Continued benefit of nusinersen initiated in the presymptomatic stage of spinal muscular atrophy: 5-year update of the NURTURE study.

INTRODUCTION/AIMS: NURTURE (NCT02386553) is an open-label study of nusinersen in children (two SMN2 copies, n = 15; three SMN2 copies, n = 10) who initiated treatment in the presymptomatic stage of spinal muscular atrophy (SMA). A prior analysis after ~3 y showed benefits on survival, respiratory outcomes, motor milestone achievement, and a favorable safety profile. An additional 2 y of follow-up (data cut: February 15, 2021) are reported. METHODS: The primary endpoint is time to death or respiratory intervention (≥6 h/day continuously for ≥7 days or tracheostomy). Secondary outcomes include overall survival, motor function, and safety. RESULTS: Median age of children was 4.9 (3.8-5.5) y at last visit. No children have discontinued the study or treatment. All were alive. No additional children utilized respiratory intervention (defined per primary endpoint) since the prior data cut. Children with three SMN2 copies achieved all World Health Organization (WHO) motor milestones, with all but one milestone in one child within normal developmental timeframes. All 15 children with two SMN2 copies achieved sitting without support, 14/15 walking with assistance, and 13/15 walking alone. Mean Hammersmith Functional Motor Scale Expanded total scores showed continued improvement. Subgroups with two SMN2 copies, minimum baseline compound muscle action potential amplitude ≥2 mV, and no baseline areflexia had better motor and nonmotor outcomes versus all children with two SMN2 copies. DISCUSSION: These results demonstrate the value of early treatment, durability of treatment effect, and favorable safety profile after ~5 y of nusinersen treatment. Inclusion/exclusion criteria and baseline characteristics should be considered when interpreting presymptomatic SMA trial data.

Fluorescent aptasensor based on conformational switch-induced hybridization for facile detection of ß-amyloid oligomers.

Aß oligomers (AßO) are a dominant biomarker for early Alzheimer's disease diagnosis. A fluorescent aptasensor coupled with conformational switch-induced hybridization was established to detect AßO. The fluorescent aptasensor is based on the interaction of fluorophore-labeled AßO-specific aptamer (FAM-Apt) against its partly complementary DNA sequence on the surface of magnetic beads (cDNA-MBs). Once the FAM-Apt binds to AßO, the conformational switch of FAM-Apt increases the tendency to be captured by cDNA-MBs. This causes a descending fluorescence of supernatant, which can be utilized to determine the levels of AßO. Thus, the base-pair matching above 12 between FAM-Apt and cDNA-MBs with increasing hybridizing free energies reached the ascending fluorescent signal equilibrium. The optimized aptasensor showed linearity from 1.7 ng mL-1 to 85.1 (R = 0.9977) with good recoveries (79.27-109.17%) in plasma. Furthermore, the established aptasensor possesses rational selectivity in the presence of monomeric Aß, fibrotic Aß, and interferences. Therefore, the developed aptasensor is capable of quantifying AßO in human plasma and possesses the potential to apply in clinical cases.

L-Carnitine ameliorates congenital myopathy in a tropomyosin 3 de novo mutation transgenic zebrafish.

BACKGROUND: Congenital myopathy (CM) is a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A de novo mutation in Tropomyosin 3-TPM3(E151G) was identified from a boy diagnosed with CM, previously TPM3(E151A) was reported to cause CM. However, the role of TPM3(E151G) in CM is unknown. METHODS: Histopathological, swimming behavior, and muscle endurance were monitored in TPM3 wild-type and mutant transgenic fish, modelling CM. Gene expression profiling of muscle of the transgenic fish were studied through RNAseq, and mitochondria respiration was investigated. RESULTS: While TPM3(WT) and TPM3(E151A) fish show normal appearance, amazingly a few TPM3(E151G) fish display either no tail, a crooked body in both F0 and F1 adults. Using histochemical staining for the muscle biopsy, we found TPM3(E151G) displays congenital fiber type disproportion and TPM3(E151A) resembles nemaline myopathy. TPM3(E151G) transgenic fish dramatically swimming slower than those in TPM3(WT) and TPM3(E151A) fish measured by DanioVision and T-maze, and exhibit weaker muscle endurance by swimming tunnel instrument. Interestingly, L-carnitine treatment on TPM3(E151G) transgenic larvae significantly improves the muscle endurance by restoring the basal respiration and ATP levels in mitochondria. With RNAseq transcriptomic analysis of the expression profiling from the muscle specimens, it surprisingly discloses large downregulation of genes involved in pathways of sodium, potassium, and calcium channels, which can be rescued by L-carnitine treatment, fatty acid metabolism was differentially dysregulated in TPM3(E151G) fish and rescued by L-carnitine treatment. CONCLUSIONS: These results demonstrate that TPM3(E151G) and TPM3(E151A) exhibit different pathogenicity, also have distinct gene regulatory profiles but the ion channels were downregulated in both mutants, and provides a potential mechanism of action of TPM3 pathophysiology. Our results shed a new light in the future development of potential treatment for TPM3-related CM.

Specific Gene Capture Combined with Restriction-Fragment Release for Directly Fluorescent Genotyping of Single-Nucleotide Polymorphisms in Diagnosing Spinal Muscular Atrophy.

In this study, a fast and simple fluorescent genotyping strategy, streptavidin magnetic beads combined with biotin-coupled PCR and restriction-fragment release, was developed for determination of nucleotide variants. This method was further applied for analyzing SMN1 gene in diagnosis of spinal muscular atrophy (SMA). After biotin-coupled PCR, the streptavidin magnetic beads would capture the biotin-labeled SMN genetic fragments, and then the restriction enzyme of HPY188I could only digest and release the fluorescent end of SMN1 genetic fragment into the supernatant. Therefore, the SMN1 gene could be easily fluorescently quantified, and SMN2 would not, for diagnosis of SMA. The copy number of the SMN1 gene could be regressed using the relative fluorescent unit versus the known copy number, and the coefficient of correlation is equal to 0.9617 ( r = 0.9617). In this research, a total of 16 blind DNA samples were analyzed, including 6 wild types, 5 carriers, and 5 SMA patients. Importantly, this fast, simple, and highly efficient method is universal for detection of all nucleotides variants by replacing the specific restriction enzyme. This technique has the potency to be served as a tool for fast and accurate diagnosis of genotypes in clinical medicine.

A Three-MicroRNA Signature as a Potential Biomarker for the Early Detection of Oral Cancer.

Oral squamous cell carcinoma (OSCC) is often diagnosed at a late stage and may be malignantly transformed from oral leukoplakia (OL). This study aimed to identify potential plasma microRNAs (miRNAs) for the early detection of oral cancer. Plasma from normal, OL, and OSCC patients were evaluated. Small RNA sequencing was used to screen the differently expressed miRNAs among the groups. Next, these miRNAs were validated with individual samples by quantitative real-time polymerase chain reaction (qRT-PCR) assays in the training phase (n = 72) and validation phase (n = 178). The possible physiological roles of the identified miRNAs were further investigated using bioinformatics analysis. Three miRNAs (miR-222-3p, miR-150-5p, and miR-423-5p) were identified as differentially expressed among groups; miR-222-3p and miR-423-5p negatively correlated with T stage, lymph node metastasis status, and clinical stage. A high diagnostic accuracy (Area under curve = 0.88) was demonstrated for discriminating OL from OSCC. Bioinformatics analysis reveals that miR-423-5p and miR-222-3p are significantly over-expressed in oral cancer tissues and involved in various cancer pathways. The three-plasma miRNA panel may be useful to monitor malignant progression from OL to OSCC and as potential biomarkers for early detection of oral cancer.

Presymptomatic Diagnosis of Spinal Muscular Atrophy Through Newborn Screening.

OBJECTIVE: To demonstrate the feasibility of presymptomatic diagnosis of spinal muscular atrophy (SMA) through newborn screening (NBS). STUDY DESIGN: We performed a screening trial to assess all newborns who underwent routine newborn metabolic screening at the National Taiwan University Hospital newborn screening center between November 2014 and September 2016. A real-time polymerase chain reaction (RT-PCR) genotyping assay for the SMN1/SMN2 intron 7 c.888+100A/G polymorphism was performed to detect homozygous SMN1 deletion using dried blood spot (DBS) samples. Then the exon 7 c.840C>T mutation and SMN2 copy number were determined by both droplet digital PCR (ddPCR) using the original screening DBS and multiplex ligation-dependent probe amplification (MLPA) using a whole blood sample. RESULTS: Of the 120 267 newborns, 15 tested positive according to the RT-PCR assay. The DBS ddPCR assay excluded 8 false-positives, and the other 7 patients were confirmed by the MLPA assay. Inclusion of the second-tier DBS ddPCR screening assay resulted in a positive prediction value of 100%. The incidence of SMA was 1 in 17 181 (95% CI, 1 in 8323 to 1 in 35 468). Two of the 3 patients with 2 copies of SMN2 and all 4 patients with 3 or 4 copies of SMN2 were asymptomatic at the time of diagnosis. Five of the 8 false-positives were caused by intragenic recombination between SMN1 and SMN2. CONCLUSION: Newborn screening can detect patients affected by SMA before symptom onset and enable early therapeutic intervention. A combination of a RT-PCR and a second-tier ddPCR can accurately diagnose SMA from DBS samples with no false-positives. TRIAL REGISTRATION: ClinicalTrials.gov NCT02123186.

Pediatric necrotizing myopathy associated with anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase antibodies.

OBJECTIVE: Antibodies against 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) have recently been associated with immune-mediated necrotizing myopathy, especially in patients with statin exposure. As the data are very limited concerning phenotypes and treatment in paediatric patients, we aimed to identify the paediatric patients positive for anti-HMGCR antibodies and clarify their features and therapeutic strategies. METHODS: We screened 62 paediatric patients who were clinically and/or pathologically suspected to have inflammatory myopathy for anti-HMGCR antibodies. We further re-assessed the clinical and histological findings and the treatment of the patients positive for anti-HMGCR antibodies. RESULTS: We identified nine paediatric patients with anti-HMGCR antibodies (15%). This was more frequent than anti-signal recognition particle antibodies (four patients, 6%) in our cohort. The onset age ranged from infancy to 13 years. Five patients were initially diagnosed with muscular dystrophy, including congenital muscular dystrophy. Most patients responded to high-dose corticosteroid therapy first but often needed adjuvant immunosuppressants to become stably controlled. CONCLUSION: Paediatric necrotizing myopathy associated with anti-HMGCR antibodies may not be very rare. Phenotypes are similar to those of adult patients, but a chronic slowly progressive course may be more frequent. Some patients share the clinicopathological features of muscular dystrophy indicating that recognizing inflammatory aetiology would be challenging without autoantibody information. On the other hand, most patients responded to treatment, especially those who were diagnosed early. Our results suggest the importance of early autoantibody testing in paediatric patients who have manifestations apparently compatible with muscular dystrophy in addition to those who have typical features of inflammatory myopathy.

Neurite growth could be impaired by ETFDH mutation but restored by mitochondrial cofactors.

INTRODUCTION: c.250G>A (p.Ala84Thr) in ETFDH is the most common mutation that causes later-onset multiple acyl-coenzyme A dehydrogenase deficiency (MADD) in the southern Chinese population. No functional study has targeted this mutation. METHODS: Using cells expressing ETFDH-wild-type (WT) or ETFDH-mutant (p.Ala84Thr), reactive oxygen species (ROS) production and neurite length were analyzed, followed by pathomechanism exploration and drug screening. RESULTS: Increased ROS production and marked neurite shortening were observed in the cells expressing the ETFDH-mutant, compared with WT. Further studies demonstrated that suberic acid, an accumulated intermediate metabolite in MADD, could significantly impair neurite outgrowth of NSC34 cells, but neurite shortening could be restored by supplementation with carnitine, riboflavin, or Coenzyme Q10. CONCLUSIONS: Neurite shortening caused by the c.250G>A mutation in ETFDH suggests that neural defects could be underdiagnosed in human patients with MADD. This impairment might be treatable with mitochondrial cofactor supplementation. Muscle Nerve 56: 479-485, 2017.

3-O-methyldopa levels in newborns: Result of newborn screening for aromatic l-amino-acid decarboxylase deficiency.

BACKGROUND: The diagnosis of aromatic l-amino-acid decarboxylase (AADC) deficiency is often delayed because a cerebrospinal fluid analysis is required to detect a neurotransmitter deficiency. We here demonstrated that an elevated concentration of l-dopa metabolite 3-O-methyldopa (3-OMD) in dried blood spots could be integrated into newborn screening program to precisely predict AADC deficiency. METHODS: After obtaining parental consent, an additional spot was punched from newborn filter paper, eluted, cleaned, and analyzed by tandem mass spectrometry. Newborns with a 3-OMD concentration exceeding 500ng/mL were referred for confirmatory testing. RESULTS: From September 2013 to December 2015, 127,987 newborns were screened for AADC deficiency. The mean 3-OMD concentration in these newborns was 88.08ng/mL (SD=27.74ng/mL). Four newborns exhibited an elevated 3-OMD concentration (range, 939-3241ng/mL). All four newborns were confirmed to carry two pathologic DDC mutations, indicating an incidence of AADC deficiency of 1:32,000. During the follow-up period, three patients developed typical symptoms of AADC deficiency. Among 16 newborns with mildly elevated 3-OMD levels, six were heterozygous for the DDC IVS6+4A>T mutation. CONCLUSION: Newborn screening of AADC deficiency was achieved with a 100% positive-predictive rate. An association for gestational age could be further elucidated.

Loganin possesses neuroprotective properties, restores SMN protein and activates protein synthesis positive regulator Akt/mTOR in experimental models of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease characterized by motor neurons degeneration and muscular atrophy. There is no effective SMA treatment. Loganin is a botanical candidate with anti-inflammatory, anti-oxidant, glucose-lowering and anti-diabetic nephropathy activities. The aim of this study is to investigate the potential protective effects of loganin on SMA using two cellular models, SMN-deficient NSC34 cells and SMA patient fibroblasts, and an animal disease model, SMAΔ7 mice. In SMN-deficient NSC34 cells, loganin increased cell viability, neurite length, and expressions of SMN, Gemin2, SMN-Gemin2 complex, p-Akt, p-GSK-3ß, p-CREB, BDNF and Bcl-2. However, both AG1024 (IGF-1 R antagonist) and IGF-1 R siRNA attenuated the protective effects of loganin on SMN level and cell viability in SMN-deficient NSC34 cells. In SMA patient fibroblasts, loganin up-regulated levels of SMN, FL-SMN2, and Gemins, increased numbers of SMN-containing nuclear gems, modulated splicing factors, and up-regulated p-Akt. Furthermore, in the brain, spinal cord and gastrocnemius muscle of SMAΔ7 mice, loganin up-regulated the expressions of SMN and p-Akt. Results from righting reflex and hind-limb suspension tests indicated loganin improved muscle strength of SMAΔ7 mice; moreover, loganin activated Akt/mTOR signal and inhibited atrogin-1/MuRF-1 signal in gastrocnemius muscle of SMAΔ7 mice. Loganin also increased body weight, but the average lifespan of loganin (20mg/kg/day)-treated SMA mice was 16.80±0.73 days, while saline-treated SMA mice was 10.91±0.96 days. In conclusion, the present results demonstrate that loganin provides benefits to SMA therapeutics via improving SMN restoration, muscle strength and body weight. IGF-1 plays an important role in loganin neuroprotection. Loganin can be therefore a valuable complementary candidate for treatment of neuromuscular diseases via regulation of muscle protein synthesis and neuroprotection.

Identification of KLHL40 mutations by targeted next-generation sequencing facilitated a prenatal diagnosis in a family with three consecutive affected fetuses with fetal akinesia deformation sequence.

BACKGROUND: Fetal akinesia deformation sequence (FADS) refers to a broad spectrum of disorder with the absent fetal movement as the unifying feature. The etiology of FADS is heterogeneous, and the majority remains unknown. Prenatal diagnosis of FADS because of neuromuscular origin has relied on clinical features and fetal muscle pathology, which can be unrevealing. The recent advance of next-generation sequencing (NGS) can provide definitive molecular diagnosis effectively. METHODS AND RESULTS: An 18-week-old fetus presented with akinesia and multiple contractures of joints. The mother had two previously aborted similarly affected fetuses. Clinical diagnosis of FADS was made. Molecular diagnosis using cord blood by NGS of genes related to neuromuscular diseases revealed two compound heterozygous mutations; c.602G > A(p.W201*) and c.1516A > C(p.T506P), in the Kelch-like 40 (KLHL40) gene. Based on this information, prenatal diagnosis was performed on the CVS of the subsequent pregnancy that resulted in an unaffected female baby, heterozygous for the c.1516A > C(p.T506P) mutation. CONCLUSION: Identification of KLHL40 mutations in one of the aborted fetuses provided a confirmative diagnosis of FADS, facilitating the prenatal diagnosis of the subsequent pregnancy. This report underscores the importance of target NGS in providing FADS families with an affordable, precise molecular diagnosis for genetic counseling and options of prenatal diagnosis. © 2016 John Wiley & Sons, Ltd.

Using CRISPR/Cas9-Mediated GLA Gene Knockout as an In Vitro Drug Screening Model for Fabry Disease.

The CRISPR/Cas9 Genome-editing system has revealed promising potential for generating gene mutation, deletion, and correction in human cells. Application of this powerful tool in Fabry disease (FD), however, still needs to be explored. Enzyme replacement therapy (ERT), a regular administration of recombinant human α Gal A (rhα-GLA), is a currently available and effective treatment to clear the accumulated Gb3 in FD patients. However, the short half-life of rhα-GLA in human body limits its application. Moreover, lack of an appropriate in vitro disease model restricted the high-throughput screening of drugs for improving ERT efficacy. Therefore, it is worth establishing a large-expanded in vitro FD model for screening potential candidates, which can enhance and prolong ERT potency. Using CRISPR/Cas9-mediated gene knockout of GLA in HEK-293T cells, we generated GLA-null cells to investigate rhα-GLA cellular pharmacokinetics. The half-life of administrated rhα-GLA was around 24 h in GLA-null cells; co-administration of proteasome inhibitor MG132 and rhα-GLA significantly restored the GLA enzyme activity by two-fold compared with rhα-GLA alone. Furthermore, co-treatment of rhα-GLA/MG132 in patient-derived fibroblasts increased Gb3 clearance by 30%, compared with rhα-GLA treatment alone. Collectively, the CRISPR/Cas9-mediated GLA-knockout HEK-293T cells provide an in vitro FD model for evaluating the intracellular pharmacokinetics of the rhα-GLA as well as for screening candidates to prolong rhα-GLA potency. Using this model, we demonstrated that MG132 prolongs rhα-GLA half-life and enhanced Gb3 clearance, shedding light on the direction of enhancing ERT efficacy in FD treatment.

Label-Free Fluorescent Copper Nanoclusters for Genotyping of Deletion and Duplication of Duchenne Muscular Dystrophy.

Real applications in clinical diagnosis of label-free fluorescent copper nanoclusters (CuNCs) are demonstrated. Double-strand DNA (dsDNA) can act as an effective template for the formation of CuNCs, which can be used to distinguish the deletion or duplication genotypes of Duchenne muscular dystrophy (DMD) due to different fluorescent intensities. After PCR, the DMD amplicons reacted with copper ion by reduction of ascorbic acid and generated fluorescence. The exons of the DMD gene were taken as the model analytes for genetic diagnosis. In this sensing system, the deletion type does not show fluorescence; on the other hand, the duplication type emits higher fluorescence than normal type. Parameters of this sensing system were optimized, including PCR conditions, levels of copper ion and ascorbate, and reaction time. The DMD-dominated exons 45, 46, and 47 were detected, and the method was applied to six samples of DMD patients. The results were consistent with those of the multiplex ligation-dependent probe amplification method. This strategy was feasible to detect all exons of this disease.

Genotyping of exons 1 to 20 in Duchenne muscular dystrophy by universal multiplex PCR and short-end capillary electrophoresis.

One rapid CE method was established to diagnose Duchenne muscular dystrophy (DMD). DMD is a severe recessive inherited disorder frequently caused by gene deletions. Among them, exons 1-20 account for nearly 30% of occurrences. In this study, the universal multiplex PCR was used to enhance the fluorescently labeling efficiency, which was performed only by one universal fluorescent primer. After PCR, a short-end injection CE (short-end CE) speeded up the genotyping of the DMD gene. This method involved no extra purification, and was completed within 9 min. The CE conditions contained a polymer solution of 1.5% hydroxylethylcellulose in 1× TBE buffer at 6 kV for separation. This method was applied to test six DMD patients and one healthy male person. The results showed good agreement with those of multiplex ligation-dependent probe amplification. This method can be applied for clinical diagnosis of DMD disease. Accurate diagnosis of the DMD gene is the best way to prevent the disease.

Creatine and L-carnitine attenuate muscular laminopathy in the LMNA mutation transgenic zebrafish.

Lamin A/C gene (LMNA) mutations contribute to severe striated muscle laminopathies, affecting cardiac and skeletal muscles, with limited treatment options. In this study, we delve into the investigations of five distinct LMNA mutations, including three novel variants and two pathogenic variants identified in patients with muscular laminopathy. Our approach employs zebrafish models to comprehensively study these variants. Transgenic zebrafish expressing wild-type LMNA and each mutation undergo extensive morphological profiling, swimming behavior assessments, muscle endurance evaluations, heartbeat measurement, and histopathological analysis of skeletal muscles. Additionally, these models serve as platform for focused drug screening. We explore the transcriptomic landscape through qPCR and RNAseq to unveil altered gene expression profiles in muscle tissues. Larvae of LMNA(L35P), LMNA(E358K), and LMNA(R453W) transgenic fish exhibit reduced swim speed compared to LMNA(WT) measured by DanioVision. All LMNA transgenic adult fish exhibit reduced swim speed compared to LMNA(WT) in T-maze. Moreover, all LMNA transgenic adult fish, except LMNA(E358K), display weaker muscle endurance than LMNA(WT) measured by swimming tunnel. Histochemical staining reveals decreased fiber size in all LMNA mutations transgenic fish, excluding LMNA(WT) fish. Interestingly, LMNA(A539V) and LMNA(E358K) exhibited elevated heartbeats. We recognize potential limitations with transgene overexpression and conducted association calculations to explore its effects on zebrafish phenotypes. Our results suggest lamin A/C overexpression may not directly impact mutant phenotypes, such as impaired swim speed, increased heart rates, or decreased muscle fiber diameter. Utilizing LMNA zebrafish models for drug screening, we identify L-carnitine treatment rescuing muscle endurance in LMNA(L35P) and creatine treatment reversing muscle endurance in LMNA(R453W) zebrafish models. Creatine activates AMPK and mTOR pathways, improving muscle endurance and swim speed in LMNA(R453W) fish. Transcriptomic profiling reveals upstream regulators and affected genes contributing to motor dysfunction, cardiac anomalies, and ion flux dysregulation in LMNA mutant transgenic fish. These findings faithfully mimic clinical manifestations of muscular laminopathies, including dysmorphism, early mortality, decreased fiber size, and muscle dysfunction in zebrafish. Furthermore, our drug screening results suggest L-carnitine and creatine treatments as potential rescuers of muscle endurance in LMNA(L35P) and LMNA(R453W) zebrafish models. Our study offers valuable insights into the future development of potential treatments for LMNA-related muscular laminopathy.

The NAT2 tag SNP rs1495741 correlates with the susceptibility of antituberculosis drug-induced hepatotoxicity.

BACKGROUND: Earlier studies have demonstrated an association between N-acetyltransferase 2 (NAT2) catalytic activity and the genotype of a recently published tag single nucleotide polymorphism (SNP), rs1495741. There have been no reports on the relationship between the rs1495741 genotype and antituberculosis drug-induced hepatotoxicity (ATDIH) to date. OBJECTIVE: The aim of the present study was to determine the frequency of the NAT2 tag SNP (rs1495741) in the Taiwanese and its relation to the incidence of ATDIH. MATERIALS AND METHODS: A total of 348 tuberculosis patients were enrolled to determine the frequency of NAT2 tag SNP rs1495741 and its relation to the incidence of ATDIH. The conventional NAT2 variants alleles have also been investigated. Furthermore, to evaluate the correlation of NAT2 activity and rs1495741 genotypes, a pharmacokinetic study of isoniazid was also conducted in healthy volunteers. RESULTS: Among the 348 tuberculosis patients, 20 (5.7%) were diagnosed with ATDIH. The frequencies of the three rs1495741 genotypes, viz., AA, AG, and GG, were 24.7, 52.3, and 23.0%, respectively. Significant differences among rs1495741 genotypes and susceptibility to hepatotoxicity were noted (odds ratio=14.068, P<0.05). Moreover, the rs1495741 genotypes showed an association with the isoniazid dosage required for induction of hepatotoxicity. In the pharmacokinetic study, NAT2 activity was strongly associated with genotype categories (P<0.001). CONCLUSION: The present study demonstrated that the three genotypes according to rs1495741 were in good accordance with conventional NAT2 alleles-inferred phenotypes and the tag SNP could be used as a proxy to determine the susceptibility to ATDIH.

Capillary electrophoresis for analysis of deletion and duplication in exon 44-55 of Duchenne muscular dystrophy gene.

In this study, a genotyping CGE method was established for analysis of Duchenne muscular dystrophy (DMD) gene deletions and duplications in exon 44-55. A total of 12 DMD exons (exon 44-55) and 2 internal standard gene fragments were simultaneously amplified by using a universal multiplex PCR (UMPCR) and determined by CGE. The conditions of UMPCR and CGE were optimized, including the kinds of polymerase, temperatures in UMPCR, separation matrix, separation temperature, and voltage. Finally, the separation was performed by 1.2% poly(ethylene oxide) in 1× TBE buffer at -6 kV and 25°C. After validation, our results showed the peak patterns for differentiation of genetic deletion or duplication in 27 DMD patients and normal subjects, according to the peak height ratios by comparison of two internal standard peaks. Among the 27 subjects, 23 cases are deletion type and four are duplication type. The data of two patients analyzed by this CGE-PCR method were different from that of multiplex ligation dependent probe amplification method, and the sequencing results demonstrated that our results were correct. This UMPCR-CGE method was considered better than the multiplex ligation dependent probe amplification method. Furthermore, this method can be used for eugenics in clinical applications.

Inhibition of histamine H1 receptor activity modulates proinflammatory cytokine production of dendritic cells through c-Rel activity.

BACKGROUND: Histamine exerts diverse effects on immune regulation through four types of histamine receptors (HRs). Among them, type 1 receptor (H1R) plays an important role in allergic inflammation. Dendritic cells (DCs), which express at least three types of HRs, are professional antigen-presenting cells controlling the development of allergic inflammation. However, the molecular mechanisms involved in H1R-mediated NF-ĸB signaling of DCs remain poorly defined. METHODS: Bone-marrow (BM)-derived DCs (BM-DCs) were treated with H1R inverse agonists to interrupt basal H1R-mediated signaling. The crosstalk of H1R-mediated signaling and the NF-ĸB pathway was examined by NF-ĸB cellular activity using a luciferase reporter assay, NF-ĸB subunit analysis using Western blotting and TNF-α promoter activity using chromatin immunoprecipitation. RESULTS: Blockage of H1R signaling by inverse agonists significantly inhibited TNF-α and IL-6 production of BM-DCs. H1R-specific agonists were able to enhance TNF-α production, but this overexpression was significantly inhibited by NF-ĸB inhibitor. The H1R inverse agonist ketotifen also suppressed cellular NF-ĸB activity, suggesting crosstalk between H1R and NF-ĸB signaling in DCs. After comprehensive analysis of NF-ĸB subunits, c-Rel protein expression was significantly down-regulated in ketotifen-treated BM-DCs, which led to inhibition of the promoter activity of TNF-α. Finally, adoptive transfer of the ketotifen-treated BM-DCs did not induce significant allergic airway inflammation compared to that of control cells in vivo. CONCLUSIONS: Our results suggest that c-Rel controls H1R-mediated proinflammatory cytokine production in DCs. This study provides a potential mechanism of H1R-mediated signaling and NF-ĸB pathway crosstalk in allergic inflammation.

Lung cancer-derived galectin-1 mediates dendritic cell anergy through inhibitor of DNA binding 3/IL-10 signaling pathway.

Lung cancer, one of the leading causes of death worldwide, is often associated with a state of immune suppression, but the molecular and functional basis remains enigmatic. Evidence is provided in this paper supporting the role of lung cancer-derived soluble lectin, galectin-1, as a culprit in dendritic cell (DC) anergy. We have shown that galectin-1 is highly expressed in lung cancer cell lines, together with the serum and surgical samples from lung cancer patients. Functionally, lung cancer-derived galectin-1 has been shown to alter the phenotypes of monocyte-derived DCs (MdDCs) and impair alloreactive T cell response, concomitant with the increase of CD4(+)CD25(+)FOXP3(+) regulatory T cells. The regulatory effect of galectin-1 is mediated, in part, through its ability to induce, in an Id3 (inhibitor of DNA binding 3)-dependent manner, the expression of IL-10 in monocytes and MdDCs. This effect is inhibited by the addition of lactose, which normalizes the phenotypic and functional alterations seen in MdDCs. Of note, significant upregulation of IL-10 was seen in tumor-infiltrating CD11c(+) DCs in human lung cancer samples. This was also noted in mice transplanted with lung cancer cells, but not in those receiving tumor cells with galectin-1 knockdown. Furthermore, a significant reduction was noted in lung cancer incidence and in the levels of IL-10-expressing, tumor-infiltrating DCs, in mice receiving galectin-1-silenced tumor cells. These results thus suggest that the galectin-1/IL-10 functional axis may be crucial in lung cancer-mediated immune suppression, and that galectin-1 may serve as a target in the development of lung cancer immunotherapy.