the isolation of patients with Clostridioides difficile infection (CDI) is imperative. We conducted a retrospective study to investigate the secondary CDI transmission rate in a hospital in South Korea, where patients with CDI were not isolated. Using data from a real-time locating system and electronic medical records, we investigated patients who had both direct and indirect contact with CDI index patients. The primary outcome was secondary CDI transmission, identified by whole-genome sequencing. Among 909 direct and 2,711 indirect contact cases, 2 instances of secondary transmission were observed (2 [0.05%] of 3,620 cases), 1 transmission via direct contact and 1 via environmental sources. A low level of direct contact (113 minutes) was required for secondary CDI transmission. Our findings support the adoption of exhaustive standard preventive measures, including environmental decontamination, rather than contact isolation of CDI patients in nonoutbreak settings.

Common genes and recurrent causative variants in 957 Asian patients with pediatric epilepsy.

OBJECTIVE: We aimed to identify common genes and recurrent causative variants in a large group of Asian patients with different epilepsy syndromes and subgroups. METHODS: Patients with unexplained pediatric-onset epilepsy were identified from the in-house Severance Neurodevelopmental Disorders and Epilepsy Database. All patients underwent either exome sequencing or multigene panels from January 2017 to December 2019, at Severance Children's Hospital in Korea. Clinical data were extracted from the medical records. RESULTS: Of the 957 patients studied, 947 (99.0%) were Korean and 570 were male (59.6%). The median age at testing was 4.91 years (interquartile range, 1.53-9.39). The overall diagnostic yield was 32.4% (310/957). Clinical exome sequencing yielded a diagnostic rate of 36.9% (134/363), whereas the epilepsy panel yielded a diagnostic rate of 29.9% (170/569). Diagnostic yield differed across epilepsy syndromes. It was high in Dravet syndrome (87.2%, 41/47) and early infantile developmental epileptic encephalopathy (60.7%, 17/28), but low in West syndrome (21.8%, 34/156) and myoclonic-atonic epilepsy (4.8%, 1/21). The most frequently implicated genes were SCN1A (n = 49), STXBP1 (n = 15), SCN2A (n = 14), KCNQ2 (n = 13), CDKL5 (n = 11), CHD2 (n = 9), SLC2A1 (n = 9), PCDH19 (n = 8), MECP2 (n = 6), SCN8A (n = 6), and PRRT2 (n = 5). The recurrent genetic abnormalities included 15q11.2 deletion/duplication (n = 9), Xq28 duplication (n = 5), PRRT2 deletion (n = 4), MECP2 duplication (n = 3), SCN1A, c.2556+3A>T (n = 3), and 2q24.3 deletion (n = 3). SIGNIFICANCE: Here we present the results of a large-scale study conducted in East Asia, where we identified several common genes and recurrent variants that varied depending on specific epilepsy syndromes. The overall genetic landscape of the Asian population aligns with findings from other populations of varying ethnicities.

Utility of Plasma Microbial Cell-Free DNA Whole-Genome Sequencing for Diagnosis of Invasive Aspergillosis in Patients With Hematologic Malignancy or COVID-19.

BACKGROUND: We evaluated the clinical accuracy and utility of whole-genome sequencing (WGS) of plasma microbial cell-free DNA (cfDNA) as a novel noninvasive method in diagnosing invasive aspergillosis (IA) in patients with hematologic malignancy (HM) or coronavirus disease 2019 (COVID-19). METHODS: Adults with HM or COVID-19 and suspected IA were recruited. IA cases were retrospectively diagnosed according to EORTC/MSG definitions and ECMM/ISHAM criteria for HM and COVID-19 patients, respectively. The results of cfDNA WGS were compared with the conventional diagnosis. RESULTS: Microbial cfDNA WGS was performed 53 times from 41 participants (19 from HM, 16 from COVID-19, and 7 from the control group). In participants with HM, Aspergillus cfDNA was detected in 100% of proven IA and 91.7% of probable IA cases. In participants with COVID-19, 50.0% of probable IA were positive for Aspergillus in cfDNA WGS. Concordance between Aspergillus cfDNA detection and proven/probable IA conventional diagnosis was significantly higher in participants with HM than in those with COVID-19. IA diagnosed using EORTC/MGS definitions showed significantly high concordance between Aspergillus cfDNA detection and proven/probable IA. CONCLUSIONS: Aspergillus cfDNA detection strongly correlated with proven/probable IA diagnosed using EORTC/MSG definitions and could be used as an additional diagnostic tool for IA.

Eif2b3 mutants recapitulate phenotypes of vanishing white matter disease and validate novel disease alleles in zebrafish.

Leukodystrophy with vanishing white matter (VWM), also called Childhood Ataxia with Central Nervous System Hypomyelination, is caused by mutations in the subunits of the eukaryotic translation initiation factor, EIF2B1, EIF2B2, EIF2B3, EIF2B4 or EIF2B5. However, little is known regarding the underlying pathogenetic mechanisms, and there is no curative treatment for VWM. In this study, we established the first EIF2B3 animal model for VWM disease in vertebrates by CRISPR mutagenesis of the highly conserved zebrafish ortholog eif2b3. Using CRISPR, we generated two mutant alleles in zebrafish eif2b3, 10- and 16-bp deletions, respectively. The eif2b3 mutants showed defects in myelin development and glial cell differentiation, and increased expression of genes in the induced stress response pathway. Interestingly, we also found ectopic angiogenesis and increased VEGF expression. Ectopic angiogenesis in the eif2b3 mutants was reduced by the administration of VEGF receptor inhibitor SU5416. Using the eif2b3 mutant zebrafish model together with in silico protein modeling analysis, we demonstrated the pathogenicity of 18 reported mutations in EIF2B3, as well as of a novel variant identified in a 19-month-old female patient: c.503 T > C (p.Leu168Pro). In summary, our zebrafish mutant model of eif2b3 provides novel insights into VWM pathogenesis and offers rapid functional analysis of human EIF2B3 gene variants.

Copy-number analysis by base-level normalization: An intuitive visualization tool for evaluating copy number variations.

Next-generation sequencing (NGS) facilitates comprehensive molecular analyses that help with diagnosing unsolved disorders. In addition to detecting single-nucleotide variations and small insertions/deletions, bioinformatics tools can identify copy number variations (CNVs) in NGS data, which improves the diagnostic yield. However, due to the possibility of false positives, subsequent confirmation tests are generally performed. Here, we introduce Copy-number Analysis by BAse-level NormAlization (CABANA), a visualization tool that allows users to intuitively identify candidate CNVs using the normalized single-base-level read depth calculated from NGS data. To demonstrate how CABANA works, NGS data were obtained from 474 patients with neuromuscular disorders. CNVs were screened using a conventional bioinformatics tool, ExomeDepth, and then we normalized and visualized those data at the single-base level using CABANA, followed by manual inspection by geneticists to filter out false positives and determine candidate CNVs. In doing so, we identified 31 candidate CNVs (7%) in 474 patients and subsequently confirmed all of them to be true using multiplex ligation-dependent probe amplification. The performance of CABANA was deemed acceptable by comparing its diagnostic yield with previous data about neuromuscular disorders. Despite some limitations, we expect CABANA to help researchers accurately identify CNVs and reduce the need for subsequent confirmation testing.

Trajectory of genetic alterations associated with colistin resistance in Acinetobacter baumannii during an in-hospital outbreak of infection.

BACKGROUND: As carbapenem-resistant Acinetobacter baumannii is dominant in clinical settings, the old polymyxin antibiotic colistin has been revived as a therapeutic option. The development of colistin resistance during treatment is becoming a growing concern. OBJECTIVES: To access low- to mid-level colistin-resistant A. baumannii blood isolates recovered from an outbreak in a tertiary care hospital from a national antimicrobial surveillance study. METHODS: The entire bacterial genome was sequenced through long-read sequencing methodology. Quantitative RT-PCR was carried out to determine the level of gene expression. Relative growth rates were determined to estimate fitness costs of each isolate caused by the genetic alterations. RESULTS: The A. baumannii isolates belonged to global clone 2 harbouring two intrinsic phosphoethanolamine transferases. Cumulative alterations continuing the colistin resistance were observed. PmrC overproduction caused by the PmrBA226T alteration was identified in A. baumannii isolates with low-level colistin resistance and an additional PmrCR109H substitution led to mid-level colistin resistance. Truncation of the PmrC enzyme by insertion of ISAba59 was compensated by ISAba10-mediated overproduction of EptA and, in the last isolate, the complete PmrAB two-component regulatory system was eliminated to restore the biological cost of the bacterial host. CONCLUSIONS: During the in-hospital outbreak, a trajectory of genetic modification in colistin-resistant A. baumannii isolates was observed for survival in the harsh conditions imposed by life-threatening drugs with the clear purpose of maintaining drug resistance above a certain level with a reasonable fitness cost.

Comparative Results of QuantiFERON-TB Gold In-Tube and QuantiFERON-TB Gold Plus Assays for Detection of Tuberculosis Infection in Clinical Samples.

The QuantiFERON-TB Gold plus (QFT-Plus) assay, an interferon gamma (IFN-γ) release assay (IGRA), was recently introduced as the next version of the QuantiFERON-TB Gold In-Tube (QFT-GIT) assay for diagnosing latent tuberculosis (TB). Whereas the QFT-GIT assay uses only one TB tube that induces a cell-mediated immune (CMI) response of CD4+ T cells, the QFT-Plus has an additional TB antigen 2 tube (TB2) for the CMI response of CD8+ T and CD4+ T cells, in addition to a TB antigen 1 (TB1) tube for the CMI response of CD4+ T cells only. We compared the results of the QFT-Plus and QFT-GIT assays as routine clinical tests for diagnosing TB. Samples from 220 patients referred for routine IGRA in various clinical departments were used. Correlations between IFN-γ levels in the QFT-GIT and QFT-Plus assays were strong and showed good agreement (kappa value = 0.69). Seven cases with positive QFT-GIT assay results and negative QFT-Plus assay results showed IFN-γ values near the cutoff value. However, 10 cases with active TB, recent TB, or immune problems showed discordance with the positive results only in the TB2 tube in QFT-Plus, unlike the negative results in TB1 and TB tubes. In these cases, IFN-γ levels in the TB2 tube were significantly higher than those in other tubes. This is the first study to compare these assays as routine IGRAs in the clinical setting. The QFT-Plus assay showed good agreement with the QFT-GIT assay and is presumably advantageous for patients with active TB, recent TB, and specific immune conditions involving CD8+ T-cell responses.

Comparison of exon-level copy number variants in CytoScan XON assay and next-generation sequencing in clinical samples.

BACKGROUND AND AIMS: Next-generation sequencing (NGS)-based copy number variants (CNVs) have high false-positive rates. The fewer the exons involved, the higher the false-positive rate. A CytoScan XON assay was developed to assess exon-level CNVs. MATERIALS AND METHODS: Twenty-three clinically relevant exon-level CNVs in 20 patient blood samples found in previous NGS studies were compared with the results from the CytoScan XON and multiplex ligation-dependent probe amplification (MLPA). RESULTS: Fifteen of the 23 exon-level CNVs were consistent with the NGS results. Among these, eight were confirmed using MLPA. In six out of eight discrepancies between the CytoScan Xon and NGS, MLPA was performed, and three were negative, indicating that the CNVs in NGS were false positives. The CytoScan XON exhibits a sensitivity of 72.7% for small exon-level CNVs, along with a specificity of 100%. The assay could not detect the three exon-level CNVs in PKD1 and TSC2 that were detected using both NGS and MLPA. This could be due to the distribution of the probes in some areas, and the CNV-calling regions containing multiple exons. CONCLUSION: The CytoScan XON assay is a promising complementary tool for the detection of exon-level CNVs, provided that the users carefully examine the distribution of probes and calling regions.

Characterization of Vestibular Phenotypes in Patients with Genetic Hearing Loss.

Background: The vestibular phenotypes of patients with genetic hearing loss are poorly understood. Methods: we performed genetic testing including exome sequencing and vestibular function tests to investigate vestibular phenotypes and functions in patients with genetic hearing loss. Results: Among 627 patients, 143 (22.8%) had vestibular symptoms. Genetic variations were confirmed in 45 (31.5%) of the 143 patients. Nineteen deafness genes were linked with vestibular symptoms; the most frequent genes in autosomal dominant and recessive individuals were COCH and SLC26A4, respectively. Vestibular symptoms were mostly of the vertigo type, recurrent, and persisted for hours in the genetically confirmed and unconfirmed groups. Decreased vestibular function in the caloric test, video head impulse test, cervical vestibular-evoked myogenic potential, and ocular vestibular-evoked myogenic potential was observed in 42.0%, 16.3%, 57.8%, and 85.0% of the patients, respectively. The caloric test revealed a significantly higher incidence of abnormal results in autosomal recessive individuals than in autosomal dominant individuals (p = 0.011). The genes, including SLC26A4, COCH, KCNQ4, MYH9, NLRP3, EYA4, MYO7A, MYO15A, and MYH9, were heterogeneously associated with abnormalities in the vestibular function test. Conclusions: In conclusion, diverse vestibular symptoms are commonly concomitant with genetic hearing loss and are easily overlooked.

Clinician-Driven Reanalysis of Exome Sequencing Data From Patients With Inherited Retinal Diseases.

Importance: Despite advances in next-generation sequencing (NGS), a significant proportion of patients with inherited retinal disease (IRD) remain undiagnosed after initial genetic testing. Exome sequencing (ES) reanalysis in the clinical setting has been suggested as one method for improving diagnosis of IRD. Objective: To investigate the association of clinician-led reanalysis of ES data, which incorporates updated clinical information and comprehensive bioinformatic analysis, with the diagnostic yield in a cohort of patients with IRDs in Korea. Design, Setting, and Participants: This was a multicenter prospective cohort study involving 264 unrelated patients with IRDs, conducted in Korea between March 2018 and February 2020. Comprehensive ophthalmologic examinations and ES analyses were performed, and ES data were reanalyzed by an IRD specialist for single nucleotide variants, copy number variants, mobile element insertions, and mitochondrial variants. Data were analyzed from March to July 2023. Main Outcomes and Measures: Diagnostic rate of conventional bioinformatic analysis and clinician-driven ES reanalysis. Results: A total of 264 participants (151 [57.2%] male; mean [SD] age at genetic testing, 33.6 [18.9] years) were enrolled, including 129 patients (48.9%) with retinitis pigmentosa and 26 patients (9.8%) with Stargardt disease or macular dystrophy. Initial bioinformatic analysis diagnosed 166 patients (62.9%). Clinician-driven reanalysis identified the molecular cause of diseases in an additional 22 patients, corresponding to an 8.3-percentage point increase in diagnostic rate. Key factors associated with new molecular diagnoses included clinical phenotype updates (4 patients) and detection of previously overlooked variation, such as structural variants (9 patients), mitochondrial variants (3 patients), filtered or not captured variants (4 patients), and noncanonical splicing variants (2 patients). Among the 22 patients, variants in 7 patients (31.8%) were observed in the initial analysis but not reported to patients, while those in the remaining 15 patients (68.2%) were newly detected by the ES reanalysis. Conclusions and Relevance: In this cohort study, clinician-centered reanalysis of ES data was associated with improved molecular diagnostic yields in patients with IRD. This approach is important for uncovering missed genetic causes of retinal disease.

Histopathologic image-based deep learning classifier for predicting platinum-based treatment responses in high-grade serous ovarian cancer.

Platinum-based chemotherapy is the cornerstone treatment for female high-grade serous ovarian carcinoma (HGSOC), but choosing an appropriate treatment for patients hinges on their responsiveness to it. Currently, no available biomarkers can promptly predict responses to platinum-based treatment. Therefore, we developed the Pathologic Risk Classifier for HGSOC (PathoRiCH), a histopathologic image-based classifier. PathoRiCH was trained on an in-house cohort (n = 394) and validated on two independent external cohorts (n = 284 and n = 136). The PathoRiCH-predicted favorable and poor response groups show significantly different platinum-free intervals in all three cohorts. Combining PathoRiCH with molecular biomarkers provides an even more powerful tool for the risk stratification of patients. The decisions of PathoRiCH are explained through visualization and a transcriptomic analysis, which bolster the reliability of our model's decisions. PathoRiCH exhibits better predictive performance than current molecular biomarkers. PathoRiCH will provide a solid foundation for developing an innovative tool to transform the current diagnostic pipeline for HGSOC.

Adult-onset MELAS syndrome in a 51-year-old woman without typical clinical manifestations: a case report.

Background: Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome is a multi-organ disorder resulting from mitochondrial DNA (mtDNA) mutations. We report a case of suspected MELAS syndrome that progressed to left ventricular dysfunction 24 years after an initial diagnosis of atrioventricular block (AVB). Case summary: A 51-year-old woman was referred to heart failure clinic because of dyspnoea on exertion and progressive cardiomegaly. She had a dual-chamber pacemaker implanted for 24 years because of a high-degree AVB. She was treated for diabetes mellitus for 23 years and used hearing aids for 12 years because of sensorineural hearing loss. Transthoracic echocardiography revealed reduced left ventricular ejection fraction (26%), with increased thickness and unusual texture of the myocardium. The absence of abnormal findings on serum and urine protein electrophoresis suggested that light-chain amyloidosis was unlikely. In addition, 99mTc-3,3-diphosphono-1,2-propanodicarboxylic acid scintigraphy revealed no definite uptake in the myocardium. Endomyocardial biopsy revealed a hypertrophy of myocytes in haematoxylin-eosin staining, and electron microscopy revealed a disarrangement of mitochondrial cristae, which were suggestive of mitochondrial cardiomyopathy. A mtDNA test detected the m.3243A > G mutation in the MT-TL1 gene. According to these findings, MELAS syndrome was the most probable diagnosis despite the absence of common symptoms such as stroke-like episodes or lactic acidosis. Discussion: The patient had progressed to heart failure with reduced ejection fraction 24 years after the first cardiac manifestation. An identification of the mutation in the MT-TL1 gene, indicative of MELAS syndrome, enabled the diagnosis of MELAS syndrome without typical manifestations.

Comparative Analysis of the Molecular Characteristics of Group B Streptococcus Isolates Collected from Pregnant Korean Women Using Whole-genome Sequencing.

Background: The incidence of early- and late-onset sepsis and meningitis in neonates due to maternal rectovaginal group B Streptococcus (GBS) colonization may differ with serotype distribution and clonal complex (CC). CC17 strains are associated with hypervirulence and poor disease outcomes. GBS serotypes are distinguished based on the polysaccharide capsule, the most important virulence factor. We determined the sequence type distribution of GBS isolates from pregnant women in Korea and validated whole-genome sequencing (WGS)-based prediction of antimicrobial susceptibility and capsular serotypes in GBS isolates. Methods: Seventy-five GBS isolates collected from pregnant Korean women visiting Wonju Severance Christian Hospital, Wonju, Korea between 2017 and 2019 were subjected to WGS using the NovaSeq 6000 system (Illumina, San Diego, CA, USA). Multilocus sequence types, serotypes, antimicrobial resistance genes, and hemolysin operon mutations were determined by WGS, and the latter three were compared with the results of conventional phenotypic methods. Results: The predominant lineage was CC1 (37.3%), followed by CC19 (32.0%), CC12 (17.3%), and CC17 (4.0%). All isolates were cps typeable (100%, (75/75), and 89.3% of cps genotypes (67/75) were concordant with serotypes obtained using latex agglutination. The cps genotypes of the 75 isolates were serotypes III (24.0%), V (22.7%), and VIII (17.3%). All isolates harboring intact ermB and tet were non-susceptible to erythromycin and tetracycline, respectively. Three non-hemolytic strains had 1-bp frameshift insertions in cylE. Conclusions: The low prevalence of CC17 GBS colonization may explain the low frequency of neonatal GBS infections. WGS is a useful tool for simultaneous genotyping and antimicrobial resistance determination.

Corrigendum: Genetic diagnosis of inborn errors of immunity using clinical exome sequencing.

[This corrects the article DOI: 10.3389/fimmu.2023.1178582.].

Circulating Tumor DNA Analysis on Metastatic Prostate Cancer with Disease Progression.

The positivity rate of circulating tumor DNA (ctDNA) next-generation sequencing (NGS) varies among patients with metastatic prostate cancer (mPC), complicating its incorporation into regular practice. This retrospective study analyzed the ctDNA sequencing results of 100 mPC patients from May 2021 to March 2023 to identify the factors associated with positive ctDNA. Three custom gene panels were used for sequencing. Overall, 63% of the patients exhibited tier I/II somatic alterations, while 12% had pathogenic/likely pathogenic germline alterations. The key genes that were altered included AR, TP53, RB1, PTEN, and APC. Mutations in BRCA1/2, either germline or somatic, were observed in 21% of the patients. Among the metastatic castration-resistant prostate cancer (mCRPC) patients, the ctDNA-positive samples generally showed higher median prostate-specific antigen (PSA) levels and were more likely to be at the radiographic and clinical progressive disease stages, although they were not significantly associated with PSA progression. Our results suggest that ctDNA analysis could detect meaningful genetic changes in mPC patients, especially during disease progression.

Genetic diagnosis of inborn errors of immunity using clinical exome sequencing.

Inborn errors of immunity (IEI) include a variety of heterogeneous genetic disorders in which defects in the immune system lead to an increased susceptibility to infections and other complications. Accurate, prompt diagnosis of IEI is crucial for treatment plan and prognostication. In this study, clinical utility of clinical exome sequencing (CES) for diagnosis of IEI was evaluated. For 37 Korean patients with suspected symptoms, signs, or laboratory abnormalities associated with IEI, CES that covers 4,894 genes including genes related to IEI was performed. Their clinical diagnosis, clinical characteristics, family history of infection, and laboratory results, as well as detected variants, were reviewed. With CES, genetic diagnosis of IEI was made in 15 out of 37 patients (40.5%). Seventeen pathogenic variants were detected from IEI-related genes, BTK, UNC13D, STAT3, IL2RG, IL10RA, NRAS, SH2D1A, GATA2, TET2, PRF1, and UBA1, of which four variants were previously unreported. Among them, somatic causative variants were identified from GATA2, TET2, and UBA1. In addition, we identified two patients incidentally diagnosed IEI by CES, which was performed to diagnose other diseases of patients with unrecognized IEI. Taken together, these results demonstrate the utility of CES for the diagnosis of IEI, which contributes to accurate diagnosis and proper treatments.

Amplification of the Chromosomal blaCTX-M-14 Gene in Escherichia coli Expanding the Spectrum of Resistance under Antimicrobial Pressure.

Various forms of adaptive evolution occur in clinical isolates in response to the presence of antimicrobial drugs. Among a total of 171 CTX-M-9 group/family extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli blood isolates recovered between 2016 and 2017 in six general hospitals, 50.3% of the isolates possessed the blaCTX-M-14-like gene in their chromosome rather than in a plasmid. Focusing on this unprecedented way of the blaCTX-M ESBL gene possession, molecular epidemiology of the isolates was assessed and the chromosomal location of the acquired cephalosporinase gene was dissected in an evolutionary point of view. Taking advantage of a complete collection of E. coli blood isolates from a limited period, clonal relatedness of the E. coli isolates carrying the blaCTX-M-14-like gene was clarified and the dominant clone, ST131 H30R, was identified. To control the level of resistance and the resistance spectrum to oxyimino-cephalosporin drugs, transcription level of the blaCTX-M-14-like gene was tuned finely through positioning the gene near the chromosomal initiation dnaA gene and amplifying numbers of the gene in a chromosome using either the copy-and-paste or the tandem amplification methods. Inconspicuous fitness cost by chromosomal location of the gene and free adjustment of the oxyimino-cephalosporin resistance would urge the dominancy of E. coli clinical isolates harboring the blaCTX-M ESBL gene in their chromosome. IMPORTANCE Increasing prevalence of E. coli producing CTX-M ESBL is a major concern in clinical settings because it significantly limits treatment options. Thus, it is important to keep watching current molecular mechanisms of resistance and the scheme for dissemination. Recently, chromosomal locations of the blaCTX-M genes are often documented in clinical settings and the bacterial strategies were needed to be dissected in an evolutionary point of view. Both main mechanisms of fine tuning the chromosomal gene expression, bacterial gene amplification either by copy-and-paste or by tandem amplification and positioning the gene near the chromosomal initiation dnaA gene, were demonstrated in the study, and the fitness cost by the chromosomal location was evaluated.

Germline Mutations Related to Primary Hyperparathyroidism Identified by Next-Generation Sequencing.

Primary hyperparathyroidism (PHPT) is characterized by overproduction of parathyroid hormone and subsequent hypercalcemia. Approximately 10% of PHPT cases are hereditary, and several genes, such as MEN1, RET, CASR, and CDC73, are responsible for the familial forms of PHPT. However, other genetic mutations involved in the etiology of PHPT are largely unknown. In this study, we identified genetic variants that might be responsible for PHPT, including familial PHPT, benign sporadic PHPT, and sporadic parathyroid cancer, using next-generation sequencing (NGS). A total of 107 patients with PHPT who underwent NGS from 2017 to 2021 at Severance Hospital were enrolled. We reviewed the pathogenic variants, likely pathogenic variants, and variants of uncertain significance (VUS) according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology criteria. Of the 107 patients (mean age: 47.6 ± 16.1 years, women 73.8%), 12 patients were diagnosed with familial PHPT, 13 with parathyroid cancer, and 82 with benign sporadic PHPT. Using NGS, we identified three pathogenic variants in two genes (CDC73 and MEN1), 10 likely pathogenic variants in six genes (CASR, CDC73, LRP5, MEN1, SDHA, and VHL), and 39 non-synonymous VUS variants that could be related to parathyroid disease. Interestingly, we identified one GCM2 variant (c.1162A>G [p.Lys388Glu]) and five APC variants that were previously reported in familial isolated hyperparathyroidism, benign sporadic PHPT, and parathyroid cancer. We also analyzed the characteristics of subjects with positive genetic test results (pathogenic or likely pathogenic variants), and 76.9% of them had at least one of the following features: 1) age < 40 years, 2) family history of PHPT, 3) multiglandular PHPT, or 4) recurrent PHPT. In this study, we analyzed the NGS data of patients with PHPT and observed variants that could possibly be related to PHPT pathogenesis. NGS screening for selected patients with PHPT might help in the diagnosis and management of the disease.

Noncanonical Splice Site and Deep Intronic FRMD7 Variants Activate Cryptic Exons in X-linked Infantile Nystagmus.

Purpose: We aim to report noncoding pathogenic variants in patients with FRMD7-related infantile nystagmus (FIN). Methods: Genome sequencing (n = 2 families) and reanalysis of targeted panel next generation sequencing (n = 2 families) was performed in genetically unsolved cases of suspected FIN. Previous sequence analysis showed no pathogenic coding variants in genes associated with infantile nystagmus. SpliceAI, SpliceRover, and Alamut consensus programs were used to annotate noncoding variants. Minigene splicing assay was performed to confirm aberrant splicing. In silico analysis of exonic splicing enhancer and silencer was also performed. Results: FRMD7 intronic variants were identified based on genome sequencing and targeted next-generation sequencing analysis. These included c.285-12A>G (pedigree 1), c.284+63T>A (pedigrees 2 and 3), and c. 383-1368A>G (pedigree 4). All variants were absent in gnomAD, and the both c.285-12A>G and c.284+63T>A variants were predicted to enhance new splicing acceptor gains with SpliceAI, SpliceRover, and Alamut consensus approaches. However, the c.383-1368 A>G variant only had a significant impact score on the SpliceRover program. The c.383-1368A>G variant was predicted to promote pseudoexon inclusion by binding of exonic splicing enhancer. Aberrant exonizations were validated through minigene constructs, and all variants were segregated in the families. Conclusions: Deep learning-based annotation of noncoding variants facilitates the discovery of hidden genetic variations in patients with FIN. This study provides evidence of effectiveness of combined deep learning-based splicing tools to identify hidden pathogenic variants in previously unsolved patients with infantile nystagmus. Translational Relevance: These results demonstrate robust analysis using two deep learning splicing predictions and in vitro functional study can lead to finding hidden genetic variations in unsolved patients.