Jung-Young Park

Estimation of Wilson's Disease Incidence and Carrier Frequency in the Korean Population by Screening ATP7B Major Mutations in Newborn Filter Papers Using the SYBR Green Intercalator Method Based on the Amplification Refractory Mutation System

Wilsons disease (WD), an autosomal recessive disorder of copper transport, is one of the most common inherited metabolic disorders in Korea. Despite its frequency, the incidence and carrier frequency of WD has not yet been estimated in the Korean population. We therefore screened for four major missense mutations (p.Arg778Leu, p.Ala874Val, p.Leu1083Phe, and p.Asn1270Ser) of the ATP7B gene in 476 newborn filter papers by real-time multiplex PCR and melting curve analysis using the SYBR Green intercalator method based on the amplification refractory mutation system test. Newborn filter papers with abnormal melting curves were subjected to subsequent sequence analysis. Three mutated alleles, one p.Arg778Leu and two p.Ala874Val, were detected among the 476 newborn filter papers (952 alleles). The carrier frequency and incidence of WD in the Korean population were determined as 1 in 88.2 and 30,778, respectively, by reversely calculating based on the Hardy-Weinberg law.

Effects of a chemical chaperone on genetic mutations in alpha-galactosidase A in Korean patients with Fabry disease.

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism that results from mutations in the gene encoding the α-galactosidase A (GLA) enzyme. We have identified 15 distinct mutations in the GLA gene in 13 unrelated patients with classic Fabry disease and 2 unrelated patients with atypical Fabry disease. Two of the identified mutations were novel (i.e., the D231G missense mutation and the L268delfsX1 deletion mutation). This study evaluated the effects of the chemical chaperones 1-deoxygalactonojirimycin (DGJ) on the function of GLA in vitro, in cells containing missense mutations in the GLA gene. Nine missense and a nonsense mutations, including one novel mutation were cloned into mammalian expression vectors. After transient expression in COS-7 cells, GLA enzyme activity and protein expression were analyzed using fluorescence spectrophotometry and Western blot analysis, respectively. DGJ enhanced GLA enzyme activity in the M42V, I91T, R112C and F113L mutants. Interestingly, the I91T and F113L mutations are associated with the atypical form of Fabry disease. However, DGJ treatment did not have any significant effect on the GLA enzyme activity and protein expression of other mutants, including C142W, D231G, D266N, and S297F. Of note, GLA enzyme activity was not detected in the novel mutant (i.e., D231G), although protein expression was similar to the wild type. In the absence of DGJ, the E66Q mutant had wild-type levels of GLA protein expression and approximately 40% GLA activity, indicating that E66Q is either a mild mutation or a functional single nucleotide polymorphism (SNP). Thus, the results of this study suggest that the chemical chaperone DGJ enhances GLA enzyme activity and protein expression in milder mutations associated with the atypical form of Fabry disease.

Identification of Novel Mutations of the DAX-1 Gene in Patients with X-Linked Adrenal Hypoplasia Congenita

Objective: X-linked adrenal hypoplasia congenita (AHC) is a condition clinically featuring adrenal insufficiency and hypogonadotropic hypogonadism caused by mutations of DAX-1. This study was undertaken to characterize the molecular defects of DAX-1 in 3 unrelated Korean patients with AHC. Patients and Methods: Patient 1 is a 6-year-old boy who presented with a salt-losing adrenal crisis in the neonatal period. Patient 2 is a 3-year-old boy who manifested aspiration pneumonia and adrenal insufficiency at the age of 1 month. Patient 3 is a 7-year-old boy who developed an adrenal crisis at the age of 3 days. In each of these patients, DAX-1 was analyzed by direct DNA sequencing after polymerase chain reaction amplification of the entire coding region. Results: Direct sequencing of DAX-1 revealed two novel mutations, 1156_1157delCT in patient 1 and another novel nonsense mutation W105X in patient 2. Patient 3 had complete deletion of DAX-1. In patient 3, serum transaminases and creatine kinase levels were elevated while the glycerol kinase activity of leukocytes was decreased. Markedly elevated glycerol excretion was detected by urine organic acid analysis. Patient 3 was diagnosed as Xp21 contiguous gene syndrome associated with deletions of the entire IL1RAPL, GK genes and the C-terminal region of DMD gene. Conclusions: Two novel mutations of DAX-1 were detected in 2 unrelated patients with AHC, and complete deletion of DAX-1 in a patient with Xp21 contiguous gene syndrome who also presented with glycerol kinase deficiency, Duchenne muscular dystrophy, and AHC.

Effects of head elevation on intraocular pressure in healthy subjects: raising bed head vs using multiple pillows

PurposeTo evaluate the effects of different methods of head elevation on intraocular pressure (IOP) in healthy young subjects.MethodsTwenty-four healthy young Korean subjects were included in this prospective observational study. The IOP measurements were taken with the subjects in the sitting position and in the supine positions with the head flat and 30° up using two different methods: (1) raising the bed head and (2) using multiple pillows. IOP was measured using Tonopen AVIA in both eyes 10 min after assuming each position in a randomized sequence. The Wilcoxon signed-rank test was used to compare the IOP by changing the methods of head elevation.ResultsMean IOP of both eyes when sitting was lower than that measured in the supine position with head flat (P=0.001). Compared with that measured in the supine position with head flat, the mean IOP was lower when measured in the supine position with the head kept 30 ° up by bed head elevation (P=0.001), whereas the mean IOP was not significantly different when measured in the supine position with the head elevated using multiple pillows (right eye, P=0.061; left eye, P=0.089).ConclusionIn normal subjects, IOP was lower when measured in the supine position with the head kept up by the bed head elevation compared with that measured when lying flat. However, such head-up position-induced IOP reduction was not found when the head was kept up using multiple pillows. These findings suggest that elevating the head using multiple pillows may not help to reduce IOP in the supine posture.

Proteomic analysis of sera of asymptomatic, early-stage patients with Wilson's disease

Wilsons disease (WD) is characterized by excessive accumulation of intracellular copper in liver and extrahepatic tissues, leading to significant oxidative stress and tissue damage. To date, several diagnostic biomarkers for WD such as serum ceruloplasmin, serum or urine copper levels and copper content in liver have been identified. However, these biomarkers may not be convincing for the diagnosis in some WD patients. To identify additional novel diagnostic biomarkers, we compared the serum protein profiles of asymptomatic childhood WD patients (n=20), without neurologic manifestation or liver cirrhosis, with normal controls (n=13). Fourteen spots, five up‐regulated and nine down‐regulated (>2‐fold), were differentially expressed in WD patients in comparison to normal control on 2‐DE. Among them, three spots were down‐regulated in both male and female WD. MS/MS analysis revealed that the three spots were complement component C3, complement factor B and alpha‐2 macroglobulin. By comparative proteome analysis, complement component C3, complement factor B and alpha‐2 macroglobulin, which are related to oxidative stress and inflammation, turned out to be good candidates for novel diagnostic biomarkers for early stages of WD.

Simple and easy DNA mapping method using peptide nucleic acid (PNA) tagging.

In this paper, we introduce a simple and easy DNA mapping method using peptide nucleic acid (PNA) on glass cover-slips based on optical detection. PNA and glass cover-slips were used as easy tagging and stretching method, separately. The PNA can be invaded lambda (A) DNA at the sequences we wished to tag without any additional materials. Alexa-488 fluorophore-conjugated PNA was designed to invade at four sites (sequence: AAGAAGAA) of lambda DNA with robust and exact binding. Fluorophore tagged lambda DNA was stretched in electrical layer coated glass cover-slips. The lambda DNA was stretched as 9.2 um in length. It is approximately 60% of theoretical length of lambda DNA and sufficient length to promise optical resolution. The sites of desired sequence tagged by PNA were well detected in stretched lambda DNA.

Single quantum dot (QD) manipulation on nanowire using dielectrophoretic (DEP) force

Au nanowires of 100 nm, 200nm and 400 nm widths with micro scale Au electrode were fabricated as electrodes to apply high electric field gradient for strong DEP force within the nanometer range. Au nanowires were fabricated on a silicon dioxide (SiO2) using lift-off process after e-beam lithography and e-beam evaporation. E-beam resister (ER) was patterned and a 50 nm thick Au layer. Photo resister (PR) was patterned to make Au microelectrode and did lift-off process. The Au nanowires with microelectrode were covered with SiO2 layer deposited with PECVD resulting in 1 um thick. Opened end of gold nanowires, the target surface for QD immobilization, were formed using etching processes. Single QD immobilization on the nanowire end-facet was accomplished through positive DEP force. Sine wave of 8 Vpp intensity and 3 MHz frequency was applied and it induced electric field of 108 V/m intensity and electric field gradient around Au nanowire to make strong positive DEP. Optical analysis confirmed the attachment of single QD on the nanowire. A single 25 nm diameter QD was manipulated on 100 nm, 200 nm and 400 nm width nanowires when 8 Vpp, 3 MHz sine wave was applied.