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Background and Objectives@#Difficulties often encountered in separating and purifying active muscle satellite cells (MSCs) from skeletal muscle tissues have limited the supply of cells for muscle therapy and artificial meat production. Here, we report an effective isolation protocol to economically and conveniently retrieve active MSCs from skeletal muscle tissues in mice. @*Methods@#and Results: We optimized an enzyme-based tissue digestion protocol for isolating skeletal muscle-derived primary cell population having a large number of active MSCs and described a method of differential plating (DP) for improving purity of active MSCs from skeletal muscle-derived primary cell population. Then, the age of the mouse appropriate to the isolation of a large number of active MSCs was elucidated. The best isolation yield of active MSCs from mouse skeletal muscle tissues was induced by the application of DP method to the primary cell population harvested from skeletal muscle tissues of 2-week-old mice digested in 0.2% (w/v) collagenase type II for 30 min at 37℃ and then in 0.1% (w/v) pronase for 5 min at 37℃. @*Conclusions@#The protocol we developed not only facilitates the isolation of MSCs but also maximizes the retrieval of active MSCs. Our expectation is that this protocol will contribute to the development of original technologies essential for muscle therapy and artificial meat industrialization in the future.
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Background/Aims@#Waldenström macroglobulinemia (WM) is a rare lymphoproliferative disorder that usually follows an indolent clinical course. However, some patients show an aggressive clinical course leading to death. We explored the risk factors predicting poor prognosis in WM patients. @*Methods@#We retrospectively analyzed 47 patients diagnosed with WM between 2000 and 2018 to explore risk factors predicting poor prognosis using various clinical and laboratory parameters and risk models including the International Prognostic Staging System for WM (IPSS-WM). @*Results@#Over a median follow-up duration of 80.4 months, 29 patients died. The main causes of death were disease progression, organ failure related to amyloidosis, and infection. The median overall survival (OS) was 55.1 months, and 14 patients, including three with amyloidosis, died within 2 years. Serum β2-microglobulin level higher than 4 mg/dL was significantly associated with poor OS. Accordingly, the IPSS-WM showed a significant association with poor prognosis compared with other risk models, and the low-risk group had better OS than intermediate- and high-risk groups. In the retrospective analysis using the results of targeted sequencing in two cases representing good and bad prognosis, different patterns of mutation profiles were observed, including mutations of MYD88, TP53, ARID1A, and JAK2 in a refractory case. @*Conclusions@#Serum β2-microglobulin could be a single biomarker strongly predictive of poor survival of WM patients, and the low-risk group of the IPSS-WM risk model including serum β2-microglobulin has better prognostic value than other risk models. Mutation analysis also might provide additional information to predict high-risk patients.
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Background/Aims@#Waldenström macroglobulinemia (WM) is a rare lymphoproliferative disorder that usually follows an indolent clinical course. However, some patients show an aggressive clinical course leading to death. We explored the risk factors predicting poor prognosis in WM patients. @*Methods@#We retrospectively analyzed 47 patients diagnosed with WM between 2000 and 2018 to explore risk factors predicting poor prognosis using various clinical and laboratory parameters and risk models including the International Prognostic Staging System for WM (IPSS-WM). @*Results@#Over a median follow-up duration of 80.4 months, 29 patients died. The main causes of death were disease progression, organ failure related to amyloidosis, and infection. The median overall survival (OS) was 55.1 months, and 14 patients, including three with amyloidosis, died within 2 years. Serum β2-microglobulin level higher than 4 mg/dL was significantly associated with poor OS. Accordingly, the IPSS-WM showed a significant association with poor prognosis compared with other risk models, and the low-risk group had better OS than intermediate- and high-risk groups. In the retrospective analysis using the results of targeted sequencing in two cases representing good and bad prognosis, different patterns of mutation profiles were observed, including mutations of MYD88, TP53, ARID1A, and JAK2 in a refractory case. @*Conclusions@#Serum β2-microglobulin could be a single biomarker strongly predictive of poor survival of WM patients, and the low-risk group of the IPSS-WM risk model including serum β2-microglobulin has better prognostic value than other risk models. Mutation analysis also might provide additional information to predict high-risk patients.
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Background and Objectives@#Outbred mice are widely used in toxicology, pharmacology, and fundamental biomedical research. However, there have been no reports of in vitro culture systems for spermatogonial stem cells (SSCs) derived from these mice. @*Methods@#As a step towards constructing a non-cellular niche supporting the in vitro maintenance of outbred mouse SSC self-renewal, we systematically investigated the types of integrin heterodimers that are expressed transcriptionally, translationally, and functionally in SSCs derived from Imprinting Control Region (ICR) mice. @*Results@#Among the genes encoding 25 integrin subunits, integrin α1, α5, α6, α9, αV, and αE, and integrin β1 and β5 had significantly higher transcriptional levels than the other subunits. Furthermore, at the translational level, integrin α5, α6, α9, αV, and αE, and β1 were localized on the surface of SSCs, but integrin α1 and β5 not. Moreover, significantly stronger translational expression than integrin α9 and αE was observed in integrin α5, α6, αV, and β1. SSCs showed significantly increased adhesion to fibronectin, laminin, tenascin C and vitronectin, and functional blocking of integrin α5β1, α6β1, α9β1 or αVβ1 significantly inhibited adhesion to these molecules. @*Conclusions@#We confirmed that integrin α5β1, α6β1, α9β1 and αVβ1 actively function on the surface of undifferentiated SSCs derived from outbred ICR mice.
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The outbreak of coronavirus disease 2019 (COVID-19), which began in December 2019, is still ongoing in Korea, with >9,000 confirmed cases as of March 25, 2020. COVID-19 is a severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) infection, and real-time reverse transcription-PCR is currently the most reliable diagnostic method for COVID-19 around the world. Korean Society for Laboratory Medicine and the Korea Centers for Disease Prevention and Control propose guidelines for diagnosing COVID-19 in clinical laboratories in Korea. These guidelines are based on other related domestic and international guidelines, as well as expert opinions and include the selection of test subjects, selection of specimens, diagnostic methods, interpretation of test results, and biosafety.
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Background@#Pheochromocytoma and paragangliomas (PPGL) are hereditary in approximately 30% to 40% cases. With the advancement of genetic analysis techniques, including next-generation sequencing (NGS), there were attempts to classify PPGL into molecular clusters. With NGS being applied to clinical settings recently, we aimed to review the results of genetic analysis, including NGS, and investigate the association with clinical characteristics in Korean PPGL patients. @*Methods@#We reviewed the medical records of PPGL patients who visited Severance hospital from 2006 to 2019. We documented the clinical phenotype of those who underwent targeted NGS or had known germline mutations of related genes. @*Results@#Among 57 PPGL patients, we found 28 pathogenic germline mutations of susceptibility genes. Before the targeted NGS was implemented, only obvious syndromic feature lead to the Sanger sequencing for the specific genes. Therefore, for the exact prevalence, only patients after the year 2017, when targeted NGS was added, were included (n=43). The positive germline mutations were found in 14 patients; thus, the incidence rate is 32.6%. Patients with germline mutations had a higher likelihood of family history. There were significant differences in the type of PPGLs, percentage of family history, metastasis rate, presence of other tumors, and biochemical profile among three molecular clusters: pseudohypoxic tricarboxylic acid cycle-related, pseudohypoxic von Hippel-Lindau (VHL)/endothelial PAS domain-containing protein 1-related, and kinase-signaling group. Germline mutations were identified in seven PPGL-related genes (SDHB, RET, VHL, NF1, MAX, SDHA, and SDHD). @*Conclusion@#We report the expected prevalence of germline mutations in Korean PPGL patients. NGS is a useful and accessible tool for genetic analysis in patients with PPGLs, and further research on molecular classification is needed for precise management.
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Background@#Pheochromocytoma and paragangliomas (PPGL) are hereditary in approximately 30% to 40% cases. With the advancement of genetic analysis techniques, including next-generation sequencing (NGS), there were attempts to classify PPGL into molecular clusters. With NGS being applied to clinical settings recently, we aimed to review the results of genetic analysis, including NGS, and investigate the association with clinical characteristics in Korean PPGL patients. @*Methods@#We reviewed the medical records of PPGL patients who visited Severance hospital from 2006 to 2019. We documented the clinical phenotype of those who underwent targeted NGS or had known germline mutations of related genes. @*Results@#Among 57 PPGL patients, we found 28 pathogenic germline mutations of susceptibility genes. Before the targeted NGS was implemented, only obvious syndromic feature lead to the Sanger sequencing for the specific genes. Therefore, for the exact prevalence, only patients after the year 2017, when targeted NGS was added, were included (n=43). The positive germline mutations were found in 14 patients; thus, the incidence rate is 32.6%. Patients with germline mutations had a higher likelihood of family history. There were significant differences in the type of PPGLs, percentage of family history, metastasis rate, presence of other tumors, and biochemical profile among three molecular clusters: pseudohypoxic tricarboxylic acid cycle-related, pseudohypoxic von Hippel-Lindau (VHL)/endothelial PAS domain-containing protein 1-related, and kinase-signaling group. Germline mutations were identified in seven PPGL-related genes (SDHB, RET, VHL, NF1, MAX, SDHA, and SDHD). @*Conclusion@#We report the expected prevalence of germline mutations in Korean PPGL patients. NGS is a useful and accessible tool for genetic analysis in patients with PPGLs, and further research on molecular classification is needed for precise management.
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Subject(s)
Classification , Consensus , Diagnosis , Hematologic Neoplasms , Hematology , Lymphoid TissueABSTRACT
This study was performed to examine the effects of various macromolecules in in vitro growth (IVG) media on the growth, maturation, and parthenogenesis (PA) of pig oocytes derived from small antral follicles (SAF). Immature oocytes were cultured for two days in IVG medium supplemented with 10% (v/v) fetal bovine serum (FBS), 10% (v/v) pig follicular fluid (PFF), 0.4% (w/v) bovine serum albumin (BSA), or 0.1% (w/v) polyvinyl alcohol (PVA) and then maintained for 44 h for maturation. After IVG, the mean diameters of the SAF treated with FBS, PVA, and no IVG-MAF (113.0–114.8 µm) were significantly larger than that of no IVG-SAF (111.8 µm). The proportion of metaphase II oocytes was higher in PFF (73.6%) than in BSA (43.5%) and PVA (53.7%) but similar to that in the FBS treatment (61.5%). FBS and PFF increased cumulus expansion significantly compared to PVA and BSA while the intraoocyte glutathione content was not influenced by the macromolecules. Blastocyst formation of PA oocytes treated with FBS (51.8%), PFF (50.4%), and PVA (45.2%) was significantly higher than that of the BSA-treated oocytes (20.6%). These results show that the PFF and FBS treatments during IVG improved the growth, maturation, and embryonic development of SAF.
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Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), which is caused by mutations in SACS gene, is a very rare neurodegenerative disorder characterized by the clinical triad of early onset cerebellar ataxia, pyramidal tract features, and sensorimotor polyneuropathy. Herein, we report a 35-year-old Korean male who presented with gait disturbance and lower extremity weakness. Neuroimaging and ophthalmologic evaluation revealed features consistent with ARSACS. Mutation in SACS gene was demonstrated in clinical exome sequence analysis and the patient was finally diagnosed as ARSACS.
Subject(s)
Adult , Humans , Male , Ataxia , Cerebellar Ataxia , Exome , Gait , Lower Extremity , Muscle Spasticity , Neurodegenerative Diseases , Neuroimaging , Polyneuropathies , Pyramidal Tracts , Sequence Analysis , Spinocerebellar DegenerationsABSTRACT
BACKGROUND AND OBJECTIVES: Treatment with mesenchymal stem cells (MSC) in spinal cord injury (SCI) has been highlighted as therapeutic candidate for SCI. Although astrogliosis is a major phenomenon after SCI, the role of astrogliosis is still controversial. In this study, we determined whether acute transplantation of MSC improves the outcome of SCI through modulating astrogliosis. METHODS: Bone marrow derived rat MSCs were induced neural differentiation and transplanted after acute SCI rats. Matrix metalloproteinase (MMP) and neuro-inflammatory pathway were analyzed for acute astrogliosis at 1, 3 and 7 d after SCI in RT-PCR- and western blot analysis. Functional outcome was assessed serially at postoperative 1 d and weekly for 4 weeks. Histopathologic analysis was undertaken at 7 and 28 d following injury in immunohistochemistry. RESULTS: Transplantation of MSCs decreased IL-1α, CXCL-2, CXCL-10, TNF-α and TGF-β in a rat model of contusive SCI. Protein level of NF-κB p65 was slightly decreased while level of STAT-3 was increased. In immunohistochemistry, MSC transplantation increased acute astrogliosis whereas attenuated scar formation with increased sparing white matter of spinal cord lesions. In RT-PCR analysis, mRNA levels of MMP2 was significantly increased in MSC transplanted rats. In BBB locomotor scale, the rats of MSC treated group exhibited improvement of functional recovery. CONCLUSIONS: Transplantation of MSC reduces the inflammatory reaction and modulates astrogliosis via MMP2/STAT3 pathway leading to improve functional recovery after SCI in rats.
Subject(s)
Animals , Rats , Blotting, Western , Bone Marrow , Cicatrix , Immunohistochemistry , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Models, Animal , RNA, Messenger , Spinal Cord Injuries , Spinal Cord , White MatterABSTRACT
PURPOSE: There has been an increasing trend in the use of contralateral prophylactic mastectomy (CPM) among women diagnosed with unilateral breast cancer or mutations in BRCA1 or BRCA2 to reduce the occurrence of contralateral breast cancer. This study aimed to examine trends in the CPM rate according to clinicopathologic and socioeconomic status at a single institution in Korea. METHODS: This study included 128 patients with mutations in BRCA1 or BRCA2. Patients were divided into a CPM group (n = 8) and a non-CPM group (n = 120) between May 2013 and March 2016. The main outcome variables, including epidemiology, clinical features, socioeconomic status, and tumor characteristics, were analyzed. RESULTS: A total of 8 CPMs were performed among 128 patients. All CPM patients were married. The proportion of professional working women was higher in the CPM group (P = 0.049). Most patients who underwent CPM graduated college, compared to less than a third of the non-CPM group (P = 0.013). The CPM group had a higher rate of visits to the Hereditary Breast and Ovarian Cancer (HBOC) clinic (P = 0.021). The risk-reducing salpingo-oophorectomy (RRSO) rate was significantly higher in the CPM group (P < 0.01). CONCLUSION: CPM rates were significantly different according to socioeconomic status. The CPM rate tends to increase in highly educated and professional working women. The socioeconomic status of patients is an important factor in the decision to participate in the HBOC clinic and undergo CPM or RRSO.
Subject(s)
Female , Humans , Breast , Breast Neoplasms , Epidemiology , Korea , Mastectomy , Ovarian Neoplasms , Social Class , Unilateral Breast Neoplasms , Women, WorkingABSTRACT
Next-generation sequencing (NGS) is currently used in the clinical setting for targeted therapies and diagnosis of hematologic malignancies. Accurate detection of somatic variants is challenging because of tumor purity, heterogeneity, and the complexity of genetic alterations, with various issues ranging from high detection design to test implementation. This article presents guidelines developed through consensus among a panel of experts from the Korean Society for Genetic Diagnostics. They are based on experiences with the validation processes of NGS-based somatic panels for hematologic malignancies, with reference to previous international recommendations. These guidelines describe basic parameters with emphasis on the design of a validation protocol for NGS-based somatic panels to be used in practice. In addition, they suggest thresholds of key metrics, including minimum coverage, mean coverage with uniformity index, and minimum variant allele frequency, for the initial diagnosis of hematologic malignancies.
Subject(s)
Clothing , Consensus , Diagnosis , Gene Frequency , Hematologic Neoplasms , Population CharacteristicsABSTRACT
No abstract available.
Subject(s)
Autoimmune Diseases , B-Lymphocytes , Bone Marrow , Lymphoma, B-CellABSTRACT
This study was performed to examine the effects of various macromolecules in in vitro growth (IVG) media on the growth, maturation, and parthenogenesis (PA) of pig oocytes derived from small antral follicles (SAF). Immature oocytes were cultured for two days in IVG medium supplemented with 10% (v/v) fetal bovine serum (FBS), 10% (v/v) pig follicular fluid (PFF), 0.4% (w/v) bovine serum albumin (BSA), or 0.1% (w/v) polyvinyl alcohol (PVA) and then maintained for 44 h for maturation. After IVG, the mean diameters of the SAF treated with FBS, PVA, and no IVG-MAF (113.0–114.8 µm) were significantly larger than that of no IVG-SAF (111.8 µm). The proportion of metaphase II oocytes was higher in PFF (73.6%) than in BSA (43.5%) and PVA (53.7%) but similar to that in the FBS treatment (61.5%). FBS and PFF increased cumulus expansion significantly compared to PVA and BSA while the intraoocyte glutathione content was not influenced by the macromolecules. Blastocyst formation of PA oocytes treated with FBS (51.8%), PFF (50.4%), and PVA (45.2%) was significantly higher than that of the BSA-treated oocytes (20.6%). These results show that the PFF and FBS treatments during IVG improved the growth, maturation, and embryonic development of SAF.
Subject(s)
Female , Pregnancy , Blastocyst , Embryonic Development , Follicular Fluid , Glutathione , In Vitro Techniques , Metaphase , Oocytes , Parthenogenesis , Polyvinyl Alcohol , Serum Albumin, BovineABSTRACT
PURPOSE@#Mutations in the cyclin-dependent kinase-like 5 (CDKL5) gene are associated with epileptic encephalopathy and severe cognitive impairment. We aim to characterize the association between this gene and treatment efficacy.@*METHODS@#We retrospectively analyzed 10 patients who were treated at Severance Children's Hospital for epileptic encephalopathy who were subsequently diagnosed with a CDKL5 mutation using next-generation sequencing.@*RESULTS@#Electroencephalography (EEG) results showed generalized pattern abnormalities in 60% (6/10) of patients with CDKL5 mutations. We analyzed the effects of three treatments, namely antiepileptic drugs (AEDs), ketogenic diet (KD), and steroids. A more than 50% reduction in seizures was observed in 12% (1/8) of patients treated with clobazam. KD treatment proved ineffective in most cases. In addition, a more than 50% reduction in seizures was observed in 57% (4/7) of patients treated with steroids. EEG analysis of patients treated effectively with steroids revealed that 75% (3/4) showed hypsarrhythmia and 25% (1/4) showed focal epileptiform.@*CONCLUSION@#In this study, as in other studies, AEDs and KD did not effectively control seizures in most patients with a CDKL5 mutation. However, steroid therapy reduced the frequency of seizures in patients who also exhibited hypsarrhythmia. This suggests that steroid treatment is helpful in cases of hypsarrhythmia with CDKL5 mutations.
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In both 2016 and 2017, the cytogenetic and molecular cytogenetic programs conducted three assessments for the Korean Association of External Quality Assessment Service. A total of six cases with chromosomal aberrations were distributed in 2016, and nine cases were examined in 2017 in the chromosome surveys. For the fluorescence in situ hybridization surveys, six cases and nine cases were assessed in 2016 and 2017, respectively. A total of 38 laboratories in 2016 and 39 laboratories in 2017 participated in the cytogenetics program. In the molecular cytogenetics program, a total of 32 laboratories participated in 2016, and 31 laboratories participated in 2017. Most of the participating laboratories showed acceptable results for the cytogenetics and molecular cytogenetics programs. For the unacceptable results, there were various annotation errors, suggesting the need for continuous education and quality control.
Subject(s)
Chromosome Aberrations , Cytogenetics , Education , Fluorescence , In Situ Hybridization , Korea , Laboratory Proficiency Testing , Quality ControlABSTRACT
PURPOSE: Next-generation sequencing (NGS) allows simultaneous sequencing of multiple cancer susceptibility genes and may represent a more efficient and less expensive approach than sequential testing. We assessed the frequency of germline mutations in individuals with epithelial ovarian cancer (EOC), using multi-gene panels and NGS. MATERIALS AND METHODS: Patients with EOC (n=117) with/without a family history of breast or ovarian cancer were recruited consecutively, from March 2016 toDecember 2016.GermlineDNAwas sequenced using 35-gene NGS panel, in order to identify mutations. Upon the detection of a genetic alteration using the panel, results were cross-validated using direct sequencing. RESULTS: Thirty-eight patients (32.5%) had 39 pathogenic or likely pathogenic mutations in eight genes, including BRCA1 (n=21), BRCA2 (n=10), BRIP1 (n=1), CHEK2 (n=2), MSH2 (n=1), POLE (n=1), RAD51C (n=2), and RAD51D (n=2). Among 64 patients with a family history of cancer, 27 (42.2%) had 27 pathogenic or likely pathogenic mutations, and six (9.3%) had mutations in genes other than BRCA1/2, such as CHECK2, MSH2, POLE, and RAD51C. Fifty-five patients (47.0%) were identified to carry only variants of uncertain significance. CONCLUSION: Using the multi-gene panel test, we found that, of all patients included in our study, 32.5% had germline cancer-predisposing mutations. NGS was confirmed to substantially improve the detection rates of a wide spectrum of mutations in EOC patients compared with those obtained with the BRCA1/2 testing alone.
Subject(s)
Humans , Breast , Germ-Line Mutation , Ovarian Neoplasms , PrevalenceABSTRACT
BACKGROUND: The aim of this study was to investigate the status of BRCA1/2 genetic testing practices in Korea in 2014. METHODS: A structured questionnaire was provided to the specialist in charge of BRCA1/2 genetic testing via e-mail between 28 July and 10 August 2015. A total of 11 genetic testing professionals from 14 organizations responded to the survey that asked about the status of BRCA1/2 genetic testing in the year 2014. RESULTS: The average number of BRCA1/2 genetic tests executed was 192; 6 organizations had executed less than 100 tests, and 5 organizations had conducted more than 100 tests. The primary testing method used was Sanger sequencing (100%), and 2 institutes performed multiplex ligation-dependent probe amplification (MLPA). The analysis software differed across the various organizations, with Sequencher (81.81%), Seqscape (27.27%), and Codoncode Aligner (9.09%) reported as utilized. We found that the guidelines for the interpretation of the genetic tests were different at each institution. CONCLUSIONS: Although this study only examined the status of the 2014 BRCA1/2 genetic testing practices of 11 institutions, it illustrates the necessity for standardized genetic testing or interpretation guidelines in Korea.