Paired analysis of tumor mutation burden calculated by targeted deep sequencing panel and whole exome sequencing in non-small cell lung cancer

( Sehhoon Park ),( Chung Lee ),( Bo Mi Ku ),( Minjae Kim ),( Woong-yang Park ),( Nayoung K. D. Kim ),( Myung-ju Ahn ) 생화학분자생물학회(구 한국생화학분자생물학회) 2021 BMB Reports Vol.54 No.7

Owing to rapid advancements in NGS (next generation sequencing), genomic alteration is now considered an essential predictive biomarkers that impact the treatment decision in many cases of cancer. Among the various predictive biomarkers, tumor mutation burden (TMB) was identified by NGS and was considered to be useful in predicting a clinical response in cancer cases treated by immunotherapy. In this study, we directly compared the lab-developed-test (LDT) results by target sequencing panel, K-MASTER panel v3.0 and whole-exome sequencing (WES) to evaluate the concordance of TMB. As an initial step, the reference materials (n = 3) with known TMB status were used as an exploratory test. To validate and evaluate TMB, we used one hundred samples that were acquired from surgically resected tissues of non-small cell lung cancer (NSCLC) patients. The TMB of each sample was tested by using both LDT and WES methods, which extracted the DNA from samples at the same time. In addition, we evaluated the impact of capture region, which might lead to different values of TMB; the evaluation of capture region was based on the size of NGS and target sequencing panels. In this pilot study, TMB was evaluated by LDT and WES by using duplicated reference samples; the results of TMB showed high concordance rate (R² = 0.887). This was also reflected in clinical samples (n = 100), which showed R² of 0.71. The difference between the coding sequence ratio (3.49%) and the ratio of mutations (4.8%) indicated that the LDT panel identified a relatively higher number of mutations. It was feasible to calculate TMB with LDT panel, which can be useful in clinical practice. Furthermore, a customized approach must be developed for calculating TMB, which differs according to cancer types and specific clinical settings. [BMB Reports 2021; 54(7): 386-391]

Unraveling of Enigmatic Hearing-Impaired <i>GJB2</i> Single Heterozygotes by Massive Parallel Sequencing: DFNB1 or Not?

Kim, So Young,Kim, Ah Reum,Kim, Nayoung K. D.,Lee, Chung,Kim, Min Young,Jeon, Eun-Hee,Park, Woong-Yang,Choi, Byung Yoon,Xie., Maohua Williams & Wilkins Co 2016 Medicine Vol.95 No.14

<▼1><P>Supplemental Digital Content is available in the text</P></▼1><▼2><P><B>Abstract</B></P><P>The molecular etiology of nonsyndromic sensorineural hearing loss (SNHL) in subjects with only one detectable autosomal recessive <I>GJB2</I> mutation is unclear. Here, we report <I>GJB2</I> single heterozygotes with various final genetic diagnoses and suggest appropriate diagnostic strategies. A total of 160 subjects with SNHL without phenotypic markers were screened for <I>GJB2</I> mutations. Single-nucleotide variants or structural variations within the DFNB1 locus or in other deafness genes were examined by Sanger sequencing, breakpoint PCR, and targeted exome sequencing (TES) of 129 deafness genes. We identified 27 subjects with two mutations and 10 subjects with only one detectable mutation in <I>GJB2.</I> The detection rate of the single <I>GJB2</I> mutation among the 160 SNHL subjects in the present study (6.25%) was higher than 2.58% in normal hearing controls in Korean. The DFNB1 was clearly excluded as a molecular etiology in four (40%) subjects: other recessive deafness genes (N = 3) accounted for SNHL and the causative gene for the other non-DFNB1 subject (N = 1) was not identified. The etiology of additional two subjects was potentially explained by digenic etiology (N = 2) of <I>GJB2</I> with <I>MITF</I> and <I>GJB3</I>, respectively. The contribution of the single <I>GJB2</I> mutation in the four remaining subjects is unclear. Comprehensive diagnostic testing including TES is prerequisite for understanding <I>GJB2</I> single heterozygotes.</P></▼2>

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

Kim, Bong Jik,Ueyama, Takehiko,Miyoshi, Takushi,Lee, Seungmin,Han, Jin Hee,Park, Hye-Rim,Kim, Ah Reum,Oh, Jayoung,Kim, Min Young,Kang, Yong Seok,Oh, Doo Yi,Yun, Jiwon,Hwang, Sang Mee,Kim, Nayoung K D BMJ Publishing Group Ltd 2019 Journal of medical genetics Vol.56 No.12

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

Clinical Application of Targeted Deep Sequencing in Solid‐Cancer Patients and Utility for Biomarker‐Selected Clinical Trials

Kim, Seung Tae,Kim, Kyoung‐,Mee,Kim, Nayoung K.D.,Park, Joon Oh,Ahn, Soomin,Yun, Jae‐,Won,Kim, Kyu‐,Tae,Park, Se Hoon,Park, Peter J.,Kim, Hee Cheol,Sohn, Tae Sung,Choi, Dong Il,Cho, AlphaMed Press 2017 The oncologist Vol.22 No.10

<P>Molecular profiling of actionable mutations in refractory cancer patients has the potential to enable 'precision medicine,' wherein individualized therapies are guided based on genomic profiling. The molecular-screening program was intended to route participants to different candidate drugs in trials based on clinical-sequencing reports. In this screening program, we used a custom target-enrichment panel consisting of cancer-related genes to interrogate single-nucleotide variants, insertions and deletions, copy number variants, and a subset of gene fusions. From August 2014 through April 2015, 654 patients consented to participate in the program at Samsung Medical Center. Of these patients, 588 passed the quality control process for the 381-gene cancer-panel test, and 418 patients were included in the final analysis as being eligible for any anticancer treatment (127 gastric cancer, 122 colorectal cancer, 62 pancreatic/biliary tract cancer, 67 sarcoma/other cancer, and 40 genitourinary cancer patients). Of the 418 patients, 55 (12%) harbored a biomarker that guided them to a biomarker-selected clinical trial, and 184 (44%) patients harbored at least one genomic alteration that was potentially targetable. This study demonstrated that the panel-based sequencing program resulted in an increased rate of trial enrollment of metastatic cancer patients into biomarker-selected clinical trials. Given the expanding list of biomarker-selected trials, the guidance percentage to matched trials is anticipated to increase.</P>

Identifying <i>SYNE1</i> ataxia and extending the mutational spectrum in Korea

Kim, Ji Sun,Kim, Ah Reum,Youn, Jinyoung,Lee, Chung,Kim, Nam-Soon,Park, Woong-Yang,Park, Jong Kyu,Kim, Nayoung K.D.,Cho, Jin Whan Elsevier 2019 Parkinsonism & related disorders Vol.58 No.-

<P><B>Abstract</B></P> <P><B>Introduction</B></P> <P>Recent advances in next generation sequencing technologies have uncovered the genetic background of various diseases. The mutations in the <I>SYNE1</I> gene was previously identified as a potential cause of pure cerebellar ataxia. Although autosomal recessive ataxias are slightly more frequent than autosomal dominant forms worldwide, autosomal recessive forms are extremely rare in Korea. In this study, we aimed to identify <I>SYNE1-</I>associated ataxia by whole exome sequencing in a Korean sample, and to review the prevalence of <I>SYNE1</I> in non-French-Canadians.</P> <P><B>Methods</B></P> <P>Patients with suspected cerebellar ataxia who visited movement disorders clinic from March 2014 to December 2017 were clinically screened. After excluding cases with acquired causes and common genetic causes in Korea, including spinocerebellar ataxia and dentatorubral-pallidoluysian atrophy, 63 undiagnosed subjects were screened for <I>SYNE1</I> mutations by next generation sequencing methods.</P> <P><B>Results</B></P> <P>We identified four novel mutations (one splicing, one truncating, and two missense mutations) distributed throughout the <I>SYNE1</I> gene in two patients. The phenotype was mainly pure cerebellar ataxia in both cases. However, axonal neuropathy, mild frontal dysfunction, and autonomic dysfunction were also revealed. The age of disease onset was relatively late and the disease course was only mildly progressive.</P> <P><B>Conclusion</B></P> <P>Our results indicate that <I>SYNE1</I> mutations are not an uncommon cause of recessive ataxia with additional clinical features in the Korean population. The results of this study should alert neurologists to request <I>SYNE1</I> testing to aid the diagnosis of undetermined adult-onset ataxia in Korean patients.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>SYNE1</I> mutation is not uncommon outside the French-Canadian founder population. </LI> <LI> We identified 4 novel mutations distributed throughout the <I>SYNE1</I> gene in 2 patients. </LI> <LI> Phenotype was mainly pure ataxia in Korean <I>SYNE1</I> ataxia. </LI> <LI> Axonal neuropathy and mild cognitive impairment was also shown in <I>SYNE1</I> ataxia. </LI> </UL> </P>

Genomic Alterations in Biliary Tract Cancer Using Targeted Sequencing ¹

Yoo, Kwai Han,Kim, Nayoung K.D.,Kwon, Woo Il,Lee, Chung,Kim, Sun Young,Jang, Jiryeon,Ahn, Jungmi,Kang, Mihyun,Jang, Hyojin,Kim, Seung Tae,Ahn, Soomin,Jang, Kee-Taek,Park, Young Suk,Park, Woong-Yang,Le Neoplasia Press 2016 Translational oncology Vol.9 No.3

<P><I>Background:</I> Biliary tract cancers (BTCs) are rare and heterogeneous group of tumors classified anatomically into intrahepatic and extrahepatic bile ducts and gallbladder adenocarcinomas. Patient-derived tumor cell (PDC) models with genome analysis can be a valuable platform to develop a method to overcome the clinical barrier on BTCs. <I>Material and Methods:</I> Between January 2012 and June 2015, 40 BTC patients’ samples were collected. PDCs were isolated and cultured from surgical specimens, biopsy tissues, or malignant effusions including ascites and pleural fluid. Genome analysis using targeted panel sequencing as well as digital multiplexed gene analysis was applied to PDCs as well as primary tumors. <I>Results:</I> Extrahepatic cholangiocarcinoma (<I>N</I> = 15, 37.5%), intrahepatic cholangiocarcinoma (<I>N</I> = 10, 25.0%), gallbladder cancer (<I>N</I> = 14, 35.0%), and ampulla of Vater cancer (<I>N</I> = 1, 2.5%) were included. We identified 15 mutations with diverse genetic alterations in 19 cases of BTC from primary tumor specimens. The most common molecular alterations were in TP53 (8/19, 42.1%), including missense mutations such as C242Y, E285K, G112S, P19T, R148T, R248Q, and R273L. We also detected two NRAS mutations (G12C and Q61L), two KRAS mutations (G12A and G12S), two ERBB2 mutations (V777L and pM774delinsMA) and amplification, and three PIK3CA mutations (N345K, E545K, and E521K). PDC models were successfully established in 27 of 40 samples (67.5%), including 22/24 from body fluids (91.7%) and 5/16 from tissue specimens (31.3%). <I>Conclusions:</I> PDC models are promising tools for uncovering driver mutations and identifying rational therapeutic strategies in BTC. Application of this model is expected to inform clinical trials of drugs for molecular-based targeted therapy.</P>

Novel KCNQ4 variants in different functional domains confer genotype- and mechanism-based therapeutics in patients with nonsyndromic hearing loss

Sang-Yeon Lee,Hyun Been Choi,Mina Park,Il Soon Choi,Jieun An,Ami Kim,Eunku Kim,Nahyun Kim,Jin Hee Han,Min young Kim,Seung min Lee,Doo-Yi Oh,Bong Jik Kim,Nayoung Yi,Nayoung, K. D. Kim,Chung Lee,Woong-Y 생화학분자생물학회 2021 Experimental and molecular medicine Vol.53 No.-

Loss-of-function variant in the gene encoding the KCNQ4 potassium channel causes autosomal dominant nonsyndromic hearing loss (DFNA2), and no effective pharmacotherapeutics have been developed to reverse channel activity impairment. Phosphatidylinositol 4,5-bisphosphate (PIP2), an obligatory phospholipid for maintaining KCNQ channel activity, confers differentialpharmacological sensitivity of channels to KCNQ openers. Through whole-exome sequencing of DFNA2 families, we identified three novel KCNQ4 variants related to diverse auditory phenotypes in the proximal C-terminus (p.Arg331Gln), the C-terminus of the S6 segment (p.Gly319Asp), and the pore region (p.Ala271_Asp272del). Potassium currents in HEK293T cells expressing each KCNQ4 variant were recorded by patch-clamp, and functional recovery by PIP2 expression or KCNQ openers was examined. In the homomeric expression setting, the three novel KCNQ4 mutant proteins lost conductance and were unresponsive to KCNQ openers or PIP2 expression. Loss of p.Arg331Gln conductance was slightly restored by a tandem concatemer channel (WT-p.R331Q), and increased PIP2 expression further increased the concatemer current to the level of the WT channel. Strikingly, an impaired homomeric p.Gly319Asp channel exhibited hyperactivity when a concatemer (WT-p.G319D), with a negative shift in the voltage dependence of activation. Correspondingly, a KCNQ inhibitor and chelation of PIP2 effectively downregulated the hyperactive WTp. G319D concatemer channel. Conversely, the pore-region variant (p.Ala271_Asp272del) was nonrescuable under any condition. Collectively, these novel KCNQ4 variants may constitute therapeutic targets that can be manipulated by the PIP2 level and KCNQregulating drugs under the physiological context of heterozygous expression. Our research contributes to the establishment of a genotype/mechanism-based therapeutic portfolio for DFNA2.

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

Kim, Ah Reum,Chung, Juyong,Kim, Nayoung K. D.,Lee, Chung,Park, Woong-Yang,Oh, Doo-Yi,Choi, Byung Yoon MDPI 2017 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.18 No.11

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

Clinical Targeted Next-Generation Sequencing Panels for Detection of Somatic Variants in Gliomas

신혜미,Jason K. Sa,배준설,구하림,진선휘,조희진,최승원,경종민,김자연,서윤지,정제균,Nayoung K.D. Kim,손대순,정종석,이태섭,공두식,최정원,설호준,이정일,서연림,박웅양,남도현 대한암학회 2020 Cancer Research and Treatment Vol.52 No.1

Purpose Targeted next-generation sequencing (NGS) panels for solid tumors have been useful in clinical framework for accurate tumor diagnosis and identifying essential molecular aberrations. However, most cancer panels have been designed to address a wide spectrum of pan-cancer models, lacking integral prognostic markers that are highly specific to gliomas. Materials and Methods To address such challenges, we have developed a glioma-specific NGS panel, termed “GliomaSCAN,” that is capable of capturing single nucleotide variations and insertion/deletion, copy number variation, and selected promoter mutations and structural variations that cover a subset of intron regions in 232 essential glioma-associated genes. We confirmed clinical concordance rate using pairwise comparison of the identified variants from whole exome sequencing (WES), immunohistochemical analysis, and fluorescence in situ hybridization. Results Our panel demonstrated high sensitivity in detecting potential genomic variants that were present in the standard materials. To ensure the accuracy of our targeted sequencing panel, we compared our targeted panel to WES. The comparison results demonstrated a high correlation. Furthermore, we evaluated clinical utility of our panel in 46 glioma patients to assess the detection capacity of potential actionable mutations. Thirty-two patients harbored at least one recurrent somatic mutation in clinically actionable gene. Conclusion We have established a glioma-specific cancer panel. GliomaSCAN highly excelled in capturing somatic variations in terms of both sensitivity and specificity and provided potential clinical implication in facilitating genome-based clinical trials. Our results could provide conceptual advance towards improving the response of genomically guided molecularly targeted therapy in glioma patients.

Identification and Clinical Implications of Novel MYO15A Mutations in a Non-consanguineous Korean Family by Targeted Exome Sequencing

장문영,김아름,Nayoung K.D. Kim,이정,이경열,전우성,구자원,오승하,박웅양,김동섭,최병윤 한국분자세포생물학회 2015 Molecules and cells Vol.38 No.9

Mutations of MYO15A are generally known to cause severe to profound hearing loss throughout all frequencies. Here, we found two novel MYO15A mutations, c.3871C>T (p.L1291F) and c.5835T>G (p.Y1945X) in an affected individual carrying congenital profound sensorineural hearing loss (SNHL) through targeted resequencing of 134 known deafness genes. The variant, p.L1291F and p.Y1945X, resided in the myosin motor and IQ2 domains, respectively. The p.L1291F variant was predicted to affect the structure of the actin-binding site from three-dimensional protein modeling, thereby interfering with the correct interaction between actin and myosin. From the literature analysis, mutations in the N-terminal domain were more frequently associated with residual hearing at low frequencies than mutations in the other regions of this gene. Therefore we suggest a hypothetical genotype-phenotype correlation whereby MYO15A mutations that affect domains other than the N-terminal domain, lead to profound SNHL throughout all frequencies and mutations that affect the N-terminal domain, result in residual hearing at low frequencies. This genotype-phenotype correlation suggests that preservation of residual hearing during auditory rehabilitation like cochlear implantation should be intended for those who carry mutations in the N-terminal domain and that individuals with mutations elsewhere in MYO15A require early cochlear implantation to timely initiate speech development.