Yuh-Jyh Jong

ETFDH mutations, CoQ10 levels, and respiratory chain activities in patients with riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a metabolic disorder due to dysfunction of electron transfer flavoprotein (ETF) or ETF-ubiquinone oxidoreductase (ETF-QO). Mutations in ETFDH, encoding ETF-QO have been associated with both riboflavin-responsive and non-responsive MADD as well as a myopathic form of CoQ(10) deficiency, although pathomechanisms responsible for these different phenotypes are not well-defined. We performed mutation analysis in four Taiwanese MADD patients. Three novel ETFDH mutations were identified in four patients and all harbored the p.A84T mutation. Muscle CoQ(10) levels and respiratory chain activities measured in two patients were normal. Three patients improved on riboflavin together with carnitine. Our results show that not all MADD patients have CoQ(10) deficiency. Based upon our data, riboflavin and carnitine may be the first-line treatment for MADD.

Arecoline;a major alkaloid of areca nut;inhibits p53;represses DNA repair;and triggers DNA damage response in human epithelial cells.

The International Agency for Research on Cancer declared that areca nut was carcinogenic to human. Areca nut is the main component of betel quid (BQ), which is commonly consumed in Asia. Epidemiological studies have shown that BQ chewing is a predominant risk factor for oral and pharyngeal cancers. It has been known that areca nut is genotoxic to human epithelial cells. However, the molecular and cellular mechanisms underlying areca nut-associated genotoxicity are not fully understood. Here we showed that arecoline, a major alkaloid of areca nut, might contribute to oral carcinogenesis through inhibiting p53 and DNA repair. We found, on the biological aspect, that arecoline could induce gamma-H2AX phosphorylation, a sensitive DNA damage marker, in KB, HEp-2, and 293 cells, suggesting that DNA damages were elicited by arecoline. This phenomenon was supported by the observations of arecoline-induced hyperphosphorylation of ATM, Nbs1, Chk1/2, p53, and Cdc25C, as well as G2/M cell cycle arrest, indicating that a cellular DNA damage response was activated. To explore the possible mechanism accounting for arecoline-elicited DNA damages, we found that arecoline could inhibit p53 by its expression and transactivation function. As a result, the expression of p53-regulated p21(WAF1) and the p53-activated DNA repair were repressed by arecoline. Finally, we showed that p53 mRNA transcripts were frequently down-regulated in BQ-associated oral cancer, suggesting that arecoline-mediated p53 inhibition might play a role in BQ-associated tumorigenesis.

Respiratory Adenoviral Infections in Children: A Study of Hospitalized Cases in Southern Taiwan in 2001–2002

Adenoviruses account for 5-10 per cent of respiratory illnesses in children. To analyse the clinical features and the temporal frequency in acute adenoviral respiratory infections in hospitalized children in southern Taiwan, a total of 4333 children who were admitted to the Department of Pediatrics, Kaohsiung Municipal Hsiaokang (KMHK) Hospital, with clinical evidences of acute respiratory infections between January 2001 and December 2002 were studied. Adenoviruses were isolated from 317 patients with an isolation rate of 7.67 per cent. Serotype analysis was performed by polymerase chain reaction (PCR) and/or PCR-restriction fragment length polymorphism (PCR-RFLP) in 186 specimens. In 2001, adenovirus type 4 was found in the majority (57 per cent), followed by type 1.5.6 (15 per cent), type 2 (13 per cent), type 14 (8 per cent), type 3 (5 per cent), and type 7 (2 per cent). In 2002, type 3 became the major type (46 per cent), whereas the previously predominant type 4 decreased to 6 per cent, and type 7 increased from 2 to 19 per cent. The symptoms and signs included fever (98.7 percent), cough (77.6 per cent), abnormal breathing sounds (crackles and/or wheezing 23.3 per cent), abdominal pain (18.9 per cent), vomiting (21.8 per cent), and diarrhea (25.2 per cent). The mean duration of fever was 4.8 days (range 0-19 days). In the 186 cases in whom serotypes were analysed, pharyngitis and tonsillitis (47.8 per cent) were the most common presentation, followed by pneumonia (25.2 per cent), bronchitis (12.9 per cent), and pharyngoconjunctival fever (PCF) (7.6 per cent). Children between 4 and 8 years old were the most common group of patients with respiratory adenoviral infections. Our patients all had good prognosis. This adenoviruses molecular epidemiological study provides information that helps physicians in clinical differential diagnosis and treatment of respiratory adenoviral infection in children in southern Taiwan.

Randomized, double-blind, placebo-controlled trial of hydroxyurea in spinal muscular atrophy.

Objective: The purpose of this study was to evaluate the safety and efficacy of hydroxyurea (HU) in spinal muscular atrophy (SMA) in a randomized, double-blind, placebo-controlled trial. Methods: Twenty-eight patients with type 2 SMA and 29 patients with type 3 SMA were randomly assigned (2:1) to receive HU or matching placebo for 18 months. HU was initiated at 10 mg/kg/day with an 8-week titration to 20 mg/kg/day. Subjects were assessed at baseline (T0) and monthly for the first 2 months (T1–T2) and then every 2 months throughout treatment (T3–T10) and posttreatment periods (T11–T13). The primary outcome measures were the Gross Motor Function Measure (GMFM), Manual Muscle Test (MMT), and serum full-length survivor motor neuron (flSMN) mRNA. The secondary outcome measures were Modified Hammersmith Functional Motor Scale and forced vital capacity (FVC). Results: Fifty-five patients completed this trial, which lasted from March 2007 to June 2009. Except for neutropenia, we found no differences in adverse events between the 2 groups. Compared with the placebo group, the HU group had −1.88 for GMFM (p = 0.11), −0.55 for MMT (p = 0.49), and 2.17 for flSMN mRNA (p = 0.13). Similarly, we found no difference in mean improvement of the secondary endpoints. Both groups had a trend toward a decline in FVC with little change in strength and motor function. Conclusion: Under the current regimen and schedule, HU brought about no improvement in patients with type 2 and 3 SMA, and its main side effect was neutropenia. Classification of evidence: This trial provides Class I evidence that HU 20 mg/kg/day does not effectively treat SMA.

The effect of hydroxyurea in spinal muscular atrophy cells and patients

BACKGROUND Spinal muscular atrophy (SMA) is a degenerative motor neuron disease caused by homozygous mutations of the survival motor neuron 1 (SMN1) gene. Effective treatment for SMA is unavailable at present. The aim of this study was to investigate the effect of hydroxyurea (HU) in SMA cells and patients. MATERIALS AND METHODS Fifteen SMA lymphoid and three fibroblast cell lines, 2 from SMA patients and 1 control, were treated with HU at different concentrations, and 33 patients (types II, III) randomized into three groups on different HU dosage, 20, 30, 40 mg/kg/day, were treated for 8 weeks and followed up for another drug-free 8 weeks. The effect of HU on SMN2 gene expression and clinical manifestations was evaluated. RESULTS After treatment, in vitro, full-length mRNA level and gems number increased significantly, and hnRNP A1 protein decreased. In vivo, there were slight increases in muscle strength scores at 4 weeks and full-length SMN mRNA at 8 weeks in 30 mg/kg/day subgroup. CONCLUSIONS Treating with HU enhanced SMN2 gene expression in SMA cells and showed slight trend towards improvement in some clinical outcome measures in SMA patients which suggests HU may be safe to use in SMA patients but larger randomized, placebo-controlled, double-blind trials are needed to further investigate its efficacy.

Prevalence of the FMR1 mutation in Taiwan assessed by large‐scale screening of newborn boys and analysis of DXS548‐FRAXAC1 haplotype

If carrier women could be identified in time and take appropriate measures, fragile X syndrome (FXS) can be prevented. Wide screening of women to be or in their early pregnancy was considered a good approach to identify carriers without misdetection. Nevertheless, we argued against the cost‐effectiveness of implementing such a screening program in Taiwan, due to the lower carrier rate found in our pilot study. To reliably estimate the prevalence of mutant FMR1 gene in Taiwan, we anonymously screened 10,046 newborn boys using bloodspot polymerase chain reaction (PCR). Among them, the sample from one boy, who was most likely had FXS, failed repeatedly in PCR amplification. The estimated prevalence of premutation (55–200 CGG repeats) and intermediate alleles (45–54 CGG repeats) was 1:1,674 (n = 6) and 1:143 (n = 70), respectively. All these estimates were constantly lower than that reported in Caucasian populations, with variable statistic significance. Furthermore, when comparing analyses of the distribution of alleles at the two most often investigated microsatellite loci, DXS548 and FRAXAC1, between 100 control and 28 unrelated fragile X chromosomes, we found no apparent founder haplotype prevalent among the fragile X patients. Because a few founder haplotypes were reportedly prevalent in two thirds of fragile X alleles in Caucasians and in Chinese from Central China, we thus suggested that lack of founder fragile X chromosomes might result in a relatively low prevalence of mutant FMR1 gene in a population, as observed in Taiwan.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) regulates the level of SMN expression through ubiquitination in primary spinal muscular atrophy fibroblasts.

BACKGROUND Spinal muscular atrophy (SMA), a lethal hereditary disease caused by mutations of the survival of motor neuron 1 (SMN1) gene, is the leading genetic cause of infant mortality. Its severity directly correlates to the expression level of SMN protein in patients with SMA, but the regulatory mechanisms of SMN protein expression remain incompletely defined. In the present study, we aimed to identify candidate proteins to distinguish SMA fibroblasts from normal fibroblasts. METHODS To identify cellular targets regulating the expression of SMN, we initially utilized a proteomics approach combining 2D electrophoresis and LC-MS/MS, wherein the total proteins extracted from type I SMA patients and normal skin fibroblast cells were compared. RESULTS Our initial proteomics analysis discovered significant increase of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in type I SMA fibroblasts when compared to normal fibroblasts. Significantly, UCHL1 proteins directly interacted with SMN protein, as determined by immunoprecipitation and immunofluorescence assays in P19 and NSC34 cells. Over-expression of UCHL1 in P19 and NSC34 cells significantly reduced the level of SMN proteins in vivo, and, in fact, purified UCHL1 was shown to be able to enhance, in a dose-dependent manner, the level of ubiquitinated SMN in vitro. Further, inhibition of UCHL1 activity by UCHL1 inhibitor (LDN-57444) increased cellular SMN protein and gems number in the nucleus in NSC34 and SMA skin fibroblasts. The same results were observed in cells with UCHL1-specific knockdown. CONCLUSIONS These results suggested that UCHL1 may be a critical regulator in controlling cellular SMN protein turnover, and that it may serve as an attractive therapeutic target for SMA.

Prenatal morphine alters the synaptic complex of postsynaptic density 95 with N-methyl-d-aspartate receptor subunit in hippocampal CA1 subregion of rat offspring leading to long-term cognitive deficits

Infants who are passively exposed to morphine or heroin through their addicted mothers usually develop neurobiological changes. The postsynaptic density 95 (PSD-95) protein, a submembranous cytoskeletal specialization, is dynamically linked with N-methyl-d-aspartate receptors (NMDARs) to form a synaptic complex in postsynaptic neurons. This complex serves important neurobiological functions, including mammalian learning and memory. However, the effects of prenatal morphine exposure on this synaptic complex are not well understood. In this study, we determined whether prenatal morphine exposure altered the synaptic complex association between PSD-95 and three major NMDAR subunits (NR1, NR2A, and NR2B), at the mRNA and protein levels, within the hippocampal CA1 subregion (an important integration area for mammalian learning and memory) of rat offspring along with the performance of long-term cognitive functions. Sprague-Dawley rat offspring from morphine-addicted mothers were studied at a younger age (postnatal day 14; P14) and at an older age (P45). Subsequently, an eight-arm radial maze task was applied to analyze the working and cued reference memory in such offspring (P45). The real-time polymerase chain reaction results showed that prenatal morphine exposure caused significant decreases in mRNA levels of the PSD-95 and three NMDAR subunits (NR1, NR2A, and NR2B) in offspring (P14 and P45). Similarly, at the protein level, immunoblotting showed that decreased whole levels of PSD-95 and NMDAR subunits were seen in offspring subjected with prenatal morphine. Furthermore, the protein interaction of the synaptic complex between the PSD-95 and NMDAR subunit, as indicated by coimmunoprecipitation, was less in prenatal morphine samples than in vehicle controls (P14 and P45). The prenatal morphine group also showed poorer performance for an eight-arm radial maze task than the vehicle-control group. These results are particularly important for a better understanding of certain opioid-mediated neurobehavioral cognitive changes in offspring associated with altered protein interaction between PSD-95 and NMDAR subunits within the developing brain.

Posttraumatic stress disorder in adolescents after Typhoon Morakot-associated mudslides.

The aims of this study were to examine prevalence rate of post-traumatic stress disorder (PTSD), its associated factors and co-occurring psychological problems in a group of displaced adolescents 3 months following Typhoon Morakot in Taiwan. The relationship of trauma dimension and PTSD was also explored. A total of 271 adolescents who had been evacuated from their homes participated in this school-based survey. Adolescents were interviewed using the Mini-International Neuropsychiatric Interview for Children and Adolescents. Subjects themselves completed the following questionnaires: an inventory of exposure experiences to Typhoon Morakot, the Chinese version of Impact of Events Scale-Revised, the Center for Epidemiological Studies Depression Scale, and the Family APGAR Index. Teachers completed the Teachers Report Form in the Achenbach system of Empirically Bases Assessment. Results revealed that the prevalence of PTSD related to Typhoon Morakot was 25.8%. Adolescents who were female, had PTSD related to previous traumatic events before Typhoon Morakot, had more exposure experiences, were physically injured, or had family member in same household died or seriously injured were more likely to have the diagnoses of PTSD. Meanwhile, adolescents with PTSD had more severe depression, internalizing, externalizing, social, thought, and attention problems than those without PTSD. Our findings indicate that specialized trauma services are needed for these youngsters to lessen prolonged vulnerabilities.

High resolution melting analysis facilitates mutation screening of ETFDH gene: Applications in riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency

BACKGROUND Multiple acyl-CoA dehydrogenase deficiency (MADD) or gluaric aciduria type II is an autosomal recessive disease caused by defects in mitochondrial electron transfer system and metabolism of fatty acid. Recently, ETFDH mutations were reported to be major causes of riboflavin-responsive MADD. The present study is aimed at screening ETFDH mutations. METHODS High resolution melting (HRM) analysis was performed to screen ETFDH mutations. Genomic DNA was extracted from peripheral blood samples of the 9 patients with MADD and normal controls. Total 13 exons of ETFDH were screened by HRM analysis. The results were subsequently confirmed by direct DNA sequencing. RESULTS This diagnostic strategy proved to be feasible in detecting 3 known (c.250G>A, c380T>A, c.524G>T) and 1 novel (c.1831G>A) ETFDH mutations. Each mutation could be readily and accurately identified in the difference plot curves. We estimated the carrier frequency of the hotspot mutation, c.250G>A, in the Taiwanese population to be 1:125 (0.8%). CONCLUSIONS HRM analysis can be successfully applied to screen ETFDH mutations. Since riboflavin-responsive MADD is often treatable, especially with mutations in ETFDH, identifying ETFDH mutations is crucial for these patients.