• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제17권4호
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• pp.257-262
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• 2014

Severe combined immunodeficiency (SCID) is a life-threatening syndrome of recurrent infections and gastro-intestinal alterations due to severe compromise of T cells and B cells. Clinically, most patients present symptoms before the age of 3 months and without intervention SCID usually results in severe infections and death by the age of 2 years. Its association with intestinal anomalies as multiple intestinal atresias (MIA) is rare and worsens the prognosis, resulting lethal. We describe the case of a four year-old boy with SCID-MIA. He presented at birth with meconium peritonitis, multiple ileal atresias and underwent several intestinal resections. A targeted Sanger sequencing revealed a homozygous 4-bp deletion ($c.313{\Delta}TATC$; p.Y105fs) in tetratricopeptide repeat domain 7A (TTC7A). He experienced surgical procedures including resection and stricturoplasty. Despite parenteral nutrition-associated liver disease, the patient is surviving at the time of writing the report. Precocious immune system assessment, scrutiny of TTC7A mutations and prompt surgical procedures are crucial in the management.

• Journal of Microbiology and Biotechnology
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• 제30권10호
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• pp.1583-1591
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• 2020

CRISPR/Cpf1 has emerged as a new CRISPR-based genome editing tool because, in comparison with CRIPSR/Cas9, it has a different T-rich PAM sequence to expand the target DNA sequence. Single-base editing in the microbial genome can be facilitated by oligonucleotide-directed mutagenesis (ODM) followed by negative selection with the CRISPR/Cpf1 system. However, single point mutations aided by Cpf1 negative selection have been rarely reported in Corynebacterium glutamicum. This study aimed to introduce an amber stop codon in crtEb encoding lycopene hydratase, through ODM and Cpf1-mediated negative selection; deficiency of this enzyme causes pink coloration due to lycopene accumulation in C. glutamicum. Consequently, on using double-, triple-, and quadruple-base-mutagenic oligonucleotides, 91.5-95.3% pink cells were obtained among the total live C. glutamicum cells. However, among the negatively selected live cells, 0.6% pink cells were obtained using single-base-mutagenic oligonucleotides, indicating that very few single-base mutations were introduced, possibly owing to mismatch tolerance. This led to the consideration of various target-mismatched crRNAs to prevent the death of single-base-edited cells. Consequently, we obtained 99.7% pink colonies after CRISPR/Cpf1-mediated negative selection using an appropriate single-mismatched crRNA. Furthermore, Sanger sequencing revealed that single-base mutations were successfully edited in the 99.7% of pink cells, while only two of nine among 0.6% of pink cells were correctly edited. The results indicate that the target-mismatched Cpf1 negative selection can assist in efficient and accurate single-base genome editing methods in C. glutamicum.

• Asian Pacific Journal of Cancer Prevention
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• 제17권3호
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• pp.1539-1546
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• 2016

Specific patterns of the hereditary breast and ovarian cancer (HBOC) syndrome are related to mutations in the BRCA1 gene. One hundred unrelated breast cancer patients were interviewed to obtain clinical symptoms and signs, pedigree and familial history of HBOC syndrome related cancer. Subsequently, data were calculated using the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) risk prediction model. Patients with high score of BOADICEA were offered genetic testing. Eleven patients with high score of BOADICEA, 2 patients with low score of BOADICEA, 2 patient's family members and 15 controls underwent BRCA1 genetic testing. Mutation screening using PCR-HRM was carried out in 22 exons (41 amplicons) of BRCA1 gene. Sanger sequencing was subjected in all samples with aberrant graph. This study identified 10 variants in the BRCA1 gene, consisting of 6 missense mutations (c.1480C>A, c.2612C>T, c.2566T>C, c.3113A>G, c.3548 A>G, c.4837 A>G), 3 synonymous mutations (c.2082 C>T, c.2311 T>C and c.4308T>C) and one intronic mutation (c.134+35 G>T). All variants tend to be polymorphisms and unclassified variants. However, no known pathogenic mutations were found.

• Asian Pacific Journal of Cancer Prevention
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• 제16권13호
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• pp.5211-5217
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• 2015

Background: We earlier used PCR-dHPLC for mutation analysis of BRCA1 and BRCA2. In this article we report application of targeted resequencing of 30 genes involved in hereditary cancers. Materials and Methods: A total of 91 patient samples were analysed using a panel of 30 genes in the Illumina HiScan SQ system. CLCBio was used for mapping reads to the reference sequences as well as for quality-based variant detection. All the deleterious mutations were then reconfirmed using Sanger sequencing. Kaplan Meier analysis was conducted to assess the effect of deleterious mutations on disease free and overall survival. Results: Seventy four of the 91 samples had been run earlier using the PCR-dHPLC and no deleterious mutations had been detected while 17 samples were tested for the first time. A total of 24 deleterious mutations were detected, 11 in BRCA1, 4 in BRCA2, 5 in p53, one each in RAD50, RAD52, ATM and TP53BP1. Some 19 deleterious mutations were seen in patients who had been tested earlier with PCR-dHPLC [19/74] and 5/17 in the samples tested for the first time, Together with our earlier detected 21 deleterious mutations in BRCA1 and BRCA2, we now had 45 mutations in 44 patients. BRCA1c.68_69delAG;p.Glu23ValfsX16 mutation was the most common, seen in 10/44 patients. Kaplan Meier survival analysis did not show any difference in disease free and overall survival in the patients with and without deleterious mutations. Conclusions: The NGS platform is more sensitive and cost effective in detecting mutations in genes involved in hereditary breast and/or ovarian cancers.

• Asian Pacific Journal of Cancer Prevention
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• 제15권24호
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• pp.10647-10652
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• 2015

Background: The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. Materials and Methods: We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. Results: It was found that the sensitivity of Sanger reached 0.5% with COLD-PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. Conclusions: It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

• 대한의생명과학회지
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• 제26권2호
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• pp.114-119
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• 2020

Of the various types of mice used for genome editing, conditional knock-out (cKO) mice serve as an important model for studying the function of genes. cKO mice can be produced using loxP knock-in (KI) mice in which loxP sequences (34 bp) are inserted on both sides of a specific region in the target gene. These mice can be used as KO mice that do not express a gene at a desired time or under a desired condition by cross-breeding with various Cre Tg mice. Genome editing has been recently made easy by the use of third-generation gene scissors, the CRISPR-Cas9 system. However, very few laboratories can produce mice for genome editing. Here we present a more efficient method for producing loxP KI mice. This method involves the use of an HDR vector as the target vector and ssODN as the donor DNA in order to induce homologous recombination for producing loxP KI mice. On injecting 20 ng/µL of ssODN, it was observed that the target exon was deleted or loxP was inserted on only one side. However, on injecting 10 ng/µL of the target HDR vector, the insertion of loxP was observed on both sides of the target region. In the first PCR, seven mice were identified to be loxP KI mice. The accuracy of their gene sequences was confirmed through Sanger sequencing. It is expected that the loxP KI mice produced in this study will serve as an important tool for identifying the function of the target gene.

• Journal of Interdisciplinary Genomics
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• 제3권1호
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• pp.21-24
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• 2021

Purpose: Activating mutations of the calcium-sensing receptor (CASR) are a rare genetic disorder, and result in autosomal dominant hypocalcemia with hypercalciuria (ADHH). ADHH exhibited varying degrees of hypocalcemia. In this study, we report the clinical and molecular characteristics of activating variants in CASR patients diagnosed in Korea. Methods: This study included three patients with activating variants of CASR confirmed by biochemical and molecular analysis of CASR. Clinical and biochemical findings were reviewed chart retrospectively. Mutation analysis of CASR was performed by Sanger sequencing. Results: Subject 1 showed severe symptoms from the neonatal period and had difficulty in controlling the medications that were administered. Subject 2 was identified as having a novel variant of CASR with hypocalcemia and a low parathyroid hormone that were found in the neonatal period. During a course without medication, hypocalcemia occurred suddenly around 2 years of age. Subject 3 was diagnosed with hypoparathyroidism with hypocalcemic seizures starting from the neonatal period. About 4 years without taking medication with any symptom. However, at 10 years old revisited by repetitive hypocalcemic seizure events. Subject 1 and 3, were heterozygous for c.2474A>T (p.Y825F), c.2395G>A (p.E799K) located in the transmembrane domain (TMD) of CASR. Subject 2 was heterozygous for c.403A>C (S430L) located in the extracellular domain (ECD) of CASR. Conclusion: We reported 3 patients who have activating CASR variant with different onset and severity of symptoms. In the future, further study is needed to determine how the protein level according to the location of the mutation of CASR affects the degree of symptoms.

• Journal of Genetic Medicine
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• 제15권2호
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• pp.64-71
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• 2018

Purpose: Overgrowth syndromes are conditions that involve generalized or localized areas of excess growth. In this study, the clinical, molecular, and genetic characteristics of Korean patients with overgrowth syndrome were analyzed. Materials and Methods: We recruited 13 patients who presented with overgrowth syndrome. All patients fulfilled inclusion criteria of overgrowth syndrome. Analysis of the clinical and molecular investigations of patients with overgrowth syndrome was performed retrospectively. Results: Among the 13 patients with overgrowth syndrome, 9 patients (69.2%) were found to have molecular and genetic causes. Among the seven patients with Sotos syndrome (SS), two had a 5q35microdeletion that was confirmed by fluorescent in situ hybridization. In two patients with SS, intragenic mutations including a novel mutation, c.5993T>A (p.M1998L), were found by Sanger sequencing. One patient had one copy deletion of NDS1 gene which was confirmed by multiplex ligation-dependent probe amplification. Among five patients with Beckwith-Wiedemann syndrome, three had aberrant imprinting control regions; 2 hypermethylation of the differentially methylated region of H19, 1 hypomethylation of the differentially methylated region of Kv. In one patient displaying overlapping clinical features of SS, a de novo heterozygous deletion in the chromosomal region 7q22.1-22.3 was found by single nucleotide polymorphism-based microarray. Conclusion: Considering high detection rate of molecular and genetic abnormalities in this study, rigorous investigations of overgrowth syndrome may be an important tool for the early diagnosis and genetic counseling. A detailed molecular analysis of the rearranged regions may supply the clues for the identification of genes involved in growth regulation.

• Laboraroty Animal Research
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• 제34권4호
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• pp.279-287
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• 2018

Placenta specific 8 (PLAC8, also known as ONZIN) is a multi-functional protein that is highly expressed in the intestine, lung, spleen, and innate immune cells, and is involved in various diseases, including cancers, obesity, and innate immune deficiency. Here, we generated a Plac8 knockout mouse using the CRISPR/Cas9 system. The Cas9 mRNA and two single guide RNAs targeting a region near the translation start codon at Plac8 exon 2 were microinjected into mouse zygotes. This successfully eliminated the conventional translation start site, as confirmed by Sanger sequencing and PCR genotyping analysis. Unlike the previous Plac8 deficient models displaying increased adipose tissue and body weights, our male Plac8 knockout mice showed rather lower body weight than sex-matched littermate controls, though the only difference between these two mouse models is genetic context. Differently from the previously constructed embryonic stem cell-derived Plac8 knockout mouse that contains a neomycin resistance cassette, this knockout mouse model is free from a negative selection marker or other external insertions, which will be useful in future studies aimed at elucidating the multi-functional and physiological roles of PLAC8 in various diseases, without interference from exogenous foreign DNA.

• Clinical and Experimental Reproductive Medicine
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• 제48권1호
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• pp.69-79
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• 2021

Objective: Poor ovarian response (POR) refers to a subnormal follicular response that leads to a decrease in the quality and quantity of the eggs retrieved after ovarian stimulation during assisted reproductive treatment (ART). The present study investigated the associations of multiple variants of the estrogen receptor 2 (ESR2) and follicle-stimulating hormone receptor (FSHR) genes with POR in infertile Jordanian women undergoing ART. Methods: Four polymorphisms, namely ESR2 rs1256049, ESR2 rs4986938, FSHR rs6165, and FSHR rs6166, were investigated in 60 infertile Jordanian women undergoing ART (the case group) and 60 age-matched fertile women (the control group), with a mean age of 33.60±6.34 years. Single-nucleotide polymorphisms (SNPs) were detected by restriction fragment length polymorphism and then validated using Sanger sequencing. Results: The p-value of the difference between the case and control groups regarding FSHR rs6166 was very close to 0.05 (p=0.054). However, no significant differences were observed between the two groups in terms of the other three SNPs, namely ESR2 rs1256049, ESR2 rs4986938, and FSHR rs6165 (p=0.561, p=0.433, and p=0.696, respectively). Conclusion: The association between FSHR rs6166 and POR was not statistically meaningful in the present study, but the near-significant result of this experiment suggests that statistical significance might be found in a future study with a larger number of patients.