• Journal of Genetic Medicine
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• 제15권2호
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• pp.87-91
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• 2018

X-linked dominant mutations in lysosome-associated membrane protein 2 (LAMP2) gene have been shown to be the cause of Danon disease, which is a rare disease associated with clinical triad of cardiomyopathy, skeletal myopathy, and mental retardation. Cardiac involvement is a common manifestation and is the leading cause of death in Danon disease. We report a case of a 24-month-old boy with hemizygous LAMP2 mutation who presented with failure to thrive and early-onset hypertrophic cardiomyopathy. We applied targeted exome sequencing and found a novel hemizygous c.692del variant in exon 5 of the LAMP2 gene, resulting a frameshift mutation p.Thr231Ilefs*11. Our study indicates that target next-generation sequencing can be used as a fast and highly sensitive screening method for inherited cardiomyopathy.

• Genomics & Informatics
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• 제20권2호
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• pp.24.1-24.8
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• 2022

Hypomyelinating leukodystrophy type 2 (HLD2), is an inherited genetic disease of the central nervous system caused by recessive mutations in the gap junction protein gamma 2 (GJC2/GJA12). HLD2 is characterized by nystagmus, developmental delay, motor impairments, ataxia, severe speech problem, and hypomyelination in the brain. The GJC2 sequence encodes connexin 47 protein (Cx47). Connexins are a group of membrane proteins that oligomerize to construct gap junctions protein. In the present study, a novel missense mutation gene c.760G>A (p.Val254Met) was identified in a patient with HLD2 by performing whole exome sequencing. Following the discovery of the new mutation in the proband, we used Sanger sequencing to analyze his affected sibling and parents. Sanger sequencing verified homozygosity of the mutation in the proband and his affected sibling. The autosomal recessive inheritance pattern was confirmed since Sanger sequencing revealed both healthy parents were heterozygous for the mutation. PolyPhen2, SIFT, PROVEAN, and CADD were used to evaluate the function prediction scores of detected mutations. Cx47 is essential for oligodendrocyte function, including adequate myelination and myelin maintenance in humans. Novel mutation p.Val254Met is located in the second extracellular domain of Cx47, both extracellular loops are highly conserved and probably induce intramolecular disulfide interactions. This novel mutation in the Cx47 gene causes oligodendrocyte dysfunction and HLD2 disorder.

• Journal of Genetic Medicine
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• 제20권1호
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• pp.1-5
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• 2023

Until now, rare disease studies have mainly been carried out by detecting simple variants such as single nucleotide substitutions and short insertions and deletions in protein-coding regions of disease-associated gene panels using diagnostic next-generation sequencing in association with patient phenotypes. However, several recent studies reported that the detection rate hardly exceeds 50% even when whole-exome sequencing is applied. Therefore, the necessity of introducing whole-genome sequencing is emerging to discover more diverse genomic variants and examine their association with rare diseases. When no diagnosis is provided by whole-genome sequencing, additional omics techniques such as RNA-seq also can be considered to further interrogate causal variants. This paper will introduce a description of these multi-omics techniques and their applications in rare disease studies.

• 생명과학회지
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• 제23권1호
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• pp.8-14
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• 2013

고추 탄저병균의 포자 발아 단계에서 발현되는 유전자를 파악하기 위해 포자 발아단계cDNA library를 제작하고, 임의로 선택된 cDNA clone들에 대한 EST sequencing을 실시하였다. 총 983개 EST를 확보하여 contig assembly를 실시한 결과, 197개 contigs와 267개 singletons으로 조합되어, 최종적으로 464개의 유전자를 동정하였다. 464개 유전자 서열에서 유추한 아미노산 서열을 이용한 상동유전자 검색을 통해 절반의 유전자가 GenBank에 기존 등록된 유전자와 유의성 있는 유사성을 보였다. 가장 높은 빈도로 발현된 유전자는 elongation factor, histone protein, ATP synthease, 14-3-3 protein, clock controlled protein을 암호화하는 유전자들이었다. 그리고 고추 탄저병균의 세포 발달과정에 관여 하는것으로 추정되는 GTP-binding protein, MAP kinase, transaldolase, ABC transporter 유전자들도 검출되었다. 또한 고추탄저병균의 병원성에 영향을 미치는 것으로 파악되는 ATP citrate lyase, CAP20, manganese-superoxide dismutase 유전자들도 검출되어, EST sequencing 을 통한 세포 발달 단계 발현 유전자 탐색이 효과적임을 알 수 있었다.

• Genes and Genomics
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• 제40권12호
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• pp.1279-1285
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• 2018

Interdigitating dendritic cell sarcoma (IDCS) is an aggressive neoplasm and is an extremely rare disease, with a challenging diagnosis. Etiology of IDCS is also unknown and most studies with only case reports. In our case, immunohistochemistry showed that the tumor cells were positive for S100, CD45, and CD68, but negative for CD1a and CD21. This study aimed to investigate the causative factors of IDCS by sequencing the protein-coding regions of IDCS. We performed whole-exome sequencing with genomic DNA from blood and sarcoma tissue of the IDCS patient using the Illumina Hiseq 2500 platform. After that, we conducted Sanger sequencing for validation of sarcoma-specific variants and gene ontology analysis using DAVID bioinformatics resources. Through comparing sequencing data of sarcoma with normal blood, we obtained 15 nonsynonymous single nucleotide polymorphisms (SNPs) as sarcoma-specific variants. Although the 15 SNPs were not validated by Sanger sequencing due to tumor heterogeneity and low sensitivity of Sanger sequencing, we examined the function of the genes in which each SNP is located. Based on previous studies and gene ontology database, we found that POLQ encoding DNA polymerase theta enzyme and FNIP1 encoding tumor suppressor folliculin-interacting protein might have contributed to the IDCS. Our study provides potential causative genetic factors of IDCS and plays a role in advancing the understanding of IDCS pathogenesis.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제30권3호
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• pp.186-192
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• 2004

Purpose: Human tooth proteins are highly heterogeneous, comprising diverse proteins derived from a number of genes. The attempts to identify protein for activity of tooth matrix proteins have been defied by several factors. First, the amount of proteins within teeth is very small relative to many extracellular matrix proteins of other tissues. Second, the bioassay system is tedious and needed for long time. Therefore we tried to find easy techniques, which increase the product rate, and an assay of small proteins, with which amino acid sequence is possible without additional procedures. Materials and Methods: Total protein were extracted from 300 g enamel removed teeth and 600 g teeth with 4 mol/L guanidine HCl and purified by gel chromatography. Aliquot of proteins was implanted into muscle pouches in Sprague-Dawley rats for bioassay. By SDS-PAGE and membrane blotting, molecular weight of each protein was estimated and a partial amino acid sequence was obtained. Each fraction blotted on the membrane was cut out and inserted in rat ectopic model. Results: In dissociative method, total tooth proteins were obtained 1mg/ml from enamel removed teeth and 3.5 mg/ml from teeth. In SDS-PAGE, four clear bands at the sites corresponding to 66, 40, 20 and 18 kD. Especially The 66 kD band was clearly exhibited. Amino acid sequencing from tooth could be possible using PVDF membrane blotting technique. In amino acid sequencing, 66 kD protein was identified as albumin. Conclusion: Compared with conventional method for extraction of teeth protein and bioassay of proteins, the methods in this study were easy, time-saving and more productive technique. The matured tooth proteins omitting additional procedure of mechanical removal of enamel were simply analyzed using blotted PVDF membrane. This method seems to make a contribution as a technique for bioassay and amino acid sequencing of protein.

• Journal of Microbiology
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• 제37권4호
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• pp.234-242
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• 1999

To obtain the recombinant circumsporozoite (CS) protein for the diagnosis of patients and seroepidemiology of Plasmodium vivax malaria which have been prevalent in northern part of Kyonggido, the CS protein gene was amplified by the polymerase chain reaction (PCR) from genomic DNA of the Korean vivax malaria patient. The gene consists of 1,123 nucleotides except signal peptide sequences and had an uninterrupted reading frame encoding a protein of 374 amino acids with a central region of 20 tandem repeats of the nonapeptide. The CS protein gene was expressed in Escherichia coli and purified, the molecular weight of recombinant CS protein was about 44 kDa (monomer) under denaturing purification and about 65 kDa (dimer) under native purification by SDS-PAGE. The purified recombinant CS protein which has antigenicity to malaria patients in Western blot analysis and Enzyme-linked immunosorbent assay, reacted only with the serum of P. vivax (PV210) infected malaria patients with no cross reaction to the P. falciparum malaria patient. The recombinant CS protein purified in this study will serve as a useful antigen to support the diagnosis of malaria patients and seroepidemiology.

• Animal Bioscience
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• 제34권9호
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• pp.1439-1450
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• 2021

Objective: With the rapid development of proteomics sequencing and RNA sequencing technology, multi-omics analysis has become a current research hotspot. Our previous study indicated that Xinjiang brown cattle have better meat quality than Kazakh cattle. In this study, Xinjiang brown cattle and Kazakh cattle were used as the research objects. Methods: Proteome sequencing and RNA sequencing technology were used to analyze the proteome and transcriptome of the longissimus dorsi muscle of the two breeds of adult steers (n = 3). Results: In this project, 22,677 transcripts and 1,874 proteins were identified through quantitative analysis of the transcriptome and proteome. By comparing the identified transcriptome and proteome, we found that 1,737 genes were identified at both the transcriptome and proteome levels. The results of the study revealed 12 differentially expressed genes and proteins: troponin I1, crystallin alpha B, cysteine, and glycine rich protein 3, phosphotriesterase-related, myosin-binding protein H, glutathione s-transferase mu 3, myosin light chain 3, nidogen 2, dihydropyrimidinase like 2, glutamate-oxaloacetic transaminase 1, receptor accessory protein 5, and aspartoacylase. We performed functional enrichment of these differentially expressed genes and proteins. The Kyoto encyclopedia of genes and genomes results showed that these differentially expressed genes and proteins are enriched in the fatty acid degradation and histidine metabolism signaling pathways. We performed parallel reaction monitoring (PRM) verification of the differentially expressed proteins, and the PRM results were consistent with the sequencing results. Conclusion: Our study provided and identified the differentially expressed genes and proteins. In addition, identifying functional genes and proteins with important breeding value will provide genetic resources and technical support for the breeding and industrialization of new genetically modified beef cattle breeds.

• 한국발생생물학회지:발생과생식
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• 제27권1호
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• pp.9-24
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• 2023

The advancement in high-throughput sequencing (HTS) technology has revolutionized the field of biology, including genomics, epigenomics, transcriptomics, and metagenomics. This technology has become a crucial tool in many areas of research, allowing scientists to generate vast amounts of genetic data at a much faster pace than traditional methods. With this increased speed and scale of data generation, researchers can now address critical questions and gain new insights into the inner workings of living organisms, as well as the underlying causes of various diseases. Although the first HTS technology have been introduced about two decades ago, it can still be challenging for those new to the field to understand and use effectively. This review aims to provide a comprehensive overview of commonly used HTS technologies these days and their applications in terms of genome sequencing, transcriptome, DNA methylation, DNA-protein interaction, chromatin accessibility, three-dimensional genome organization, and microbiome.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제22권6호
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• pp.581-587
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• 2019

Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome is a rare autosomal recessive multisystemic disease that is associated with the liver, kidney, skin, and central nervous and musculoskeletal systems. ARC occurs as a result of mutations in the VPS33B (Vacuolar protein sorting 33 homolog B) or VIPAR (VPS33B interacting protein, apical-basolateral polarity regulator) genes. A female infant presented with neonatal cholestasis with a severe clinical outcome. She was diagnosed with ARC syndrome using targeted exome sequencing (TES). Exome sequencing revealed compound heterozygous mutations, c.707A>T and c.239+5G>A, in VPS33B, where c.707A>T was a novel variant; the resultant functional protein defects were predicted via in silico analysis. c.239+5G>A, a pathogenic mutation that affects splicing, is found in less than 0.1% of the general population. Invasive techniques, such as liver biopsies, did not contribute to a differential diagnosis of ARC syndrome; thus, early TES together with clinical presentations constituted an apparently accurate diagnostic procedure.