Identification of novel susceptibility genes associated with bone density and osteoporosis in Korean women

Purpose@#Osteoporosis is a common calcium and metabolic skeletal disease which is characterized by decreased bone mass, microarchitectural deterioration of bone tissue and impaired bone strength, thereby leading to enhanced risk of bone fragility.In this study, we aimed to identify novel genes for susceptibility to osteoporosis and/or bone density. @*Materials and Methods@#To identify differentially expressed genes (DEGs) between control and osteoporosis-induced cells, annealing control primer-based differential display reverse-transcription polymerase chain reaction (RT-PCR) was carried out in pre-osteoblast MC3T3-E1 cells. Expression levels of the identified DEGs were evaluated by quantitative RT-PCR. Association studies for the quantitative bone density analysis and osteoporosis case-control analysis of single nucleotide polymorphism (SNPs) were performed in Korean women (3,570 subjects) from the Korean Association REsource (KARE) study cohort. @*Results@#Comparison analysis of expression levels of the identified DEGs by quantitative RT-PCR found seven genes, Anxa6, Col5a1, Col6a2, Eno1, Myof, Nfib, and Scara5, that showed significantly different expression between the dexamethasontreated and untreated MC3T3-E1 cells and between the ovariectomized osteoporosis-induced mice and sham mice. Association studies revealed that there was a significant association between the SNPs in the five genes, ANXA6, COL5A1, ENO1, MYOF, and SCARA5, and bone density and/or osteoporosis. @*Conclusion@#Using a whole-genome comparative expression analysis, gene expression evaluation analysis, and association analysis, we found five genes that were significantly associated with bone density and/or osteoporosis. Notably, the association P-values of the SNPs in the ANXA6 and COL5A1 genes were below the Bonferroni-corrected significance level.

A Korean case of neurofibromatosis type 1 with an exonic splicing enhancer site mutation

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease characterized by neurological, cutaneous, and ophthalmological manifestations. A 33-year-old woman with typical symptoms of NF1 visited Ajou University Hospital. Screening of the whole-messenger RNA region of NF1 at the complementary DNA level by polymerase chain reaction-direct sequencing confirmed the presence of an NF1 mutation at the genomic level. The mutation analysis revealed an in-frame skipping of exon 46 (c.6757_6858del) caused by a point mutation (c. 6792C>A) in exon 46. In this report, we have described the first Korean case of a proband with NF1 that carries an allele with an exon 46 deletion caused by an exonic splicing enhancer site mutation, leading to the skipping of the whole of exon 46 (c.6757_6858del).

Genetic and Clinical Characteristics of Korean Patients with Isolated Hypoparathyroidism: From the Korean Hypopara Registry Study

Isolated hypoparathyroidism (IH) shows heterogeneous phenotypes and can be caused by defects in a variety of genes. The goal of our study was to determine the clinical features and to analyze gene mutations in a large cohort of Korean patients with sporadic or familial IH. We recruited 23 patients. They showed a broad range of onset age and various values of biochemical data. Whole exome sequencing was performed on two affected cases and one unaffected individual in a family. All coding exons and exon-intron borders of GCMB, CASR, and prepro-PTH were sequenced using PCR-amplified DNA. In one family who underwent the whole exome sequencing analysis, approximately 300 single nucleotide changes emerged as candidates for genetic alteration. Among them, we identified a functional mutation in exon 2 of GCMB (C106R) in two affected cases. Besides, heterozygous gain-of-function mutations in the CASR gene were found in other subjects; D410E and P221L. We also found one single nucleotide polymorphism (SNP) in the prepro-PTH gene, five SNPs in the CASR gene, and four SNPs in the GCMB gene. The current study represents a variety of biochemical phenotypes in IH patients with the molecular genetic diagnosis of IH.

Association Analysis of Reactive Oxygen Species-Hypertension Genes Discovered by Literature Mining

Oxidative stress, which results in an excessive product of reactive oxygen species (ROS), is one of the fundamental mechanisms of the development of hypertension. In the vascular system, ROS have physical and pathophysiological roles in vascular remodeling and endothelial dysfunction. In this study, ROS-hypertension-related genes were collected by the biological literature-mining tools, such as SciMiner and gene2pubmed, in order to identify the genes that would cause hypertension through ROS. Further, single nucleotide polymorphisms (SNPs) located within these gene regions were examined statistically for their association with hypertension in 6,419 Korean individuals, and pathway enrichment analysis using the associated genes was performed. The 2,945 SNPs of 237 ROS-hypertension genes were analyzed, and 68 genes were significantly associated with hypertension (p < 0.05). The most significant SNP was rs2889611 within MAPK8 (p = 2.70 x 10(-5); odds ratio, 0.82; confidence interval, 0.75 to 0.90). This study demonstrates that a text mining approach combined with association analysis may be useful to identify the candidate genes that cause hypertension through ROS or oxidative stress.

Identification of Genetic Variations in CBL, SORBS1, CRK, and RHOQ,Key Modulators in the CAP/TC10 Pathway of Insulin Signal Transduction and Their Association with Type 2 Diabetes Mellitus in the Korean Population

Recent evidence has strongly suggested that the CAP/TC10 pathway is involved in the trafficking, docking,and fusion of vesicles containing the insulin- responsive glucose transporter Glut4 to the plasma membrane. However, little is known about how the genes employed in the CAP/TC10 pathway are associated with the development of type 2 diabetes mellitus. In this study, we sequenced 4 genes of the CAP/TC10 pathway [SORBS1, CBL, CRK, and RHOQ] in 24 individuals to identify genetic variations in these loci. A total of 48 sequence variants were identified, including 23 novel variations. To investigate the possible association with type 2 diabetes mellitus, 3 single nucleotide polymorphisms from SORBS1, 3 from CBL , and 4 from RHOQ were genotyped in 1122 Korean type 2 diabetic patients and 1138 nondiabetic controls. Using logistic regression analysis, 1 significant association between SNP rs1376405 in RHOQ and type 2 diabetes mellitus [OR = 8.714 (C.I. 1.714-44.29), p = 0.009] was found in the recessive model. Our data demonstrate a positive association of the RHOQ gene in the CAP/TC10 pathway with T2DM in the Korean population.

Haplotype analysis at R778L mutation of ATP7B gene in Korean patients with Wilson disease / 대한간학회지

No abstract available.

Proximal caries detection using digital subtraction radiography in the artificial caries activity model / 대한구강악안면방사선학회지

PURPOSE: The purpose of the experiment was to evaluating the diagnostic ability of dental caries detection using digital subtraction in the artificial caries activity model. MATERIALS AND METHODS: Digital radiographies of five teeth with 8 proximal surfaces were obtained by CCD sensor (Kodak RVG 6100 using a size #2). The digital radiographic images and subtraction images from artificial proximal caries were examined and interpreted. In this study, we proposed novel caries detection method which could diagnose the dental proximal caries from single digital radiographic image. RESULTS: In artificial caries activity model, the range of lesional depth was 572-1,374 micrometer and the range of lesional area was 36.95-138.52mm2. The lesional depth and the area were significantly increased with demineralization time (p<0.001). Furthermore, the proximal caries detection using digital subtraction radiography showed high detection rate compared to the proximal caries examination using simple digital radiograph. CONCLUSION: The results demonstrated that the digital subtraction radiography from single radiographic image of artificial caries was highly efficient in the detection of dental caries compared to the data from simple digital radiograph.

The Effect of Extracellular Glutamate Release on Repetitive Transient Ischemic Injury in Global Ischemia Model

During operations, neurosurgeons usually perform multiple temporary occlusions of parental artery, possibly resulting in the neuronal damage. It is generally thought that neuronal damage by cerebral ischemia is associated with extracellular concentrations of the excitatory amino acids. In this study, we measured the dynamics of extracellular glutamate release in 11 vessel occlusion (VO) model to compare between single occlusion and repeated transient occlusions within short interval. Changes in cerebral blood flow were monitored by laser-Doppler flowmetry simultaneously with cortical glutamate level measured by amperometric biosensor. From real time monitoring of glutamate release in 11 VO model, the change of extracellular glutamate level in repeated transient occlusion group was smaller than that of single occlusion group, and the onset time of glutamate release in the second ischemic episode of repeated occlusion group was delayed compared to the first ischemic episode which was similar to that of single 10 min ischemic episode. These results suggested that repeated transient occlusion induces less glutamate release from neuronal cell than single occlusion, and the delayed onset time of glutamate release is attributed to endogeneous protective mechanism of ischemic tolerance.

Protein Tyrosine Phosphatase N1 Gene Variants Associated with Type 2 Diabetes Mellitus and Its Related Phenotypes in the Korean Population

Protein phosphorylation at tyrosine residues is a key regulatory event that modulates insulin signal transduction. We studied the PTPN1 gene with regard to susceptibility to Korean type 2 diabetes mellitus (T2DM) and its related quantitative traits. A total of seven SNPs [g.36171G>A (rs941798), g.58166G>A (rs3787343), g.58208A>G (rs2909270), g.64840C>T (rs754118), g.69560C>G (rs6020612), g.69866G>A (rs718050), and g.69934T>G (rs3787343)] were selected based on frequency (>0.05), linkage disequilibrium (LD) status, and haplotype tagging status. We studied the seven SNPs in 483 unrelated patients with type 2 diabetes (age: 64+/-2.8 years, onset age: 56+/-8.1 years; 206 men, 277 women) and 1138 nondiabetic control subjects (age: 64+/-2.9; 516 men, 622 women). The SNP rs941798 had protective effects against T2DM with an odds ratio of 0.726 (C.I. 0.541~0.975) and p-value=0.034, but none of the remaining six SNPs was associated with T2DM. Also, rs941798 was associated with blood pressure, HDL cholesterol, insulin sensitivity. rs941798 also has been associated with T2DM in previous reports of Caucasian-American and Hispanic-American populations. This is the first report that shows an association between PTPN1 and T2DM in the Korean as well as Asian population.

Association between Prostaglandin-endoperoxide Synthase 2 (PTGS2) Polymorphisms and Blood Pressure in Korean Population

Blood pressure refers to the force exerted by circulating blood on the walls of blood vessels, and chronical elevation of blood pressure is known as hypertension. Although hypertension is affected by genetic and environmental factors, the genetic background of hypertension is not fully understood. One of the candidate genetic factors, Prostaglandin-endoperoxide synthase 2 (PTGS2), is a membrane-bound enzyme, catalyzing the conversion of arachidonic acid to prostaglandin, and recently SNPs of PTGS2 gene was associated with hypertension in Japanese population. Therefore the association of PTGS2 polymorphisms was investigated with blood pressure in healthy Korean subjects, 470 unrelated individuals randomly selected from Ansung and Ansan cohorts. The 25 SNPs of PTGS2 gene were identified by the sequencing analysis of 24 Korean samples. Among identified polymorphisms, three SNPs (rs689466, -1329A>G; rs5275, +6365T>C; rs4648308, +8806G> A) were selected for further association analysis, and rs689466 located in promoter region was associated with blood pressure as well as triglyceride level in the blood. By in silico analysis, rs689466 locates in v-Myb transcription factor binding site, and the v-Myb site disappears when the SNP is changed from A to G nucleotide. Individuals with A/G and G/G genotype in rs689466 have higher blood pressure than those with A/A genotype, and the regression p-value is 0.008 for systolic and 0.004 for diastolic blood pressure. In summary, the PTGS2 polymorphism (rs689466) is associated with blood pressure in Asian populations based on this and Japanese studies, shedding light on it as a genetic risk marker of hypertension.

Indirect Anthropometry on Cast Model of Cleft Lip Nose: Comparison with Direct Anthropometry

PURPOSE: Anthropometry can be divided into two methods, direct anthropometry and indirect anthropometry. The most ideal and accurate method is a direct anthropometry. However, it is difficult to measure in the case of children because of poor cooperation, and it lacks re-productivity. Cast model has advantages of three dimensional featuring, inexpensive and easy fabrication. This study is conducted to find out an accuracy of indirect anthropometry on cast model by comparing it with direct anthropometry. METHODS: Total 48 cleft lip nasal deformity patients (unilateral, 40; bilateral, 8) were included in this study. Cast models were made before surgery under general anesthesia with alginate impression material and model plaster. Eleven linear measurements among 7 landmarks were taken as direct anthropometry before surgery with Castroviejo spreading caliper. At the same time, indirect anthropometry on cast model was done at the same linear distances as well. RESULTS: Of the total 11 linear measurements, both ala lengths, both columella lengths, nose width, projective distance between facial insertion points of the ala, projective distance between the alar base points, right nostril floor width, and columella width were statistically correlated between indirect anthropometry on cast model and direct anthropometry. However, the nasal tip protrusion and the left nostril floor width were not statistically correlated. CONCLUSION: Accuracy of indirect anthropometry on cast model can be influenced by cast model fabrication techniques and correct identification of landmarks. Nasal tip protrusion could be reduced by compression of the nasal tip in the process of cast model fabrication and nostril floor width can be varied by muscle relaxation of anesthetics and incorrect identification of subalare in cleft lip nasal deformity. If sufficient care is taken to make cast model and to define landmarks exactly, indirect anthropometry on cast model can be a reliable method as direct anthropometry.

Indirect Anthropometry on Cast Model of Cleft Lip Nose: Comparison with Direct Anthropometry

PURPOSE: Anthropometry can be divided into two methods, direct anthropometry and indirect anthropometry. The most ideal and accurate method is a direct anthropometry. However, it is difficult to measure in the case of children because of poor cooperation, and it lacks re-productivity. Cast model has advantages of three dimensional featuring, inexpensive and easy fabrication. This study is conducted to find out an accuracy of indirect anthropometry on cast model by comparing it with direct anthropometry. METHODS: Total 48 cleft lip nasal deformity patients (unilateral, 40; bilateral, 8) were included in this study. Cast models were made before surgery under general anesthesia with alginate impression material and model plaster. Eleven linear measurements among 7 landmarks were taken as direct anthropometry before surgery with Castroviejo spreading caliper. At the same time, indirect anthropometry on cast model was done at the same linear distances as well. RESULTS: Of the total 11 linear measurements, both ala lengths, both columella lengths, nose width, projective distance between facial insertion points of the ala, projective distance between the alar base points, right nostril floor width, and columella width were statistically correlated between indirect anthropometry on cast model and direct anthropometry. However, the nasal tip protrusion and the left nostril floor width were not statistically correlated. CONCLUSION: Accuracy of indirect anthropometry on cast model can be influenced by cast model fabrication techniques and correct identification of landmarks. Nasal tip protrusion could be reduced by compression of the nasal tip in the process of cast model fabrication and nostril floor width can be varied by muscle relaxation of anesthetics and incorrect identification of subalare in cleft lip nasal deformity. If sufficient care is taken to make cast model and to define landmarks exactly, indirect anthropometry on cast model can be a reliable method as direct anthropometry.

Quantitative Analysis of SMN1 Gene and Estimation of SMN1 Deletion Carrier Frequency in Korean Population based on Real-Time PCR

Spinal muscular atrophy (SMA) is an autosomal recessive disorder, caused by homozygous absence of the survival motor neuron gene (SMN1) in approximately 94% of patients. Since most carriers have only one SMN1 gene copy, several SMN1 quantitative analyses have been used for the SMA carrier detection. We developed a reliable quantitative real-time PCR with SYBR Green I dye and studied 13 patients with SMA and their 24 parents, as well as 326 healthy normal individuals. The copy number of the SMN1 gene was determined by the comparative threshold cycle (Ct) method and albumin was used as a reference gene. The homozygous SMN1 deletion ratio of patients was 0.00 and the hemizygous SMN1 deletion ratio of parents ranged from 0.39 to 0.59. The delta delta Ct ratios of 7 persons among 326 normal individuals were within the carrier range, 0.41-0.57. According to these data, we estimated the carrier and disease prevalence of SMA at 1/47 and 1/8,496 in Korean population, respectively. These data indicated that there would be no much difference in disease prevalence of SMA compared with western countries. Since the prevalence of SMA is higher than other autosomal recessive disorders, the carrier detection method using real-time PCR could be a useful tool for genetic counseling.

Rapid Detection of Duplication/Deletion of the PMP22 Gene in Patients with Charcot-Marie-Tooth Disease Type 1A and Hereditary Neuropathy with Liability to Pressure Palsy by Real-time Quantitative PCR using SYBR Green I Dye

Mutations and altered gene dosage of the peripheral myelin protein (PMP22) gene in chromosome 17p11.2-12 are the main causes for hereditary neuropathies, accounting for approximately 70% of all cases. Patients with duplication of the PMP22 develop Charcot-Marie-Tooth disease type 1A (CMT1A) and deletion of one PMP22 allele leads to hereditary neuropathy with liability to pressure palsy (HNPP). Twenty patients with CMT1A, 17 patients with HNPP, and 18 normal family members and 28 normal controls were studied by real-time quantitative PCR using SYBR Green I on the ABI 7700 Sequence Detection System. The copy number of the PMP22 gene was determined by the comparative threshold cycle method and the albumin was used as a reference gene. The PMP22 duplication ratio ranged from 1.45 to 2.06 and the PMP22 deletion ratio ranged from 0.42 to 0.64. The PMP22 ratio in normal controls, including normal family members, ranged from 0.85 to 1.26. No overlap was found between patients with CMT1A or patients with HNPP and normal controls. This method is fast, highly sensitive, specific, and reproducible in detecting PMP22 duplication and deletion in CMT1A and HNPP patients, respectively.

A novel PCR primers HPU185 and HPL826 based on 16S rRNA gene for detection of Helicobacter pylori

The PCR primer set JW21-JW22 of Weiss et al. (19), which was reported to amplify a 139-bp fragment of the 16S rRNA gene of Helicobacter pylori, has been recently used for the detection of H. pylori in clinical specimens. However, when we applied JW21-JW22 PCR to other members of the genus Helicobacter and unrelated microorganisms, all of these bacteria produced a 139-bp PCR product. Therefore, we designed a novel primer set, HPU185-HPL826, which produced a 642-bp amplicon of the 16S rRNA gene of H. pylori. Then we further examined the specificity of the novel PCR assay using Southern blot hybridization with an internal probe, HPP225. The PCR assay described in this study was shown to be highly sensitive and specific only to the H. pylori 16S rRNA gene sequences.