Subject(s)
Mitochondrial Proton-Translocating ATPases , Retinal Diseases , Whole Genome Sequencing , Humans , Retinal Diseases/genetics , Retinal Diseases/diagnosis , Mitochondrial Proton-Translocating ATPases/genetics , Mitochondrial Diseases/genetics , Mitochondrial Diseases/diagnosis , Male , Mutation , DNA, Mitochondrial/genetics , Tomography, Optical Coherence/methods , Female , Fluorescein Angiography/methodsABSTRACT
AIM: Short tandem repeats (STRs) are repetitive DNA sequences and highly mutable in various human disorders. While the involvement of STRs in various genetic disorders has been extensively studied, their role in autism spectrum disorder (ASD) remains largely unexplored. In this study, we aimed to investigate genetic association of STR expansions with ASD using whole genome sequencing (WGS) and identify risk loci associated with ASD phenotypes. METHODS: We analyzed WGS data of 634 ASD families and performed genome-wide evaluation for 12,929 STR loci. We found rare STR expansions that exceeded normal repeat lengths in autism cases compared to unaffected controls. By integrating single cell RNA and ATAC sequencing datasets of human postmortem brains, we prioritized STR loci in genes specifically expressed in cortical development stages. A deep learning method was used to predict functionality of ASD-associated STR loci. RESULTS: In ASD cases, rare STR expansions predominantly occurred in early cortical layer-specific genes involved in neurodevelopment, highlighting the cellular specificity of STR-associated genes in ASD risk. Leveraging deep learning prediction models, we demonstrated that these STR expansions disrupted the regulatory activity of enhancers and promoters, suggesting a potential mechanism through which they contribute to ASD pathogenesis. We found that individuals with ASD-associated STR expansions exhibited more severe ASD phenotypes and diminished adaptability compared to non-carriers. CONCLUSION: Short tandem repeat expansions in cortical layer-specific genes are associated with ASD and could potentially be a risk genetic factor for ASD. Our study is the first to show evidence of STR expansion associated with ASD in an under-investigated population.
Subject(s)
Autism Spectrum Disorder , Microsatellite Repeats , Humans , Autism Spectrum Disorder/genetics , Microsatellite Repeats/genetics , Male , Female , Cerebral Cortex/pathology , Phenotype , Child , Whole Genome Sequencing , Deep Learning , Severity of Illness Index , Adult , DNA Repeat Expansion/geneticsABSTRACT
RATIONALE: Cartilage-hair hypoplasia (CHH, OMIM # 250250) is a rare autosomal recessive disorder, which includes cartilage-hair hypoplasia-anauxetic dysplasia (CHH-AD) spectrum disorders. CHH-AD is caused by homozygous or compound heterozygous mutations in the RNA component of the mitochondrial RNA-processing Endoribonuclease (RMRP) gene. PATIENT CONCERNS: Here, we report 2 cases of Korean children with CHH-AD. DIAGNOSES: In the first case, the patient had metaphyseal dysplasia without hypotrichosis, diagnosed by whole exome sequencing (WES), and exhibited only skeletal dysplasia and lacked extraskeletal manifestations, such as hair hypoplasia and immunodeficiency. In the second case, the patient had skeletal dysplasia, hair hypoplasia, and immunodeficiency, which were identified by WES. INTERVENTIONS: The second case is the first CHH reported in Korea. The patients in both cases received regular immune and lung function checkups. OUTCOMES: Our cases suggest that children with extremely short stature from birth, with or without extraskeletal manifestations, should include CHH-AD as a differential diagnosis. LESSONS SUBSECTIONS: Clinical suspicion is the most important and RMRP sequencing should be considered for the diagnosis of CHH-AD.
Subject(s)
Hair , Hirschsprung Disease , Mutation , Osteochondrodysplasias , Humans , Republic of Korea , Osteochondrodysplasias/genetics , Osteochondrodysplasias/diagnosis , Male , Female , Hair/abnormalities , Hirschsprung Disease/genetics , Hirschsprung Disease/diagnosis , Dwarfism/genetics , Dwarfism/diagnosis , Primary Immunodeficiency Diseases/genetics , Primary Immunodeficiency Diseases/diagnosis , Hypotrichosis/genetics , Hypotrichosis/diagnosis , Exome Sequencing , Infant , Child, Preschool , Endoribonucleases/genetics , Child , RNA, Long NoncodingABSTRACT
Krabbe disease (KD) is an autosomal recessive neurodegenerative disorder caused by deficiency of the galactocerebrosidase (GALC) due to variants in the GALC gene. Here, we provide the first and the largest comprehensive analysis of clinical and genetic characteristics, and genotype-phenotype correlations of KD in Korean in comparison with other ethnic groups. From June 2010 to June 2023, 10 patients were diagnosed with KD through sequencing of GALC. Clinical features, and results of GALC sequencing, biochemical test, neuroimaging, and neurophysiologic test were obtained from medical records. An additional nine previously reported Korean KD patients were included for review. In Korean KD patients, the median age of onset was 2 years (3 months-34 years) and the most common phenotype was adult-onset (33%, 6/18) KD, followed by infantile KD (28%, 5/18). The most frequent variants were c.683_694delinsCTC (23%) and c.1901T>C (23%), while the 30-kb deletion was absent. Having two heterozygous pathogenic missense variants was associated with later-onset phenotype. Clinical features were similar to those of other ethnic groups. In Korean KD patients, the most common phenotype was the adult-onset type and the GALC variant spectrum was different from that of the Caucasian population. This study would further our understanding of KD.
Subject(s)
Galactosylceramidase , Genetic Association Studies , Leukodystrophy, Globoid Cell , Phenotype , Humans , Leukodystrophy, Globoid Cell/genetics , Leukodystrophy, Globoid Cell/pathology , Leukodystrophy, Globoid Cell/diagnosis , Leukodystrophy, Globoid Cell/physiopathology , Galactosylceramidase/genetics , Male , Female , Republic of Korea/epidemiology , Child, Preschool , Adult , Infant , Child , Adolescent , Young Adult , Mutation/genetics , Genotype , Genetic Predisposition to Disease , Age of OnsetABSTRACT
The aim of this study was to compare the performance of the newly developed SMG HHV-6 Q Real-Time PCR Kit (SMG assay) with the RealStar HHV-6 PCR Kit (RealStar assay). The analytical sensitivity and specificity, linearity, and precision of the SMG assay were evaluated. The clinical performance of the SMG assay was assessed and compared with that of the RealStar assay using 207 clinical specimens (HHV-6A positive, n = 51; HHV-6B positive, n = 64; HHV-6A/B negative, n = 92). The limit of detection of the SMG assay was 2.92 log10 copies/mL for HHV-6A DNA and 2.88 log10 copies/mL for HHV-6B DNA. The linear range was determined to be 3.40-9.00 log10 copies/mL for both viruses. Intra- and inter-assay variability were below 5% at concentrations ranging from 4 to 9 log10 copies/mL. No cross-reactivity was observed with the 25 microorganisms included in the specificity panel. The clinical sensitivity and specificity of the SMG and RealStar assays compared to in-house polymerase chain reaction and sequencing were as follows: SMG assay, 98.0% and 100% for HHV-6A DNA, respectively, and 96.9% and 100% for HHV-6B DNA, respectively; RealStar assay, 98.0% and 100% for HHV-6A DNA, respectively, and 90.6% and 100% for HHV-6B DNA, respectively. The correlation coefficients between viral loads measured by the two assays were 0.948 and 0.975, with mean differences of 0.62 and 0.32 log10 copies/mL for HHV-6A and HHV-6B DNA, respectively. These results demonstrate that the SMG assay is a sensitive and reliable tool for the quantitative detection and differentiation of HHV-6A and HHV-6B DNA.IMPORTANCEQuantitative real-time PCR (qPCR) that can distinguish between HHV-6A and HHV-6B DNA is recommended for diagnosis of active infection. The SMG HHV-6 Q Real-Time PCR Kit (SMG assay) is a newly developed qPCR assay that can differentiate between HHV-6A and HHV-6B DNA; however, little is known about its performance. In this study, we assessed the performance of the SMG assay and compared it with that of a commercially available qPCR assay, the RealStar HHV-6 PCR Kit (RealStar assay). The SMG assay demonstrated excellent analytical sensitivity and specificity, precision, and linearity. Furthermore, the viral loads measured by the SMG assay were highly correlated with those measured by the RealStar assay. Our results suggest that the SMG assay is a useful diagnostic tool for quantitative detection and differentiation of HHV-6A and HHV-6B DNA.
Subject(s)
Herpesvirus 6, Human , Roseolovirus Infections , Humans , Real-Time Polymerase Chain Reaction/methods , Herpesvirus 6, Human/genetics , DNA, Viral/genetics , Sensitivity and Specificity , Viral Load/methods , Roseolovirus Infections/diagnosisABSTRACT
OBJECTIVE: We assessed the performance of the Humasis COVID-19 AgHS Test (Humasis, Korea), a novel antigen rapid diagnostic test (Ag-RDT) based on lateral flow immunoassay. METHODS: 85 SARS-CoV-2-positive and 155 SARS-CoV-2-negative nasopharyngeal swab specimens confirmed by rRT-PCR were tested using the Humasis and PBCheck Ag-RDTs. The analytical specificity of the Humasis Ag-RDT was evaluated using 27 strains of human respiratory pathogens. RESULTS: The overall sensitivity and specificity were 72.9% and 99.4% for the Humasis Ag-RDT and 64.7% and 100% for the PBCheck Ag-RDT, respectively. The sensitivity for specimens with Ct≤25 was 100% for both Ag-RDTs. The Humasis Ag-RDT showed no cross-reactivity with other respiratory pathogens. CONCLUSION: Our data suggests that the Humasis Ag-RDT can be a useful diagnostic tool for the detection of SARS-CoV-2 infection.
Subject(s)
COVID-19 , Humans , COVID-19/diagnosis , Rapid Diagnostic Tests , SARS-CoV-2 , Communication , Sensitivity and Specificity , Antigens, Viral , COVID-19 TestingABSTRACT
The utility of the next-generation sequencing (NGS) panel could be increased in hereditary peripheral neuropathies, given that the duplication of PMP22 is a major abnormality. In the present study, the analytical performance of an algorithm for detecting PMP22 copy number variation (CNV) from the NGS panel data was evaluated. The NGS panel covers 141 genes, including PMP22 and five genes within 1.5-megabase duplicated region at 17p11.2. CNV calling was performed using a laboratory-developed algorithm. Among the 92 cases subjected to targeted NGS panel from March 2018 to January 2021, 26 were suggestive of PMP22 CNV. Multiplex ligation-dependent probe amplification analysis was performed in 58 cases, and the results were 100% concordant with the NGS data (23 duplications, 2 deletions, and 33 negatives). Analytical performance of the pipeline was further validated by another blind data set, including 14 positive and 20 negative samples. Reliable detection of PMP22 CNV was possible by analyzing not only PMP22 but also the adjacent genes within the 1.5-megabase region of 17p11.2. On the basis of the high accuracy of CNV calling for PMP22, the testing strategy for diagnosis of peripheral polyneuropathies could be simplified by reducing the need for multiplex ligation-dependent probe amplification.
Subject(s)
Peripheral Nervous System Diseases , Humans , Peripheral Nervous System Diseases/genetics , DNA Copy Number Variations/genetics , Reproducibility of Results , Genetic Testing/methods , Myelin Proteins/geneticsABSTRACT
Circulating tumor DNA (ctDNA) has emerged as a promising tool for various clinical applications, including early diagnosis, therapeutic target identification, treatment response monitoring, prognosis evaluation, and minimal residual disease detection. Consequently, ctDNA assays have been incorporated into clinical practice. In this review, we offer an in-depth exploration of the clinical implementation of ctDNA assays. Notably, we examined existing evidence related to pre-analytical procedures, analytical components in current technologies, and result interpretation and reporting processes. The primary objective of this guidelines is to provide recommendations for the clinical utilization of ctDNA assays.
Subject(s)
Circulating Tumor DNA , Humans , Circulating Tumor DNA/genetics , Biomarkers, Tumor/genetics , Prognosis , Neoplasm, Residual/genetics , Mutation , High-Throughput Nucleotide SequencingABSTRACT
BACKGROUND: Marfan syndrome (MFS), caused by pathogenic variants of FBN1 (fibrillin-1), is a systemic connective tissue disorder with variable phenotypes and treatment responsiveness depending on the variant. However, a significant number of individuals with MFS remain genetically unexplained. In this study, we report novel pathogenic intronic variants in FBN1 in two unrelated families with MFS. METHODS: We evaluated subjects with suspected MFS from two unrelated families using Sanger sequencing or multiplex ligation-dependent probe amplification of FBN1 and/or panel-based next-generation sequencing. As no pathogenic variants were identified, whole-genome sequencing was performed. Identified variants were analyzed by reverse transcription-PCR and targeted sequencing of FBN1 mRNA harvested from peripheral blood or skin fibroblasts obtained from affected probands. RESULTS: We found causative deep intronic variants, c.6163+1484A>T and c.5788+36C>A, in FBN1. The splicing analysis revealed an insertion of in-frame or out-of-frame intronic sequences of the FBN1 transcript predicted to alter function of calcium-binding epidermal growth factor protein domain. Family members carrying c.6163+1484A>T had high systemic scores including prominent skeletal features and aortic dissection with lesser aortic dilatation. Family members carrying c.5788+36C>A had more severe aortic root dilatation without aortic dissection. Both families had ectopia lentis. CONCLUSION: Variable penetrance of the phenotype and negative genetic testing in MFS families should raise the possibility of deep intronic FBN1 variants and the need for additional molecular studies. This study expands the mutation spectrum of FBN1 and points out the importance of intronic sequence analysis and the need for integrative functional studies in MFS diagnosis.
Subject(s)
Aortic Diseases , Aortic Dissection , Marfan Syndrome , Humans , Fibrillin-1/genetics , Mutation/genetics , Marfan Syndrome/genetics , Marfan Syndrome/complications , Marfan Syndrome/diagnosis , Genetic Testing , Adipokines/geneticsABSTRACT
Missense mutations in the alpha-B crystallin gene (CRYAB) have been reported in desmin-related myopathies with or without cardiomyopathy and have also been reported in families with only a cataract phenotype. Dilated cardiomyopathy (DCM) is a disorder with a highly heterogeneous genetic etiology involving more than 60 causative genes, hindering genetic diagnosis. In this study, we performed whole genome sequencing on 159 unrelated patients with DCM and identified an unusual stop-loss pathogenic variant in NM_001289808.2:c.527A>G of CRYAB in one patient. The mutant alpha-B crystallin protein is predicted to have an extended strand with addition of 19 amino acid residues, p.(Ter176TrpextTer19), which may contribute to aggregation and increased hydrophobicity of alpha-B crystallin. The proband, diagnosed with DCM at age 32, had a history of bilateral congenital cataracts but had no evidence of myopathy or associated symptoms. He also has a 10-year-old child diagnosed with bilateral congenital cataracts with the same CRYAB variant. This study expands the mutational spectrum of CRYAB and deepens our understanding of the complex phenotypes of alpha-B crystallinopathies.
Subject(s)
Cardiomyopathies , Cardiomyopathy, Dilated , Cataract , Muscular Diseases , Male , Child , Humans , Adult , Cardiomyopathy, Dilated/genetics , Mutation , Cataract/genetics , Phenotype , Pedigree , alpha-Crystallin B Chain/geneticsABSTRACT
BACKGROUND: Null phenotypes are characterized by complete absence of all antigens within a blood group system and caused by null variants (e.g., nonsense, frameshift, initiation codon, and canonical splice site variants) in the genes encoding the antigens. Knowing the prevalence and molecular basis of null phenotypes is essential to establish a rare donor program, and the aim of this study was to reveal the prevalence and molecular basis of null phenotypes using the Korean Reference Genome Database (KRGDB) containing whole-genome sequences of 1722 Korean individuals. STUDY DESIGN AND METHODS: Population allele frequencies of null alleles in 39 blood group systems except ABO, MNS, Rh, Lewis, and FORS were obtained from the KRGDB. The prevalence of null phenotypes was calculated using Hardy-Weinberg equation. RESULTS: The prevalence of null phenotypes were estimated to be less than 0.001% in all blood group systems except JR and SID. The prevalence of the Jr(a-) and Sd(a-) phenotypes were estimated to be 0.0453% and 0.2323%, respectively. The most frequent null allele of the JR system was ABCG2*01N.01, accounting for approximately 85% of null alleles. DISCUSSION: Our approach using a public database allowed us to investigate the prevalence and molecular basis of null phenotypes in the Korean population, which will serve as a guide for establishing a rare donor program in Korea. Considering the clinical significance, Jr(a-) is an important null phenotype that should be typed in the Korean population, and molecular assays targeting the most frequent allele ABCG2*01N.01 may be useful in detecting this phenotype.
Subject(s)
Blood Group Antigens , Neoplasm Proteins , Humans , Prevalence , Neoplasm Proteins/genetics , Blood Group Antigens/genetics , Phenotype , Alleles , Republic of Korea/epidemiologyABSTRACT
This scientific report aims to comprehensively describe the genetic and clinical characteristics of PROM1-related retinal degeneration in Korean patients. Medical records of patients diagnosed with retinal dystrophy who underwent comprehensive ophthalmologic examination and genetic testing at Samsung Medical Center between January 2016 and April 2023 were retrospectively reviewed. Genetic testing included targeted gene panel sequencing and Sanger sequencing, with diagnosis based on the presence of a "Likely Pathogenic" or "Pathogenic Variant" in the PROM1 gene, as determined by the ACMG criteria. The study identified seven patients from five unrelated families with PROM1-related retinal degeneration, all carrying the autosomal dominant variant PROM1 p.R373C; no other PROM1 gene variants were detected. All patients exhibited degenerative retinal area within the macula, with peripheral retinal degeneration observed in five patients. Substantial interfamilial and intrafamilial variability was observed in the extent of macular and peripheral degeneration. Ultra-widefield autofluorescence imaging and fluorescein angiography aided in the detection of mild peripheral degeneration in one case. In conclusion, the autosomal dominant variant PROM1 p.R373C constitutes a significant proportion of PROM1-related retinal degeneration cases in the Korean population. The observed clinical heterogeneity may suggests the potential influence of additional genetic, epigenetic, and environmental factors on disease phenotypes.
Subject(s)
Retinal Degeneration , Retinal Dystrophies , Humans , Retinal Degeneration/genetics , Retrospective Studies , Retinal Dystrophies/genetics , Phenotype , Genetic Testing , Pedigree , Republic of Korea , Mutation , AC133 Antigen/geneticsABSTRACT
In the ongoing global fight against coronavirus disease 2019 (COVID-19), the sample preparation process for real-time reverse transcription polymerase chain reaction (rRT-PCR) faces challenges due to time-consuming steps, labor-intensive procedures, contamination risks, resource demands, and environmental implications. However, optimized strategies for sample preparation have been poorly investigated, and the combination of RNase inhibitors and Proteinase K has been rarely considered. Hence, we investigated combinations of several extraction-free protocols incorporating heat treatment, sample dilution, and Proteinase K and RNase inhibitors, and validated the effectiveness using 120 SARS-CoV-2 positive and 62 negative clinical samples. Combining sample dilution and heat treatment with Proteinase K and RNase inhibitors addition exhibited the highest sensitivity (84.26%) with a mean increase in cycle threshold (Ct) value of + 3.8. Meanwhile, combined sample dilution and heat treatment exhibited a sensitivity of 79.63%, accounting for a 38% increase compared to heat treatment alone. Our findings highlight that the incorporation of Proteinase K and RNase inhibitors with sample dilution and heat treatment contributed only marginally to the improvement without yielding statistically significant differences. Sample dilution significantly impacts SARS-CoV-2 detection, and sample conditions play a crucial role in the efficiency of extraction-free methods. Our findings may provide insights for streamlining diagnostic testing, enhancing its accessibility, cost-effectiveness, and sustainability.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/diagnosis , Reverse Transcriptase Polymerase Chain Reaction , COVID-19 Testing , Endopeptidase K , Clinical Laboratory Techniques/methods , Ribonucleases , Sensitivity and Specificity , RNA, Viral/genetics , RNA, Viral/analysisABSTRACT
Introduction: RNA sequence analysis can be effectively used to identify aberrant splicing, and tumor suppressor genes are adequate targets considering their loss-of-function mechanisms. Sanger sequencing is the simplest method for RNA sequence analysis; however, because of its insufficient sensitivity in cases with nonsense-mediated mRNA decay (NMD), the use of cultured specimens with NMD inhibition has been recommended, hindering its wide adoption. Method: The results of Sanger sequencing of peripheral blood RNA without NMD inhibition performed on potential splicing variants of tumor suppressor genes were retrospectively reviewed. For negative cases, in which no change was identified in the transcript, the possibility of false negativity caused by NMD was assessed through a review of the up-to-date literature. Results: Eleven potential splice variants of various tumor suppressor genes were reviewed. Six variants were classified as pathogenic or likely pathogenic based on the nullifying effect identified by Sanger RNA sequencing. Four variants remained as variants of uncertain significance because of identified in-frame changes or normal expression of both alleles. The result of one variant was suspected to be a false negative caused by NMD after reviewing a recent study that reported the same variant as causing a nullifying effect on the affected transcript. Conclusion: Although RNA changes found in the majority of cases were expected to undergo NMD by canonical rules, most cases (10/11) were interpretable by Sanger RNA sequencing without NMD inhibition due to incomplete NMD efficiency or allele-specific expression despite highly efficient NMD.
ABSTRACT
Herein, we describe the case of a 43-month-old girl who presented with clinical manifestations of dyskinetic cerebral palsy (CP), classified as the Gross Motor Function Classification System (GMFCS) V. The patient had no family history of neurological or perinatal disorders. Despite early rehabilitation, serial assessments using the Gross Motor Function Measure (GMFM) showed no significant improvements in gross motor function. Brain magnetic resonance imaging showed nonspecific findings that could not account for developmental delay or dystonia. Whole-genome sequencing identified a heterozygous NM_002074.5(GNB1):c.239T>C (p.Ile80Thr) mutation in guanine nucleotide-binding protein beta 1 (GNB1) gene. Considering this case and previous studies, genetic testing for the etiology of dyskinetic CP is recommended for children without relevant or with nonspecific brain lesions.
ABSTRACT
Standardized protocols have been designed and developed specifically for clinical information collection and obtaining trio genomic information from infants affected with congenital anomalies (CA) and their parents, as well as securing human biological resources. The protocols include clinical and genomic information collection on multiple CA that were difficult to diagnose using pre-existing screening methods. We obtained human-derived resources and genomic information from 138 cases, including 45 families of infants with CA and their parent trios. For the clinical information collection protocol, criteria for target patient selection and a consent system for collecting and utilizing research resources are crucial. Whole genome sequencing data were generated for all participants, and standardized protocols were developed for resource collection and manufacturing. We recorded the phenotype information according to the Human Phenotype Ontology term, and epidemiological information was collected through an environmental factor questionnaire. Updating and recording of clinical symptoms and genetic information that have been newly added or changed over time are significant. The protocols enabled long-term tracking by including the growth and development status that reflect the important characteristics of newborns. Using these clinical and genetic information collection protocols for CA, an essential platform for early genetic diagnosis and diagnostic research can be established, and new genetic diagnostic guidelines can be presented in the near future.
ABSTRACT
BACKGROUND: Neurodevelopmental disorders (NDDs) have diverse phenotypes. Their genetic diagnoses are often challenged by difficulties of targeting causative genes due to heterogeneous genetic etiologies. The objective of this study was to perform genetic diagnosis of children with NDDs using whole genome sequencing. METHODS: This study included 78 pediatric patients with NDDs and their 152 family members for whole genome sequencing (WGS). All cases except one were families with at least two members. Seventy-five patients had previously undergone other genetic tests besides WGS. Detected variants were classified according to the guidelines of the American College of Medical Genetics and Genomics. RESULTS: Among 78 probands, 26 patients were genetically diagnosed with NDDs through WGS, showing a diagnostic rate of 33.3%. Of them, 22 cases had de novo variants (DNVs) identified through trio analysis. Of these DNVs, half were novel variants. Three structural variants, including a multiexon deletion, a contiguous gene deletion involving 13 Mb, and a retrotransposon insertion, were revealed by WGS. All cases except one had defects in different genes, consistent with the phenotypically diverse nature of NDDs. In addition, three patients were inconclusive, two of them had one likely pathogenic variant in a gene associated with autosomal recessive disease and the other one had no clinical phenotypes associated with the detected DNV. CONCLUSIONS: Our experience demonstrates the advantage of WGS in the diagnosis of NDDs, including detection of copy number variations and also the advantage of trio sequencing for interpretation of DNVs.
Subject(s)
DNA Copy Number Variations , Neurodevelopmental Disorders , Humans , Child , Neurodevelopmental Disorders/diagnosis , Neurodevelopmental Disorders/genetics , Whole Genome Sequencing , Genetic Testing , PhenotypeABSTRACT
BACKGROUND: Despite the 2015 American College of Medical Genetics and Genomics (ACMG) and Association of Molecular Pathology (AMP) guideline, many variants of FBN1 gene remain inconclusive. In line with publication of the FBN1-specific variant interpretation guideline by ClinGen in 2022, we reassessed variants of uncertain significance (VUS) in FBN1 gene found in our institution. METHODS: VUS found in the course of FBN1 sequencing between December 2015 and April 2022 were reassessed based on FBN1-specific variant interpretation guideline, review of updated literatures and additional genetic tests including family study and/or RNA study if available. RESULTS: Out of 695 patients who underwent FBN1 sequencing, 61 VUS were found in 69 patients. Among them, 38 VUS in 43 patients (62.3%) were reclassified as pathogenic and likely pathogenic variant ((L)PV), including 20 novel (L)PV. Major causes of reclassification were: (1) gene-specific modification of ACMG/AMP criteria, (2) updated literatures and (3) additional genetic tests. The most important evidence for reclassification was clarification of critical amino acid residues. CONCLUSIONS: After reassessing FBN1 variants according to FBN1-specific guideline and up-to-date database, a significant number of VUS was reclassified. Clinical laboratories are encouraged to perform variant reassessment at regular intervals or when there is a major change in the principle of variant interpretation.
Subject(s)
Genetic Testing , Genetic Variation , Humans , Genetic Variation/genetics , Genomics , Sequence Analysis, DNA , High-Throughput Nucleotide Sequencing , Fibrillin-1/genetics , Adipokines/geneticsABSTRACT
BACKGROUND: Vascular Ehlers-Danlos syndrome (vEDS) is an autosomal dominant disease caused by aberrations in COL3A1, which encodes type III collagen. Sanger sequencing has limitations for diagnosis since exon deletion/duplication and splicing alterations are not uncommon in COL3A1. We report 2 patients with vEDS who were not diagnosed by conventional Sanger sequencing. METHODS: We performed either targeted panel or whole-genome sequencing. Complementary DNA (cDNA) sequencing was performed using cultured skin fibroblasts. Sanger sequencing of DNA was performed for the confirmation of breakpoints in the case of exon deletion. We also evaluated the sensitivity of the splicing prediction tool, SpliceAI. RESULTS: An exon 27 deletion was suspected on targeted panel sequencing of 1 patient. The deletion was confirmed using cDNA sequencing (r.1870_1923del) and breakpoints were confirmed (c.1870-109_1923+10del). On targeted panel sequencing in the other patient, we found a novel intronic variant of c.1149+6T>C that leads to skipping of exon 16 (r.1051_1149del) by cDNA sequencing. SpliceAI showed 98.8% sensitivity for known splicing variants in COL3A1. CONCLUSION: Our study highlights the necessity of a comprehensive approach to the genetic diagnosis of vEDS. In addition, cDNA sequencing was useful as an auxiliary method, especially considering the limited sensitivity of the splicing prediction tool.
Subject(s)
Ehlers-Danlos Syndrome, Type IV , Ehlers-Danlos Syndrome , Humans , Ehlers-Danlos Syndrome/genetics , Ehlers-Danlos Syndrome/diagnosis , DNA, Complementary , Mutation , Collagen Type III/geneticsABSTRACT
Whole-genome sequencing is the most comprehensive form of next-generation sequencing method. We aimed to assess the additional diagnostic yield of whole-genome sequencing in patients with clinically diagnosed Charcot-Marie-Tooth disease when compared with whole-exome sequencing, which has not been reported in the literature. Whole-genome sequencing was performed on 72 families whose genetic cause of clinically diagnosed Charcot-Marie-Tooth disease was not revealed after the whole-exome sequencing and 17p12 duplication screening. Among the included families, 14 (19.4%) acquired genetic diagnoses that were compatible with their phenotypes. The most common factor that led to the additional diagnosis in the whole-genome sequencing was genotype-driven analysis (four families, 4/14), in which a wider range of genes, not limited to peripheral neuropathy-related genes, were analysed. Another four families acquired diagnosis due to the inherent advantage of whole-genome sequencing such as better coverage than the whole-exome sequencing (two families, 2/14), structural variants (one family, 1/14) and non-coding variants (one family, 1/14). In conclusion, an evident gain in diagnostic yield was obtained from whole-genome sequencing of the whole-exome sequencing-negative cases. A wide range of genes, not limited to inherited peripheral neuropathy-related genes, should be targeted during whole-genome sequencing.