Chanin Limwongse

Orientia tsutsugamushi stimulates an original gene expression program in monocytes: relationship with gene expression in patients with scrub typhus.

Orientia tsutsugamushi is the causal agent of scrub typhus, a public health problem in the Asia-Pacific region and a life-threatening disease. O. tsutsugamushi is an obligate intracellular bacterium that mainly infects endothelial cells. We demonstrated here that O. tsutsugamushi also replicated in monocytes isolated from healthy donors. In addition, O. tsutsugamushi altered the expression of more than 4,500 genes, as demonstrated by microarray analysis. The expression of type I interferon, interferon-stimulated genes and genes associated with the M1 polarization of macrophages was significantly upregulated. O. tsutsugamushi also induced the expression of apoptosis-related genes and promoted cell death in a small percentage of monocytes. Live organisms were indispensable to the type I interferon response and apoptosis and enhanced the expression of M1-associated cytokines. These data were related to the transcriptional changes detected in mononuclear cells isolated from patients with scrub typhus. Here, the microarray analyses revealed the upregulation of 613 genes, which included interferon-related genes, and some features of M1 polarization were observed in these patients, similar to what was observed in O. tsutsugamushi-stimulated monocytes in vitro. This is the first report demonstrating that monocytes are clearly polarized in vitro and ex vivo following exposure to O. tsutsugamushi. These results would improve our understanding of the pathogenesis of scrub typhus, during which interferon-mediated activation of monocytes and their subsequent polarization into an M1 phenotype appear critical. This study may give us a clue of new tools for the diagnosis of patients with scrub typhus.

Whole Exome Sequencing in Thai Patients With Retinitis Pigmentosa Reveals Novel Mutations in Six Genes

PURPOSE To identify disease-causing mutations and describe genotype-phenotype correlations in Thai patients with nonsyndromic retinitis pigmentosa (RP). METHODS Whole exome sequencing was performed in 20 unrelated patients. Eighty-six genes associated with RP, Leber congenital amaurosis, and cone-rod dystrophy were analyzed for variant detection. RESULTS Seventeen variants (13 novel and 4 known) in 13 genes were identified in 11 patients. These variants include 10 missense substitutions, 2 nonsense mutations, 3 deletions, 1 insertion, and 1 splice site change. Nine patients with identified inheritance patterns carried a total of 10 potentially pathogenic mutations located in genes CRB1, C8orf37, EYS, PROM1, RP2, and USH2A. Three of the nine patients also demonstrated additional heterozygous variants in genes ABCA4, GUCY2D, RD3, ROM1, and TULP1. In addition, two patients carried variants of uncertain significance in genes FSCN2 and NR2E3. The RP phenotypes of our patients were consistent with previous reports. CONCLUSIONS This is the first report of mutations in Thai RP patients. These findings are useful for genotype-phenotype comparisons among different ethnic groups.

Variable-length haplotype construction for geneߝgene interaction studies

Genetic epidemiology is a research field that aims to identify genetic polymorphisms that are involved in disease susceptibility. In this article, a variable-length haplotype construction for gene-gene interaction (VarHAP) technique is proposed. The technique will involve nonparametric classification where haplotypes inferred from multiple single nucleotide polymorphism (SNP) data are the classifier inputs.

Detecting purely epistatic multi-locus interactions by an omnibus permutation test on ensembles of two-locus analyses

BackgroundPurely epistatic multi-locus interactions cannot generally be detected via single-locus analysis in case-control studies of complex diseases. Recently, many two-locus and multi-locus analysis techniques have been shown to be promising for the epistasis detection. However, exhaustive multi-locus analysis requires prohibitively large computational efforts when problems involve large-scale or genome-wide data. Furthermore, there is no explicit proof that a combination of multiple two-locus analyses can lead to the correct identification of multi-locus interactions.ResultsThe proposed 2LOmb algorithm performs an omnibus permutation test on ensembles of two-locus analyses. The algorithm consists of four main steps: two-locus analysis, a permutation test, global p-value determination and a progressive search for the best ensemble. 2LOmb is benchmarked against an exhaustive two-locus analysis technique, a set association approach, a correlation-based feature selection (CFS) technique and a tuned ReliefF (TuRF) technique. The simulation results indicate that 2LOmb produces a low false-positive error. Moreover, 2LOmb has the best performance in terms of an ability to identify all causative single nucleotide polymorphisms (SNPs) and a low number of output SNPs in purely epistatic two-, three- and four-locus interaction problems. The interaction models constructed from the 2LOmb outputs via a multifactor dimensionality reduction (MDR) method are also included for the confirmation of epistasis detection. 2LOmb is subsequently applied to a type 2 diabetes mellitus (T2D) data set, which is obtained as a part of the UK genome-wide genetic epidemiology study by the Wellcome Trust Case Control Consortium (WTCCC). After primarily screening for SNPs that locate within or near 372 candidate genes and exhibit no marginal single-locus effects, the T2D data set is reduced to 7,065 SNPs from 370 genes. The 2LOmb search in the reduced T2D data reveals that four intronic SNPs in PGM1 (phosphoglucomutase 1), two intronic SNPs in LMX1A (LIM homeobox transcription factor 1, alpha), two intronic SNPs in PARK2 (Parkinson disease (autosomal recessive, juvenile) 2, parkin) and three intronic SNPs in GYS2 (glycogen synthase 2 (liver)) are associated with the disease. The 2LOmb result suggests that there is no interaction between each pair of the identified genes that can be described by purely epistatic two-locus interaction models. Moreover, there are no interactions between these four genes that can be described by purely epistatic multi-locus interaction models with marginal two-locus effects. The findings provide an alternative explanation for the aetiology of T2D in a UK population.ConclusionAn omnibus permutation test on ensembles of two-locus analyses can detect purely epistatic multi-locus interactions with marginal two-locus effects. The study also reveals that SNPs from large-scale or genome-wide case-control data which are discarded after single-locus analysis detects no association can still be useful for genetic epidemiology studies.

Mutation analysis of the GNE gene in distal myopathy with rimmed vacuoles (DMRV) patients in Thailand

Distal myopathy with rimmed vacuoles (DMRV) is an early‐adult‐onset, distal myopathy caused by a mutation of the UDP‐N‐acetylglucosamine 2 epimerase/N‐acetylmannosamine kinase (GNE) gene. We herein report four Thai patients with DMRV who carried compound heterozygous mutations of the GNE gene including three novel (p.G89R, p.P511T, and p.I656N) and two known mutations (p.A524V and p.V696M). All patients shared p.V696M in one allele. Our study demonstrates the mutation spectrum of the GNE gene in Thai patients with DMRV. Muscle Nerve, 2006

Pharmacokinetics and safety of a new paediatric fixed-dose combination of zidovudine/lamivudine/nevirapine in HIV-infected children.

BACKGROUND Alternatives to the available stavudine-containing paediatric fixed-dose combination (FDC) tablets are rapidly needed due to concerns regarding the cumulative toxicity of long-term stavudine exposure. We report the bioavailability and short-term safety of a novel paediatric FDC tablet of zidovudine (ZDV)/lamivudine (3TC)/nevirapine (NVP; 30/15/28 mg) in HIV-infected children. METHODS In this Phase I/II open-label pharmacokinetic study, 42 children weighing 6-30 kg treated with NVP-based HAART for ≥4 weeks were randomized to receive the FDC tablets (GPO-VIR Z30) or the liquid formulations. Dosing was weight-based. Intensive 12-h blood sampling was performed after 2 weeks; subjects then crossed-over to the alternate formulation at equal doses and sampling repeated 2 weeks later. Pharmacokinetic parameters were determined by non-compartmental analysis. Buccal-swab samples were collected for cytochrome P450 (CYP)2B6 polymorphism analysis. RESULTS With the FDC tablet, the geometric mean (90% CI) area under the curve (AUC) for ZDV, 3TC and NVP was 1.58 (1.49-1.68), 7.78 (7.38-8.19) and 68.88 (62.13-76.36) μg•h/ml, respectively. Rules for NVP therapeutic inadequacy were defined a priori, and despite lower NVP exposure with the tablet (P<0.001), the levels remained therapeutically adequate. ZDV AUC was similar between formulations. 3TC exposure was significantly higher with the tablet but comparable to historical data in adults and children taking branded tablets. While receiving the tablet, NVP AUC in children with CYP2B 516 GG (45%), GT (45%) and TT (10%) genotypes were 67.0, 74.5 and 106.4 μg•h/ml, respectively (P=0.04). CONCLUSIONS Disparities in drug exposure between formulations were observed; however, the FDC tablet delivered therapeutically adequate exposures of each drug and could well play an important role in simplifying antiretroviral treatment for children.

Six novel ATP7B mutations in Thai patients with Wilson disease.

WD is an autosomal recessive disorder of copper transport resulting in excessive copper deposition in the liver and brain. It is caused by defects of ATP7B encoding a copper transporting P-type ATPase. To identify the mutations in ATP7B in Thai patients with WD, DHPLC analysis was applied to detect mutations and polymorphisms of the entire ATP7B gene in 19 Thai patients with WD. Mutations in ATP7B were identified in 14 of 19 patients: 2 homozygotes, 8 compound heterozygotes and 4 heterozygotes. Eighteen mutations distributed throughout the entire coding region of ATP7B gene including 11 missense, 3 nonsense, 1 splice-site, 1 deletion and 2 insertions. Of 18 different mutations identified, 6 were found to be novel. Twelve single nucleotide polymorphisms (SNPs) were also identified and two SNPs have not yet previously been reported. Segregation analysis using DHPLC analysis showed mutation transmission patterns within each family of Thai patients with WD. Mutations in ATP7B in Thai patients with WD are worth adding into the public database for genetic epidemiology and population genetics.

Contiguous gene syndrome of holoprosencephaly and hypotrichosis simplex: Association with an 18p11.3 deletion

We report a patient with a unique combination of features, including microcephaly; mental retardation; poorly developed frontal lobes; hypoplastic pituitary gland; hypothyroidism; alopecia universalis; single maxillary central incisor; taurodontism; median palatal ridge; longitudinally grooved nails; and scoliosis. His unbalanced karyotype was found to be 45,XY,der(15;18)(q10;q10). The constellation of anomalies appears to represent a contiguous gene syndrome caused, at least in part, by deletion of TGIF and the gene responsible for hereditary hypotrichosis simplex. The phenotype of our patient differs other reported patients with del(18p). Possible explanations include (1) the effects of a different deleted region, (2) a positional effect caused by a gene close by, or (3) by interruption of a different gene resulting from chromosomal translocation.

Simple, Efficient, and Cost-Effective Multiplex Genotyping with Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry of Hemoglobin Beta Gene Mutations

A number of common mutations in the hemoglobin beta (HBB) gene cause beta-thalassemia, a monogenic disease with high prevalence in certain ethnic groups. As there are 30 HBB variants that cover more than 99.5% of HBB mutant alleles in the Thai population, an efficient and cost-effective screening method is required. Three panels of multiplex primer extensions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were developed. The first panel simultaneously detected 21 of the most common HBB mutations, while the second panel screened nine additional mutations, plus seven of the first panel for confirmation; the third panel was used to confirm three HBB mutations, yielding a 9-Da mass difference that could not be clearly distinguished by the previous two panels. The protocol was both standardized using 40 samples of known genotypes and subsequently validated in 162 blind samples with 27 different genotypes (including a normal control), comprising heterozygous, compound heterozygous, and homozygous beta-thalassemia. Results were in complete agreement with those from the genotyping results, conducted using three different methods overall. The method developed here permitted the detection of mutations missed using a single genotyping procedure. The procedure should serve as the method of choice for HBB genotyping due to its accuracy, sensitivity, and cost-effectiveness, and can be applied to studies of other gene variants that are potential disease biomarkers.

Novel and De-novo Truncating PAX6 Mutations and Ocular Phenotypes in Thai Aniridia Patients

Purpose: To describe the ophthalmic findings and mutation analyses of the PAX6 gene in Thai aniridia patients. Methods: Ten patients from six unrelated families underwent a comprehensive ophthalmic examination. Mutations in the PAX6 gene were screened by single-strand conformational polymorphism (SSCP) and direct DNA sequencing of the SSCP variants. Results: Seven patients developed cataracts and six developed glaucoma. Mutation analysis demonstrated four different truncating mutations, two of which were de novo. These included one novel insertion/deletion mutation (c.474del12insGA in exon 5) and three nonsense mutations. R203X and R240X are common recurrent mutations, while Q277X in exon 10 is novel. All mutations resulted in loss of function of the PAX6 protein. Conclusion: Our data confirm inter- and intrafamilial variable phenotypic manifestations of which the underlying mechanisms may be haploinsufficiency or dominant-negative mutation.