Characteristics of chronic enteropathy associated with SLCO2A1 gene (CEAS) in children, a unique type of monogenic very early-onset inflammatory bowel disease.

BACKGROUND:  Chronic enteropathy associated with SLCO2A1 gene (CEAS) is a unique type of inflammatory bowel disease. CEAS is monogenic disease and is thought to develop from childhood, but studies on pediatric CEAS are scarce. We analyzed characteristics of pediatric CEAS. METHODS: Eleven patients diagnosed with CEAS at Seoul National University Children's Hospital were identified and analyzed. Clinical data of patients were collected. Sanger sequencing of SLCO2A1 was performed on all patients. RESULTS: Patients were diagnosed at a median age of 16.0 years (IQR 11.0 ~ 20.0), and the median age at symptoms onset was only 4.0 years (IQR 2.5 ~ 6.0). Growth delay was observed at the time of diagnosis. Patients showed multiple ulcers or strictures in the small intestine, while the esophagus and colon were unaffected in any patients. Almost half of the patients underwent small intestine resection. The major laboratory features of pediatric CEAS include iron deficiency anemia (IDA), hypoalbuminemia, and near-normal levels of C-reactive protein (CRP). Two novel mutations of SLCO2A1 were identified. The most prevalent symptoms were abdominal pain and pale face. None of the immunomodulatory drugs showed a significant effect on CEAS. CONCLUSIONS: Pediatric CEAS typically develop from very young age, suggesting it as one type of monogenic very early onset inflammatory bowel disease. CEAS can cause growth delay in children but there is no effective treatment currently. We recommend screening for SLCO2A1 mutations to pediatric patients with chronic IDA from a young age and small intestine ulcers without elevation of CRP levels.

Utility of Next-Generation Sequencing-Based Chimerism Analysis for Early Relapse Prediction following Allogenic Hematopoietic Cell Transplantation.

Chimerism monitoring following allogeneic hematopoietic cell transplantation (HCT) plays a pivotal role in evaluating engraftment status and identifying early indicators of relapse. Recent advancements in next-generation sequencing (NGS) technology have introduced AlloSeq HCT as a more sensitive alternative to short tandem repeat (STR) analysis. This study aimed to compare AlloSeq HCT with STR, focusing on the prediction of early relapse post-allogeneic HCT. Chimerism levels in 29 HCT recipients were assessed using both STR and NGS, employing a total of 125 whole blood or bone marrow aspirate samples (68 post-HCT and 57 pre-HCT samples from recipients or donors). AlloSeq HCT exhibited high concordance with STR and demonstrated the potential for early detection of chimeric changes, particularly at extremely low levels. The combined advantages of high sensitivity and automated data analysis offered by AlloSeq HCT substantiate its clinical adoption for effective chimerism monitoring.

ITDetect: a method to detect internal tandem duplication of FMS-like tyrosine kinase (FLT3) from next-generation sequencing data with high sensitivity and clinical application.

Internal tandem duplication (ITD) of the FMS-like tyrosine kinase (FLT3) gene is associated with poor clinical outcomes in patients with acute myeloid leukemia. Although recent methods for detecting FLT3-ITD from next-generation sequencing (NGS) data have replaced traditional ITD detection approaches such as conventional PCR or fragment analysis, their use in the clinical field is still limited and requires further information. Here, we introduce ITDetect, an efficient FLT3-ITD detection approach that uses NGS data. Our proposed method allows for more precise detection and provides more detailed information than existing in silico methods. Further, it enables FLT3-ITD detection from exome sequencing or targeted panel sequencing data, thereby improving its clinical application. We validated the performance of ITDetect using NGS-based and experimental ITD detection methods and successfully demonstrated that ITDetect provides the highest concordance with the experimental methods. The program and data underlying this study are available in a public repository.

Prevalence and founder effect of DRC1 exon 1-4 deletion in Korean patients with primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder affecting ciliary structure and function. PCD exhibiting dynein regulatory complex subunit 1 (DRC1) exon 1-4 deletion has been reported in several Japanese PCD patients; however, no large scale studies have been performed. Here, we aimed to determine the prevalence and founder effect of this variant in the Korean population. Using an in-house copy number variation tool, we screened for DRC1 exon 1-4 deletion in 20 patients with PCD and exome data of 1435 patients in the Seoul National University Hospital repository. In cases of suspected DRC1 deletion, confirmatory gap-PCR was performed. In a PCD cohort, three of 20 (15%) patients were positive for DRC1 exon 1-4 deletion (NM_145038.5(DRC1): c.1-3952_540 + 1331del27748-bp) while pathogenic variants were found in CCDC39 (N = 1), DNAAF6 (N = 1), DNAH9 (N = 1). In the 1,435-sample exome data, seven patients (0.49%) were confirmed to have DRC1 exon 1-4 deletion. A chimeric sequence including the junction was searched from the 1000 Genomes Project data repository. One Japanese patient (0.96%) was found to have the same DRC1 exon 1-4 deletion, which was absent in other populations. This study demonstrated that the DRC1 exon 1-4 deletion is a founder mutation based on haplotype analysis. In summary, the prevalence of PCD based on DRC1 exon 1-4 deletion is particularly high in Korean and Japanese populations, which is attributed to the founder effect. Genetic testing for DRC1 exon 1-4 deletion should be considered as an initial screening tool for Korean and Japanese patients with PCD.

Characteristics of germline mutations in Korean patients with pheochromocytoma/paraganglioma.

BACKGROUND: Pheochromocytomas and paragangliomas (PPGLs) are catecholamine-producing neuroendocrine tumours. PPGLs are a rare but important cause of secondary hypertension owing to their high morbidity and mortality. Patients with PPGL exhibit an increased prevalence of mutations in one of the PPGL susceptibility genes according to previous studies. We aimed to investigate the characteristics of germline mutations in the largest number of Korean patients with PPGL. METHODS: In this study, 161 patients with PPGL were evaluated. Phenotype data, including biochemical, pathological and anatomical imaging results, were collected. Germline mutations in 10 PPGL-related genes were tested by targeted next-generation sequencing (NGS), Sanger sequencing and multiplex ligation-dependent probe amplification. RESULTS: Approximately 21% of apparently sporadic PPGLs harboured germline mutations of the PPGL-related genes. The mutation carriers were younger at the first diagnosis and had more bilateral (28.6% vs 4.0%, p<0.001) and multifocal (11.4% vs 1.6%, p=0.027) PPGLs, but showed no metastatic risk (17.1% vs 11.1%, p=0.504), than non-mutation carriers. Missense mutation of SDHD p.V111I was found in this cohort of Asian patients, which was associated with unilateral pheochromocytoma with dominantly epinephrine production. CONCLUSION: This study covered the largest number of Korean patients with PPGL. To our knowledge, it is the first to compare results of targeted NGS panel with those of conventional sequencing methods in Asia. We demonstrated that the variant type, as well as the mutated gene, may determine the phenotype and prognosis of PPGLs.

Transcriptome-based variant calling and aberrant mRNA discovery enhance diagnostic efficiency for neuromuscular diseases.

BACKGROUND: Whole-exome sequencing-based diagnosis of rare diseases typically yields 40%-50% of success rate. Precise diagnosis of the patients with neuromuscular disorders (NMDs) has been hampered by locus heterogeneity or phenotypic heterogeneity. We evaluated the utility of transcriptome sequencing as an independent approach in diagnosing NMDs. METHODS: The RNA sequencing (RNA-Seq) of muscle tissues from 117 Korean patients with suspected Mendelian NMD was performed to evaluate the ability to detect pathogenic variants. Aberrant splicing and CNVs were inspected to identify additional causal genetic factors for NMD. Aberrant splicing events in Dystrophin (DMD) were investigated by using antisense oligonucleotides (ASOs). A non-negative matrix factorisation analysis of the transcriptome data followed by cell type deconvolution was performed to cluster samples by expression-based signatures and identify cluster-specific gene ontologies. RESULTS: Our pipeline called 38.1% of pathogenic variants exclusively from the muscle transcriptomes, demonstrating a higher diagnostic rate than that achieved via exome analysis (34.9%). The discovery of variants causing aberrant splicing allowed the application of ASOs to the patient-derived cells, providing a therapeutic approach tailored to individual patients. RNA-Seq data further enabled sample clustering by distinct gene expression profiles that corresponded to clinical parameters, conferring additional advantages over exome sequencing. CONCLUSION: The RNA-Seq-based diagnosis of NMDs achieves an increased diagnostic rate and provided pathogenic status information, which is not easily accessible through exome analysis.

Consistent count region-copy number variation (CCR-CNV): an expandable and robust tool for clinical diagnosis of copy number variation at the exon level using next-generation sequencing data.

PURPOSE: Despite the importance of exonic copy number variations (CNVs) in human genetic diseases, reliable next-generation sequencing-based methods for detecting them are unavailable. We developed an expandable and robust exonic CNV detection tool called consistent count region (CCR)-CNV. METHODS: In total, about 1000 samples of the truth set were used for validating CCR-CNV. We compared CCR-CNV performance with 2 well-known CNV tools. Finally, to overcome the limitations of CCR-CNV, we devised a combined approach. RESULTS: The mean sensitivity and specificity of CCR-CNV alone were above 95%, which was superior to that of other CNV tools, such as DECoN and Atlas-CNV. However, low covered region and positive predictive value and high false discovery rate act as obstacles to its use in clinical settings. The combined approach showed much improved performance than CCR-CNV alone. CONCLUSION: In this study, we present a novel diagnostic tool that allows the identification of exonic CNVs with high confidence using various reagents and clinical next-generation sequencing platforms. We validated this method using the largest multiple ligation-dependent probe amplification-confirmed data set, including sufficient copy normal control data. The approach, combined with existing CNV tools, allows the implementation of CCR-CNV in clinical settings.

Intronic LINE-1 insertion in SLCO1B3 as a highly prevalent cause of rotor syndrome in East Asian population.

Rotor syndrome is caused by digenic loss-of-function variants in SLCO1B1 and SLCO1B3 but only a few studies have reported co-occurring inactivating variants from both genes. A rotor syndrome-causing long interspersed element-1 (LINE-1) insertion in SLCO1B3 had been reported to be highly prevalent in the Japanese population but there has been no additional report. In spite of its known association with various human diseases, LINE-1 is hard to detect with current sequencing technologies. In this study, we aimed to devise a method to screen the LINE-1 insertion variant and investigate the frequency of this variant in various populations. A chimeric sequence, that was generated by concatenating the reference sequence at the junction and a part of inserted LINE-1 sequence, was searched from 725 raw sequencing data files. In cases containing the chimeric sequence, confirmatory long-range PCR and gap-PCR were performed. In total, 95 (13.1%) of 725 patients were positive for the chimeric sequence, and all were confirmed to have the SLCO1B3 LINE-1 insertion by PCR-based tests. The same chimeric sequence was searched from the 1000 Genomes Project data repository and the carrier frequency was remarkably high in the East Asian populations (10.1%), especially in Southern Han Chinese (18.5%), but almost absent in other populations. This SLCO1B3 LINE-1 insertion should be screened in a population-specific manner under suspicion of Rotor syndrome and the methods proposed in this study would enable this in a simple way.

Longitudinal proteomic profiling provides insights into host response and proteome dynamics in COVID-19 progression.

In managing patients with coronavirus disease 2019 (COVID-19), early identification of those at high risk and real-time monitoring of disease progression to severe COVID-19 is a major challenge. We aimed to identify potential early prognostic protein markers and to expand understanding of proteome dynamics during clinical progression of the disease. We performed in-depth proteome profiling on 137 sera, longitudinally collected from 25 patients with COVID-19 (non-severe patients, n = 13; patients who progressed to severe COVID-19, n = 12). We identified 11 potential biomarkers, including the novel markers IGLV3-19 and BNC2, as early potential prognostic indicators of severe COVID-19. These potential biomarkers are mainly involved in biological processes associated with humoral immune response, interferon signalling, acute phase response, lipid metabolism, and platelet degranulation. We further revealed that the longitudinal changes of 40 proteins persistently increased or decreased as the disease progressed to severe COVID-19. These 40 potential biomarkers could effectively reflect the clinical progression of the disease. Our findings provide some new insights into host response to SARS-CoV-2 infection, which are valuable for understanding of COVID-19 disease progression. This study also identified potential biomarkers that could be further validated, which may support better predicting and monitoring progression to severe COVID-19.

Evidence of Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection After Recovery from Mild Coronavirus Disease 2019.

BACKGROUND: Positive results from real-time reverse-transcription polymerase chain reaction (rRT-PCR) in recovered patients raise concern that patients who recover from coronavirus disease 2019 (COVID-19) may be at risk of reinfection. Currently, however, evidence that supports reinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not been reported. METHODS: We conducted whole-genome sequencing of the viral RNA from clinical specimens at the initial infection and at the positive retest from 6 patients who recovered from COVID-19 and retested positive for SARS-CoV-2 via rRT-PCR after recovery. A total of 13 viral RNAs from the patients' respiratory specimens were consecutively obtained, which enabled us to characterize the difference in viral genomes between initial infection and positive retest. RESULTS: At the time of the positive retest, we were able to acquire a complete genome sequence from patient 1, a 21-year-old previously healthy woman. In this patient, through the phylogenetic analysis, we confirmed that the viral RNA of positive retest was clustered into a subgroup distinct from that of the initial infection, suggesting that there was a reinfection of SARS-CoV-2 with a subtype that was different from that of the primary strain. The spike protein D614G substitution that defines the clade "G" emerged in reinfection, while mutations that characterize the clade "V" (ie, nsp6 L37F and ORF3a G251V) were present at initial infection. CONCLUSIONS: Reinfection with a genetically distinct SARS-CoV-2 strain may occur in an immunocompetent patient shortly after recovery from mild COVID-19. SARS-CoV-2 infection may not confer immunity against a different SARS-CoV-2 strain.

Importance of extracutaneous organ involvement in determining the clinical severity and prognosis of incontinentia pigmenti caused by mutations in the IKBKG gene.

Incontinentia pigmenti (IP) is a rare X-linked skin disease caused by mutations in the IKBKG gene, which is required for activation of the nuclear factor-kappa B signalling pathway. Multiple systems can be affected with highly variable phenotypic expressivity. We aimed to clarify the clinical characteristics observed in molecularly confirmed Korean IP patients. The medical records of 25 females confirmed as IP by molecular genetic analysis were retrospectively reviewed. The phenotypic score of extracutaneous manifestations was calculated to assess the disease severity. The IKBKG gene partial deletion or intragenic mutations were investigated using long-range PCR, multiplex ligation-dependent probe amplification and direct sequencing methods. Among the 25 individuals, 18 (72%) were sporadic cases. All patients showed typical skin manifestations at birth or during the neonatal period. Extracutaneous findings were noted in 17 (68%) patients; ocular manifestations (28%), neurological abnormalities (28%), hair abnormalities (20%), dental anomalies (12%), nail dystrophy (8%). The common exon 4-10 IKBKG deletion was observed in 20 (80%) patients. In addition, five intragenic sequence variants were identified, including three novel variants. The phenotype scores were highly variable, ranging from abnormal skin pigmentation only to one or more extracutaneous features, although no significant difference was observed for each clinical characteristic between the group with sequence variants and that with common large deletion. Our cohort with IP showed heterogeneity of extracutaneous manifestations and high incidence of sporadic cases. Long-term monitoring with multidisciplinary management is essential for evaluating the clinical status, providing adequate genetic counselling and understanding the genotype-phenotype correlation in IP.

Transcatheter Dynamic Contrast-Enhanced MR Lymphangiography for Nontraumatic Lymphatic Disorders: Technical Feasibility and Imaging Findings.

This study aimed to evaluate the feasibility and added value of transcatheter dynamic contrast-enhanced magnetic resonance (MR) lymphangiography for nontraumatic lymphatic disorders. Five patients (2 males and 3 females; median age, 16.0 years; range, 3-74 years) who underwent both intranodal and transcatheter dynamic contrast-enhanced MR lymphangiography for suspected nontraumatic lymphatic leakages from June 2017 to January 2020 were included in this retrospective study. The imaging findings of both dynamic contrast-enhanced MR lymphangiography techniques were assessed for the presence of chylolymphatic reflux or direct sign of leakage. Intranodal dynamic contrast-enhanced MR lymphangiography demonstrated chylolymphatic reflux into the thoracic area in 2 patients (40%) but no direct evidence of leakage in any of the 5 patients. Transcatheter dynamic contrast-enhanced MR lymphangiography revealed chylolymphatic reflux and extravasation of the contrast agent in all 5 patients (100%). In conclusion, transcatheter dynamic contrast-enhanced MR lymphangiography may reveal additional signs of reflux and extravasation even when the findings of intranodal dynamic contrast-enhanced MR lymphangiography are negative.

Replication-Based Rearrangements Are a Common Mechanism for SNCA Duplication in Parkinson's Disease.

BACKGROUND: SNCA multiplication is a genomic cause of familial PD, showing dosage-dependent toxicity. Until now, nonallelic homologous recombination was suggested as the mechanism of SNCA duplication, based on various types of repetitive elements found in the spanning region of the breakpoints. However, the sequence at the breakpoint was analyzed only for 1 case. OBJECTIVES: We have analyzed the breakpoint sequences of 6 patients with PD who had duplicated SNCA using whole-genome sequencing data to elucidate the mechanism of SNCA duplication. METHODS: Six patient samples with SNCA duplication underwent whole-genome sequencing. The duplicated regions were defined with nucleotide-resolution breakpoints, which were confirmed by junction polymerase chain reaction and Sanger sequencing. The search for potential non-B DNA-forming sequences and stem-loop structure predictions was conducted. RESULTS: Duplicated regions ranged from the smallest region of 718.3 kb to the largest one of 4,162 kb. Repetitive elements were found at 8 of the 12 breakpoint sequences on each side of the junction, but none of the pairs shared overt homologies. Five of these six junctions had microhomologies (2-4 bp) at the breakpoint, and a short stretch of sequences was inserted in 3 cases. All except one junction were located within or next to stem-loop structures. CONCLUSION: Our study has determined that homologous recombination mechanisms involving repetitive elements are not the main cause of the duplication of SNCA. The presence of microhomology at the junctions and their position within stem-loop structures suggest that replication-based rearrangements may be a common mechanism for SNCA amplification. © 2020 International Parkinson and Movement Disorder Society.

Importance of early diagnosis in LMNA-related muscular dystrophy for cardiac surveillance.

INTRODUCTION: The identification of LMNA-related muscular dystrophy is important because it poses life-threatening cardiac complications. However, diagnosis of LMNA-related muscular dystrophy based on clinical features is challenging. METHODS: We reviewed the clinical phenotypes of 14 children with LMNA variants, focusing on the cardiac function and genotypes. RESULTS: Most patients presented with motor developmental delay or gait abnormalities. Eight (57%) patients had prominent neck extensor weakness or contractures. All patients showed ankle contractures at an early stage. Regular cardiac surveillance allowed for the detection of dysrhythmias in 57% of patients at a mean age of 14 years (range, 5-26). All patients had missense variants; however, there were no clear phenotype-genotype correlations. DISCUSSION: Early diagnosis of LMNA-related muscular dystrophy provides an opportunity for cardiac surveillance, potentially leading to the prevention of cardiac mortality in children.

Reclassification of BRCA1 and BRCA2 variants of uncertain significance: a multifactorial analysis of multicentre prospective cohort.

BACKGROUND: BRCA1 and BRCA2 (BRCA1/2) variants classified ambiguously as variants of uncertain significance (VUS) are a major challenge for clinical genetic testing in breast cancer; their relevance to the cancer risk is unclear and the association with the response to specific BRCA1/2-targeted agents is uncertain. To minimise the proportion of VUS in BRCA1/2, we performed the multifactorial likelihood analysis and validated this method using an independent cohort of patients with breast cancer. METHODS: We used a data set of 2115 patients with breast cancer from the nationwide multicentre prospective Korean Hereditary Breast Cancer study. In total, 83 BRCA1/2 VUSs (BRCA1, n=26; BRCA2, n=57) were analysed. The multifactorial probability was estimated by combining the prior probability with the overall likelihood ratio derived from co-occurrence of each VUS with pathogenic variants, personal and family history, and tumour characteristics. The classification was compared with the interpretation according to the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG/AMP) guidelines. An external validation was conducted using independent data set of 810 patients. RESULTS: We were able to redefine 38 VUSs (BRCA1, n=10; BRCA2, n=28). The revised classification was highly correlated with the ACMG/AMP guideline-based interpretation (BRCA1, p for trend=0.015; BRCA2, p=0.001). Our approach reduced the proportion of VUS from 19% (154/810) to 8.9% (72/810) in the retrospective validation data set. CONCLUSION: The classification in this study would minimise the 'uncertainty' in clinical interpretation, and this validated multifactorial model can be used for the reliable annotation of BRCA1/2 VUSs.

Erratum: Correction of Table: Genetic Mutation Profiles in Korean Patients with Inherited Retinal Diseases.

This corrects the article on e161 in vol. 34, PMID: 31144483.

Genetic Mutation Profiles in Korean Patients with Inherited Retinal Diseases.

BACKGROUND: Because of genetically and phenotypically heterogenous features, identification of causative genes for inherited retinal diseases (IRD) is essential for diagnosis and treatment in coming gene therapy era. To date, there are no large-scale data of the genes responsible for IRD in Korea. The aim of this study was to identify the distribution of genetic defects in IRD patients in Korea. METHODS: Medical records and DNA samples from 86 clinically diagnosed IRD patients were consecutively collected between July 2011 and May 2015. We applied the next-generation sequencing strategy (gene panel) for screening 204 known pathogenic genes associated with IRD. RESULTS: Molecular diagnoses were made in 38/86 (44.2%) IRD patients: 18/44 (40.9%) retinitis pigmentosa (RP), 8/22 (36.4%) cone dystrophy, 6/7 (85.7%) Stargardt disease, 1/1 (100%) Best disease, 1/1 (100%) Bardet-Biedl syndrome, 1/1 (100%) congenital stationary night blindness, 1/1 (100%) choroideremia, and 2/8 (25%) other macular dystrophies. ABCA4 was the most common causative gene associated with IRD and was responsible for causing Stargardt disease (n = 6), RP (n = 1), and cone dystrophy (n = 1). In particular, mutations in EYS were found in 4 of 14 autosomal recessive RP (29%). All cases of Stargardt disease had a mutation in the ABCA4 gene with an autosomal recessive trait. CONCLUSION: This study provided the distribution of genetic mutations responsible for causing IRD in the Korean patients. This data will serve as a reference for future genetic screening and treatment for Korean IRD patients.

Viral RNA in Blood as Indicator of Severe Outcome in Middle East Respiratory Syndrome Coronavirus Infection.

We evaluated the diagnostic and clinical usefulness of blood specimens to detect Middle East respiratory syndrome coronavirus infection in 21 patients from the 2015 outbreak in South Korea. Viral RNA was detected in blood from 33% of patients at initial diagnosis, and the detection preceded a worse clinical course.

Microevolution of Outbreak-Associated Middle East Respiratory Syndrome Coronavirus, South Korea, 2015.

During the 2015 Middle East respiratory syndrome coronavirus outbreak in South Korea, we sequenced full viral genomes of strains isolated from 4 patients early and late during infection. Patients represented at least 4 generations of transmission. We found no evidence of changes in the evolutionary rate and no reason to suspect adaptive changes in viral proteins.