External Quality Assessment of MERS-CoV Molecular Diagnostics During the 2015 Korean Outbreak

성문우,이승준,조성임,고경필,김미나,성흥섭,김재석,안지수,유병수,김택수,김의종,박성섭 대한진단검사의학회 2016 Annals of Laboratory Medicine Vol.36 No.3

Background: The largest outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infection outside Middle East Asia in 2015 has necessitated the rapid expansion of laboratories that conduct MERS-CoV molecular testing in Korea, together with external quality assessment (EQA) to evaluate the assays used. Methods: The EQA program consisted of two phases; self-validation and blind assessment. For the first EQA phase, in vitro transcribed upstream region of the envelope gene (upE) and the open reading frame (ORF)1a RNAs were used at a concentration of 1,000 copies/μL. The test panel for the second EQA phase consisted of RNA extracts from three samples, which were obtained from two MERS-CoV positive patients and one MERS-CoV negative patient. Results: The first EQA phase results for 46 participants showed a linear relationship between the threshold cycle (CT) values of RNA materials and the logarithmic concentrations for both upE and ORF1a gene targets (R2=0.73 and 0.75, respectively). The mean CT value for each concentration was different depending on which commercial kit was used for the assay. Among the three commonly used kits, PowerChek MERS Real-Time PCR kit (KogeneBiotech, Korea) showed the lowest CT values at all concentrations of upE and most concentrations of ORF1a. The second EQA phase results for 47 participants were 100% correct for all tested samples. Conclusions: This EQA survey demonstrates that the MERS-CoV molecular testing performed in Korea during the 2015 outbreak is of robust capability. However, careful establishment and validation of a cut-off value are recommended to ensure good analytical sensitivity.

만성진행외안근마비의 사립체 유전체의 이상과 분자진단법에 대한 연구

성문우,황정민,김지연,고현수,박성섭 대한안과학회 2005 대한안과학회지 Vol.46 No.2

Purpose: To evaluate the spectrum of mitochondrial DNA (mtDNA) aberrations in patients with suspected chronic progressive external ophthalmoplegia (CPEO) and to establish the molecular diagnostic method for CPEO in Koreans. Methods: We performed mtDNA analyses for single deletions with long-range PCR and direct sequencing, and for the nine important point mutations including 3243A>G and 8344A>G with PCR/RFLP in muscles, bloods and paraffin-embedded muscle sections of 16 Korean patients with suspected CPEO. Results: Three novel single mtDNA deletions were identified in three patients’ muscles: 3159bp deletion from np 6657 to np 9815, 7591bp from np 8429 to np 16019, and 6191bp from np 7799 to np 13989. In addition, multiple mtDNA deletions were found in one patient. None of the blood specimen had mtDNA deletions even in the patients with mtDNA deletion in muscle. All single deletion junctions were flanked by direct repeats of 6-8 bp. None of the nine mtDNA point mutations were found in muscles, bloods or paraffin-embedded muscle sections. Conclusions: We identified three novel single deletions by mtDNA analyses in the muscles of 3 patients with CPEO. However, point mutations were not found. Furthermore, we established a molecular diagnostic method for CPEO in Korea. Long-range PCR and direct sequencing of the muscles were appropriate as a molecular diagnostic method for CPEO in Koreans.

[Symposium] Molecular genetics of pheochromocytoma

성문우 대한외과학회 2011 대한외과학회 학술대회 초록집 Vol.2011 No.11

Leber씨 유전성시신경병증에서 사립체 DNA양과 MTND4 발현양상

성문우,김지연,고현수,황정민,박성섭 대한진단검사의학회 2004 Annals of Laboratory Medicine Vol.24 No.6

Background : Leber's hereditary optic neuropathy (LHON) is characterized by acute or subacute bilateral visual loss, primarily caused by a mitochondrial DNA (mtDNA) point mutation. However the pathogenic mechanism of visual loss has not been clearly unraveled. We investigated the pathogenetic mechanism of LHON by the analysis of mtDNA content and the MTND4 expression. Methods : mtDNA contents were absolutely quantified in 17 patients, 8 carriers and 47 normal subjects using real-time PCR. All patients and carriers had the 11778G>A mutation of mtDNA. For real-time PCR assay of mtDNA content, the reference ranges were established by age groups and the reproducibility was assessed by intra-run and inter-run assays. We also quantified the expression of two mitochondrial genes (MTND4 harboring 11778G>A and MTCYB) relative to a nuclear gene (GAPDH) in the three subject groups. Results : The mean mtDNA contents in the patients, carriers and normal subjects were 894.9 (±186.9), 848.5 (±221.7) and 1148.6 (±406.9) copies/cell, respectively. Patients and carriers had significantly lower mtDNA contents than normal subjects (P=0.001 and P=0.048, respectively). The RNA expression of both MTND4 and MTCYB tended to be lower in patients and carriers than in normal subjects (statistically insignificant), but MTND4/MTCYB ratios were similar among the three groups. Conclusions : mtDNA content and the MTND4 expression are decreased in LHON patients and carriers, and it may be caused by mitochondrial depletion. The mitochondrial depletion may be an additional cause of respiratory defect. (Korean J Lab Med 2004; 24: 439-45) 배경 : Leber씨 유전성 시신경병증(LHON)은 급성 또는 아급성으로 양안의 시력감소가 초래되는 질환이다. LHON의 일차적인 발생원인은 호흡사슬복합체를 생성하는 사립체 DNA의 돌연변이이나, 시력 소실의 발병 기전은 mtDNA의 돌연변이 만으로설명되지 않고 있다. 본 연구에서는 LHON 환자 및 보인자에서mtDNA양과 MTND4 유전자의 발현을 정량 분석함으로써 LHON444 성문우김지연고현수 외 2인의 발병 기전을 연구하였다.방법 : mtDNA 11778G >A 돌연변이를 가진 LHON 환자 17명과 보인자 8명, 그리고 정상인 47명의 말초혈액에서 세포당 mt-DNA양을 real-time PCR을 이용하여 절대정량하였다. Real-timePCR에 기반한 mtDNA 정량검사법을 이용하기 위해, 각 연령대별mtDNA양의 참고치를 산정하였고, intra-run 및 inter-run assay로 정밀도를 확인하였다. 또한 세 군에서 사립체의 두 유전자(11778G>A를 갖는 MTND4와 MTCYB) 및 핵유전자(GAPDH)의 상대적인 RNA 발현 양상을 정량하였다.결과 : mtDNA양은 환자, 보인자 및 정상인에서 각각 894.9(± 186.9), 848.5 (± 221.7), 1148.6 (± 406.9) copies/cell로, 환자와 보인자 군이 모두 정상인에 비해 유의하게 감소되었다(각P=0.001, P=0.048). MTND4 와 MTCYB 유전자발현은 정상인에 비해 환자 및 보인자에서 감소되는 경향을 보였으나, 통계적으로 유의하지는 않았으며, 세 군 간에MTND4/MTCYB 의 비 는유사하였다. 결론 : LHON 환자와 보인자에서 mtDNA양과 MTND4 발현은 감소되어 있었으며, 이는 사립체 자체의 감소와 관련된다. mtDNA 돌연변이 뿐만 아니라 사립체의 감소도 사립체 호흡기 능저하의 중요한 원인이 될 수 있을 것으로 보인다.

Molecular Characterization of the NF2 Gene in Korean Patients with Neurofibromatosis Type 2: A Report of Four Novel Mutations

성문우,여임경,조성임,박철기,김승기,백선하,김동규,정희원,박현웅,김소연,김지연,박성섭 대한진단검사의학회 2010 Annals of Laboratory Medicine Vol.30 No.2

Background : Neurofibromatosis type 2 (NF2) is an autosomal dominant syndrome caused by the NF2 tumor suppressor gene. However, the NF2 mutation characteristics in Korean patients are not sufficiently understood. In this study, we conducted a comprehensive mutational analysis in 7Korean NF2 patients by performing direct sequencing and gene-dosage assessment. Methods : We analyzed all exons and flanking regions of NF2 by direct sequencing and screened the deletions or duplications involving NF2 by multiplex ligation-dependent probe amplification. Results : Four novel NF2 mutations, including 2 splice-site mutations (c.364-1G>A and c.886-3C>G), 1 frameshift mutation (c.524delA), and 1 missense mutation (c.397T>C; p.Cys133Arg), were identified in our patients. No large deletion or duplication was identified in our series. Subsequently,we identified an abnormal splicing product by using reverse transcription-PCR and direct sequencing in 2 patients with a novel splice-site mutation. The missense mutation c.397T>C was predicted to have harmful effects on protein function. Conclusions : The detection rate of NF2 mutations in Korean patients (57%) is similar to those in other populations. Our results provided a greater insight into the mutational spectrum of the NF2 gene in Korean subjects. (Korean J Lab Med 2010;30:190-4)

Genotyping Influenza Virus by Next-Generation Deep Sequencing in Clinical Specimens

성문우,조성임,박현웅,서수현,이승준,김의종,박성섭 대한진단검사의학회 2016 Annals of Laboratory Medicine Vol.36 No.3

Rapid and accurate identification of an influenza outbreak is essential for patient care and treatment. We describe a next-generation sequencing (NGS)-based, unbiased deep sequencing method in clinical specimens to investigate an influenza outbreak. Nasopharyngeal swabs from patients were collected for molecular epidemiological analysis. Total RNA was sequenced by using the NGS technology as paired-end 250 bp reads. Total of 7 to 12 million reads were obtained. After mapping to the human reference genome, we analyzed the 3-4% of reads that originated from a non-human source. A BLAST search of the contigs reconstructed de novo revealed high sequence similarity with that of the pandemic H1N1 virus. In the phylogenetic analysis, the HA gene of our samples clustered closely with that of A/Senegal/VR785/2010(H1N1), A/Wisconsin/11/2013(H1N1), and A/Korea/ 01/2009(H1N1), and the NA gene of our samples clustered closely with A/Wisconsin/11/ 2013(H1N1). This study suggests that NGS-based unbiased sequencing can be effectively applied to investigate molecular characteristics of nosocomial influenza outbreak by using clinical specimens such as nasopharyngeal swabs.

Three-Year Experience of an External Proficieny Testing Survey for ­Next-Generation Sequencing-Based Testing for Germline Mutation

성문우,Manjin Kim,Ho-Seob Shin,Sung Im Cho,Sung Sup Park 대한임상검사정도관리협회 2020 Journal of Laboratory Medicine And Quality Assuran Vol.42 No.1

Background: Next-generation sequencing is a powerful technology that allows simultaneous analysis of several genes but also demands a well-designed quality management system owing to its complexity. We aimed to analyze the results of next-generation sequencing (NGS)-germline proficiency testing (PT) survey performed by the Korean Association of External Quality Assessment Service during 2017–2019 to assess the current status of the NGS-based genetic testing in Korea.Methods: The recent 3-year results from the PT survey were investigated. During this period, PT survey was performed twice every year with two or three challenges per round. Correct results (%) were calculated from all tested regions; the trend by year and variation type was analyzed and the probable causes estimated.Results: During this period, the number of participating laboratories increased from 5 to 22. The correct results were 97.2% in average and showed a gradual increase with year. The most common ‘unacceptable’ results were false-negative or false-positive, followed by inappropriate nomenclature and zygosity assignment.Conclusions: The PT survey shows that the overall performance of NGS laboratories in Korea is highly confident, although some improvements may be needed. A method-based PT survey for the NGS test serves as a useful approach to assess the performance of NGS laboratories.

Effect of Next-Generation Exome Sequencing Depth for Discovery of Diagnostic Variants

김경,성문우,정우형,박성섭,임상섭,박원,김지현,이기영,박래웅,김남신 한국유전체학회 2015 Genomics & informatics Vol.13 No.2

Sequencing depth, which is directly related to the cost and time required for the generation, processing, and maintenance of next-generation sequencing data, is an important factor in the practical utilization of such data in clinical fields. Unfortunately, identifying an exome sequencing depth adequate for clinical use is a challenge that has not been addressed extensively. Here, we investigate the effect of exome sequencing depth on the discovery of sequence variants for clinical use. Toward this, we sequenced ten germ-line blood samples from breast cancer patients on the Illumina platform GAII(x) at a high depth of ∼200×. We observed that most function-related diverse variants in the human exonic regions could be detected at a sequencing depth of 120×. Furthermore, investigation using a diagnostic gene set showed that the number of clinical variants identified using exome sequencing reached a plateau at an average sequencing depth of about 120×. Moreover, the phenomena were consistent across the breast cancer samples.

폐결핵 의심 환자에서 기관지 내시경 검체를 이용한 Xpert MTB/RIF 검사의 진단적 유용성

이하연,성문우,박성섭,황승식,이진우,박영식,이창훈,이상민,유철규,김영환,한성구,임재준 대한결핵 및 호흡기학회 2012 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.114 No.-

목적: Xpert MTB/RIF검사는 결핵 진단에 대해 높은 민감도와 특이도를 가진다. 한편, 도말 음성 혹은 객담을 배출하지 못하는 환자에서 기관지내시경 검사를 시행하였을 경우 폐결핵의 진단율을 높인다는 보고들이 있다. 이 연구의 목적은 폐결핵 의심 환자에서 기관지내시경 검체로 Xpert MTB/RIF검사를 시행하였을 때 진단에 얼마나 도움이 되는지를 확인하는 것이다. 방법: 2011년 9월부터 2012년 6월까지 서울대병원을 내원한 폐결핵이 의심되어 기관지내시경 검체를 이용한 Xpert MTB/RIF 검사가 시행된 환자들을 대상으로 하였다. 기관지내시경 검체로 시행한 Xpert MTB/RIF검사와 항산균 도말 검사 및 배양 검사의 민감도, 특이도, 양성예측도, 음성예측도를 구하여 분석하였다. 폐결핵은 배양검사 결과, 영상의학적, 병리적, 임상 소견을 모두 종합하여 진단하였다. 성적: 전체 132명을 분석하였고, 이중 50명이 폐결핵으로 진단되었다. 기관지내시경 검체를 이용하여 Xpert MTB/RIF 검사를 시행하였을 때 민감도, 특이도, 양성예측도, 음성예측도는 각각 70%, 100%, 100%, 84.5%였다. 항산균 도말 검사의 민감도와 특이도는 10.0%와 98.8% 였으며, 결핵균 배양 검사의 민감도 와 특이도는 62.0%와 100%였다. 결론: 기관지 내시경 검체를 이용한 Xpert MTB/RIF 검사는 도말 음성이거나 객담을 배출할 수 없는 환자에게서 빠르고 정확하게 폐결핵을 진단하는데 도움을 준다.

Clinical Characteristics of Diabetes in People with Mitochondrial DNA 3243A>G Mutation in Korea

노은후,성문우,채종희,박경수,곽수헌 대한내과학회 2022 대한내과학회 추계학술대회 Vol.2022 No.2