• Clinical and Experimental Pediatrics
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• v.57 no.9
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• pp.416-419
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• 2014

X-linked adrenoleukodystrophy (X-ALD) is a rare peroxisomal disorder, that is rapidly progressive, neurodegenerative, and recessive, and characteristically primary affects the central nervous system white matter and the adrenal cortex. X-ALD is diagnosed basaed on clinical, radiological, and serological parameters, including elevated plasma levels of very long chain fatty acids (VLCFA), such as C24:0 and C26:0, and high C24:0/C22:0 and C26:0/C22:0 ratios. These tests are complemented with genetic analyses. A 7.5-year-old boy was admitted to Department of Pediatrics, Chungnam National University Hospital with progressive weakness of the bilateral lower extremities. Brain magnetic resonance imaging confirmed clinically suspected ALD. A low dose adrenocorticotropic hormone stimulation test revealed parital adrenal insufficiency. His fasting plasma levels of VLCFA showed that his C24:0/C22:0 and C26:0/C22:0 ratios were significantly elevated to 1.609 (normal, 0-1.390) and 0.075 (normal, 0-0.023), respectively. Genomic DNA was extracted from peripheral whole blood samples collected from the patient and his family. All exons of ABCD1 gene were amplified by polymerase chain reaction (PCR) using specific primers. Amplified PCR products were sequenced using the same primer pairs according to the manufacturer's instructions. We identified a missense mutation (p.Arg163Leu) in the ABCD1 gene of the proband caused by the nucleotide change 488G>T in exon 1. His asymptomatic mother carried the same mutation. We have reported an unpublished mutation in the ABCD1 gene in a patient with X-ALD, who showed increased ratio of C24:0/C22:0 and C26:0/C22:0, despite a normal VLCFA concentrations.

• Korean Journal of Head & Neck Oncology
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• v.27 no.1
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• pp.84-87
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• 2011

Branchio-otic syndrome(BOS) is a relatively uncommon genetic malformation associated with dysmorphogenesis of the first and second branchial arches and is characterized by branchial fistulae, congenital preauricular fistulae, and anomalies of the pinnae, external, middle, and inner ears, accompanied by hearing loss. Recently, we experienced a case of BOS in a 10 years old female patient and report this case with a review of literature. 10-year-old girl presented with hearing impairment, bilateral preauricular fistula and cervical fistula. The pure tone audiometry revealed that she had 60dB sensorineural hearing loss on right side and 90dB mixed hearing loss on left. Bilateral branchial fistula was found on the neck CT scan and bilateral ossicular and cochlear abnormality combined with enlarged internal auditory canal was noted on the temporal bone CT scan. To investigate the association with EYA1 gene, we performed DNA sequncing with peripheral white blood cell and found the point mutations on Exon 7, 12 and 16 of EYA1 gene. The preauricular fistula and branchial fistula was excised surgically and hearing aid was applied on her left side. There was no sign of fistula recurrence for seven years after the surgery.

• Journal of Genetic Medicine
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• v.13 no.1
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• pp.41-45
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• 2016

Marfan syndrome (MFS) is an inherited connective tissue disorder with a mutation in the fibrillin-1 (FBN1) gene. Fibrillin is a major building block of microfibrils, which constitute the structural component of the connective tissues. A 10-year-old girl visited our hospital with the chief complaint of precocious puberty. According to her medical history, she had a pulmonary wedge resection for a pneumothorax at 9 years of age. There was no family history of MFS. Mid parental height was 161.5 cm. The patient's height was 162 cm (>97th percentile), and her weight was 40 kg (75th-90th percentile). At the time of initial presentation, her bone age was approximately 11 years. From the ophthalmologic examination, there were no abnormal findings except myopia. There was no wrist sign. At the age of 14 years, she revisited the hospital with the chief complaint of scoliosis. Her height and weight were 170 cm and 50 kg, respectively, and she had arachnodactyly and wrist sign. We performed an echocardiograph and a test for the FBN1 gene mutation with direct sequencing of 65 coding exons, suspecting MFS. There were no cardiac abnormalities including mitral valve prolapse. A cytosine residue deletion in exon 7 (c.660delC) was detected. This is a novel mutation causing a frameshift in protein synthesis and predicted to create a premature stop codon. We report the case of a patient with MFS with a novel FBN1 gene missense mutation and a history of pneumothorax at a young age without cardiac abnormalities during her teenage years.

• Journal of Life Science
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• v.20 no.3
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• pp.453-456
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• 2010

Leukemia is the abnormal increase of hematopoietic progenitor cells in tissues, resulting in anemia, increased susceptibility to infection and impaired blood clotting. The adenosine deaminase (ADA) gene is an important druggable target for the treatment of leukemia patients. Genetic and molecular analyses were performed to determine the effects of ADA gene mutations in 20 leukemia patients in the Korean population. To analyze the relationship between genotype and phenotype, the ADA genomic DNAs - including 1,092 bp of 12 exons and partial intron sequences flanking each exon - were sequenced and compared. In this study, the known mutations in other diseases, more than 50 mutations already reported in patients with severe combined immunodeficiency disease (SCID) and autism, were not found, but two novel mutations in leukemia patients were discovered. They include one nonsense mutation (A>C at nt position 478, F101F) and one missense mutation (G>A at nt position 778, E260K). One missense mutation (G>A at nt position 22, D8Y) was also detected in 20 normal control patients (allelic frequency of 7.5%). Interestingly, subjects in the Korean population retained 2 bp insertion at the intron 6 (IVS6-52insGC), something that has never been shown in other populations. The genetic study to find out the correlation between the mutant alleles and leukemia patients revealed no association statistically (p>0.05). The mutation found in leukemia needs further study to determine its possibility as a molecular marker for the diagnosis of leukemia.

• Applied Biological Chemistry
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• v.42 no.1
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• pp.34-38
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• 1999

We observed the structure of phenylalanine ammonia-lyase gene (PAL) which is one of the best studied plant defense-related genes responding to pathogen infection by producing suberin, lignin, and phytoalexins. In tomato, at least 5 different genetic loci have been identified by genomic southern blot hybridization and nucleotide sequence analyses of partially cloned gene fragments (Lee et al. 1992). However, our results suggest that two other isoforms designated as PAL X1 and PAL X2 are located on the chromosome in tomato plant. Furthermore, the preliminary results obtained from southern blot hybridization analyses of subcloned fragment digested with several restriction endonuclease indicated that PAL X1 and PAL X2 clones contain at least two copies of PAL gene and partial nucleotide sequence analyses of each subcloned fragment with the same primer taken from known nucleotide sequence of PAL5 gene indicated that they are located side by side on the same chromosome.

• Environmental Mutagens and Carcinogens
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• v.20 no.1
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• pp.26-33
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• 2000

Genetic polymorphisms of metabolizing enzymes to chemical carcinogens have been recognized as a major important host factors in human cancers. To datermine the frequencies of genotypes of CYP1A1 and GSTM1 metabolizing enzymes in healthy controls and head and neck cancer patients in Korean and to identify the relative high risk genotypes of these metabolizing enzymes to head and neck cancer, we have analyzed 133 head and neck cancer patients and corresponding healthy controls matched in age and sex using polymerase chain reaction-restriction fragment length polymorphism (PCR-RELP). In analysis of CYP1A1, the Val/Val genotype of exon 7 polymorphism and m2/m2 genotype of Msp 1 polymorphism were associated with higher relative risks to head and neck cancers (Odds ratio : 2.34, 95% CI : 0.79-6.96 and 1.27, 95% CI : 0.59-2.73, respectively). In combined genotyping of CYP1A1 and GSTMI enzymes polymorphisms, the patients with Val/Val ad GSTM1(-), and m1/m21 and GSTM1(-) combined genotypes had higher relative risks than the patients with each base genotype of combined genotypes (Odds ratio : 4.57, 95% CI : 0.5-41.25 and 1.65, 95% CI L 0.73-3.77, respectively). These results sugget the combined genotyping of metabolizing enzymes could be useful for predicting individual genetic susceptibility and screening the high risk subpopulation to head and neck cancer in Korea.

• Journal of Korean Neurosurgical Society
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• v.59 no.1
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• pp.26-36
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• 2016

Objective : This study investigated whether pyrosequencing can be used to determine the methylation status of the MGMT promoter as a clinical biomarker using relatively old archival tissue samples of glioblastoma. We also examined other prognostic factors for survival of glioblastoma patients. Methods : The available study set included formalin-fixed paraffin-embedded (FFPE) tissue from 104 patients at two institutes from 1997 to 2012, all of which were diagnosed histopathologically as glioblastoma. Clinicopathologic data were collected by review of medical records. For pyrosequencing analysis, the PyroMark Q96 CpG MGMT kit (Qiagen, Hilden, Germany) was used to detect the level of methylation at exon 1 positions 17-39 of the MGMT gene, which contains 5 CpGs. Results : Methylation of the MGMT promoter was detected in 43 (41.3%) of 104 samples. The average percentage methylation was $14.0{\pm}16.8%$ overall and $39.0{\pm}14.7%$ for methylated cases. There was no significant pattern of linear increase or decrease according to the age of the FFPE block (p=0.687). In multivariate analysis, age, performance status, extent of surgery, method of adjuvant therapy, and methylation status estimated by pyrosequencing were independently associated with overall survival. Additionally, patients with a high level of methylation survived longer than those with low methylation (p=0.016). Conclusion : In this study, the status and extent of methylation of the MGMT promoter analyzed by pyrosequencing were associated with overall survival in glioblastoma patients. Pyrosequencing is a quantitative method that overcomes the problems of MSP and a simple technique for accurate analysis of DNA sequences.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.6
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• pp.2787-2793
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• 2016

Background: Urinary bladder cancer is a common malignancy in the West and ranks as the $7^{th}$ most common cancer in our region of Kashmir, India. FGFR3 mutations are frequent in superficial urothelial carcinoma (UC) differing from the RAS gene mutational pattern. The aim of this study was to analyze the frequency and association of FGFR3 and RAS gene mutations in UC cases. Materials and Methods: Paired tumor and adjacent normal tissue specimens of 65 consecutive UC patients were examined. DNA preparations were evaluated for the occurrence of FGFR3 and RAS gene mutations by PCR-SCCP and DNA sequencing. Results: Somatic point mutations of FGFR3 were identified in 32.3% (21 of 65). The pattern and distribution were significantly associated with low grade/stage (p<0.05). The overall mutations in exon 1 and 2 in all the forms of RAS genes aggregated to 21.5% and showed no association with any clinic-pathological parameters. In total, 53.8% (35 of 65) of the tumors studied had mutations in either a RAS or FGFR3 gene, but these were totally mutually exclusive in and none of the samples showed both the mutational events in mutually exclusive RAS and FGFR3. Conclusions: We conclude that RAS and FGFR3 mutations in UC are mutually exclusive and non-overlapping events which reflect activation of oncogenic pathways through different elements.

• Journal of Ginseng Research
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• v.46 no.4
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• pp.505-514
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• 2022

Background: The roots of Panax ginseng contain two types of tetracyclic triterpenoid saponins, namely, protopanaxadiol (PPD)-type saponins and protopanaxatiol (PPT)-type saponins. In P. ginseng, the protopanaxadiol 6-hydroxylase (PPT synthase) enzyme catalyses protopanaxatriol (PPT) production from protopanaxadiol (PPD). In this study, we constructed homozygous mutant lines of ginseng by CRISPR/Cas9-mediated mutagenesis of the PPT synthase gene and obtained the mutant ginseng root lines having complete depletion of the PPT-type ginsenosides. Methods: Two sgRNAs (single guide RNAs) were designed for target mutations in the exon sequences of the two PPT synthase genes (both PPTa and PPTg sequences) with the CRISPR/Cas9 system. Transgenic ginseng roots were generated through Agrobacterium-mediated transformation. The mutant lines were screened by ginsenoside analysis and DNA sequencing. Result: Ginsenoside analysis revealed the complete depletion of PPT-type ginsenosides in three putative mutant lines (Cr4, Cr7, and Cr14). The reduction of PPT-type ginsenosides in mutant lines led to increased accumulation of PPD-type ginsenosides. The gene editing in the selected mutant lines was confirmed by targeted deep sequencing. Conclusion: We have established the genome editing protocol by CRISPR/Cas9 system in P. ginseng and demonstrated the mutated roots producing only PPD-type ginsenosides by depleting PPT-type ginsenosides. Because the pharmacological activity of PPD-group ginsenosides is significantly different from that of PPT-group ginsenosides, the new type of ginseng mutant producing only PPD-group ginsenosides may have new pharmacological characteristics compared to wild-type ginseng. This is the first report to generate target-induced mutations for the modification of saponin biosynthesis in Panax species using CRISPR-Cas9 system.

• Genomics & Informatics
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• v.20 no.3
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• pp.30.1-30.9
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• 2022

Hereditary spastic paraplegia is a not common inherited neurological disorder with heterogeneous clinical expressions. ALDH18A1 (located on 10q24.1) gene-related spastic paraplegias (SPG9A and SPG9B) are rare metabolic disorders caused by dominant and recessive mutations that have been found recently. Autosomal recessive hereditary spastic paraplegia is a common and clinical type of familial spastic paraplegia linked to the SPG11 locus (locates on 15q21.1). There are different symptoms of spastic paraplegia, such as muscle atrophy, moderate mental retardation, short stature, balance problem, and lower limb weakness. Our first proband involves a 45 years old man and our second proband involves a 20 years old woman both are affected by spastic paraplegia disease. Genomic DNA was extracted from the peripheral blood of the patients, their parents, and their siblings using a filter-based methodology and quantified and used for molecular analysis and sequencing. Sequencing libraries were generated using Agilent SureSelect Human All ExonV7 kit, and the qualified libraries are fed into NovaSeq 6000 Illumina sequencers. Sanger sequencing was performed by an ABI prism 3730 sequencer. Here, for the first time, we report two cases, the first one which contains likely pathogenic NM_002860: c.475C>T: p.R159X mutation of the ALDH18A1 and the second one has likely pathogenic NM_001160227.2: c.5454dupA: p.Glu1819Argfs Ter11 mutation of the SPG11 gene and also was identified by the whole-exome sequencing and confirmed by Sanger sequencing. Our aim with this study was to confirm that these two novel variants are direct causes of spastic paraplegia.