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author:("gediga, Roger")
1.  Fluticasone Propionate Pharmacogenetics: CYP3A4*22 Polymorphism and Pediatric Asthma Control 
The Journal of pediatrics  2013;162(6):1222-1227.e2.
To determine the relationship between allelic variations in genes involved in fluticasone propionate (FP) metabolism and asthma control among children with asthma managed with inhaled FP.
Study design
The relationship between variability in asthma control scores and genetic variation in drug metabolism was assessed by genotyping nine single nucleotide polymorphisms (SNPs) in CYP3A4, CYP3A5, and CYP3A7. Genotype information was compared with asthma control scores (0 = well-controlled to 15 = poorly-controlled), determined by using a questionnaire modified from the National Heart Lung and Blood Institute Expert Panel 3 guidelines.
Our study cohort was comprised of 734 children with asthma (mean age 8.8 ± 4.3 years), who were predominantly male (61%) and non-Hispanic Whites (53%); 413 children (56%) were receiving inhaled glucocorticoids daily, of which FP was prescribed most frequently (65%). Among the children receiving daily FP, SNPs in the genes CYP3A5 and CYP3A7 were not associated with asthma control scores. In contrast, asthma control scores were significantly improved among 20 (7%) children with the CYP3A4*22 allele (median 3, range 0-6), as compared with the 201 patients without the CYP3A4*22 allele (median 4, range 0-15) (P=0.02). The presence of CYP3A4*22 was associated with improved asthma control scores by 2.1 points (95% CI: 0.5-3.8).
The presence of CYP3A4*22, which is associated with decreased hepatic CYP3A4 expression and activity, was accompanied by improved asthma control among FP treated children. Decreased CYP3A4 activity may improve asthma control with inhaled FP.
PMCID: PMC3620714  PMID: 23290512
asthma; fluticasone; inhaled glucocorticoids; corticosteroids; children; pharmacogenetics
2.  High-resolution melt analysis to detect sequence variations in highly homologous gene regions: application to CYP2B6 
Pharmacogenomics  2013;14(8):10.2217/pgs.13.66.
High-resolution melt (HRM) analysis using ‘release-on-demand’ dyes, such as EvaGreen® has the potential to resolve complex genotypes in situations where genotype interpretation is complicated by the presence of pseudogenes or allelic variants in close proximity to the locus of interest. We explored the utility of HRM to genotype a SNP (785A>G, K262R, rs2279343) that is located within exon 5 of the CYP2B6 gene, which contributes to the metabolism of a number of clinically used drugs. Testing of 785A>G is challenging, but crucial for accurate genotype determination. This SNP is part of multiple known CYP2B6 haplotypes and located in a region that is identical to CYP2B7, a nonfunctional pseudogene. Because small CYP2B6-specific PCR amplicons bracketing 785A>G cannot be generated, we simultaneously amplified both genes. A panel of 235 liver tissue DNAs and five Coriell samples were assessed. Eight CYP2B6/CYP2B7 diplotype combinations were found and a novel variant 769G>A (D257N) was discovered. The frequency of 785G corresponded to those reported for Caucasians and African–Americans. Assay performance was confirmed by CYP2B6 and/or CYP2B7 sequence analysis in a subset of samples, using a preamplified CYP2B6-specific long-range-PCR amplicon as HRM template. Inclusion rather than exclusion of a homologous pseudogene allowed us to devise a sensitive, reliable and affordable assay to test this CYP2B6 SNP. This assay design may be utilized to overcome the challenges and limitations of other methods. Owing to the flexibility of HRM, this assay design can easily be adapted to other gene loci of interest.
PMCID: PMC3866959  PMID: 23746185
CYP2B6; CYP2B7; genotyping; high-resolution melt analysis; HRM; SNPs
3.  Vitamin D related genes in lung development and asthma pathogenesis 
BMC Medical Genomics  2013;6:47.
Poor maternal vitamin D intake is a risk factor for subsequent childhood asthma, suggesting that in utero changes related to vitamin D responsive genes might play a crucial role in later disease susceptibility. We hypothesized that vitamin D pathway genes are developmentally active in the fetal lung and that these developmental genes would be associated with asthma susceptibility and regulation in asthma.
Vitamin D pathway genes were derived from PubMed and Gene Ontology surveys. Principal component analysis was used to identify characteristic lung development genes.
Vitamin D regulated genes were markedly over-represented in normal human (odds ratio OR 2.15, 95% confidence interval CI: 1.69-2.74) and mouse (OR 2.68, 95% CI: 2.12-3.39) developing lung transcriptomes. 38 vitamin D pathway genes were in both developing lung transcriptomes with >63% of genes more highly expressed in the later than earlier stages of development. In immortalized B-cells derived from 95 asthmatics and their unaffected siblings, 12 of the 38 (31.6%) vitamin D pathway lung development genes were significantly differentially expressed (OR 3.00, 95% CI: 1.43-6.21), whereas 11 (29%) genes were significantly differentially expressed in 43 control versus vitamin D treated immortalized B-cells from Childhood Asthma Management Program subjects (OR 2.62, 95% CI: 1.22-5.50). 4 genes, LAMP3, PIP5K1B, SCARB2 and TXNIP were identified in both groups; each displays significant biologic plausibility for a role in asthma.
Our findings demonstrate a significant association between early lung development and asthma–related phenotypes for vitamin D pathway genes, supporting a genomic mechanistic basis for the epidemiologic observations relating maternal vitamin D intake and childhood asthma susceptibility.
PMCID: PMC4228235  PMID: 24188128
Vitamin D; Cholecalciferol; Lung development; Asthma; Fetal programming
4.  PharmGKB summary: methotrexate pathway 
Pharmacogenetics and Genomics  2011;21(10):679-686.
PMCID: PMC3139712  PMID: 21317831
methotrexate; 5,10-methylenetetrahydrofolate reductase; pathway; pharmacogenomic; SLC19A1; thymidylate synthetase
5.  Global tests of P-values for multifactor dimensionality reduction models in selection of optimal number of target genes 
BioData Mining  2012;5:3.
Multifactor Dimensionality Reduction (MDR) is a popular and successful data mining method developed to characterize and detect nonlinear complex gene-gene interactions (epistasis) that are associated with disease susceptibility. Because MDR uses a combinatorial search strategy to detect interaction, several filtration techniques have been developed to remove genes (SNPs) that have no interactive effects prior to analysis. However, the cutoff values implemented for these filtration methods are arbitrary, therefore different choices of cutoff values will lead to different selections of genes (SNPs).
We suggest incorporating a global test of p-values to filtration procedures to identify the optimal number of genes/SNPs for further MDR analysis and demonstrate this approach using a ReliefF filter technique. We compare the performance of different global testing procedures in this context, including the Kolmogorov-Smirnov test, the inverse chi-square test, the inverse normal test, the logit test, the Wilcoxon test and Tippett’s test. Additionally we demonstrate the approach on a real data application with a candidate gene study of drug response in Juvenile Idiopathic Arthritis.
Extensive simulation of correlated p-values show that the inverse chi-square test is the most appropriate approach to be incorporated with the screening approach to determine the optimal number of SNPs for the final MDR analysis. The Kolmogorov-Smirnov test has high inflation of Type I errors when p-values are highly correlated or when p-values peak near the center of histogram. Tippett’s test has very low power when the effect size of GxG interactions is small.
The proposed global tests can serve as a screening approach prior to individual tests to prevent false discovery. Strong power in small sample sizes and well controlled Type I error in absence of GxG interactions make global tests highly recommended in epistasis studies.
PMCID: PMC3508622  PMID: 22616673
P-value; Global tests; ReliefF; Multifactor dimensionality reduction
6.  Selective Toll-Like Receptor Expression in Human Fetal Lung 
Pediatric research  2010;68(4):335-338.
Toll-like receptors (TLRs) are critical components of the innate immune system, acting as pattern recognition molecules and triggering an inflammatory response. TLR associated gene products are of interest in modulating inflammatory related pulmonary diseases of the neonate. The ontogeny of TLR related genes in human fetal lung has not been previously described and could elucidate additional functions and identify strategies for attenuating the effects of fetal inflammation. We examined the expression of 84 TLR related genes on 23 human fetal lung samples from three groups with estimated ages of 60 (57-59d), 90 (89-91d), and 130 (117-154d) days. Using a false detection rate algorithm, we identified 32 genes displaying developmental regulation with TLR2 having the greatest up-regulation of TLR genes (9.2 fold increase) and TLR4 unchanged. We confirmed the TLR2 up-regulation by examining an additional 133 fetal lung tissue samples with a fluorogenic polymerase chain reaction assay (TaqMan®) and found an exponential best-fit curve over the time studied. The best-fit curve predicts a 6.1 fold increase from 60d to 130d. We conclude that TLR2 is developmentally expressed from the early pseudoglandular stage of lung development to the canalicular stage.
PMCID: PMC2967298  PMID: 20581745
7.  Expression analysis of asthma candidate genes during human and murine lung development 
Respiratory Research  2011;12(1):86.
Little is known about the role of most asthma susceptibility genes during human lung development. Genetic determinants for normal lung development are not only important early in life, but also for later lung function.
To investigate the role of expression patterns of well-defined asthma susceptibility genes during human and murine lung development. We hypothesized that genes influencing normal airways development would be over-represented by genes associated with asthma.
Asthma genes were first identified via comprehensive search of the current literature. Next, we analyzed their expression patterns in the developing human lung during the pseudoglandular (gestational age, 7-16 weeks) and canalicular (17-26 weeks) stages of development, and in the complete developing lung time series of 3 mouse strains: A/J, SW, C57BL6.
In total, 96 genes with association to asthma in at least two human populations were identified in the literature. Overall, there was no significant over-representation of the asthma genes among genes differentially expressed during lung development, although trends were seen in the human (Odds ratio, OR 1.22, confidence interval, CI 0.90-1.62) and C57BL6 mouse (OR 1.41, CI 0.92-2.11) data. However, differential expression of some asthma genes was consistent in both developing human and murine lung, e.g. NOD1, EDN1, CCL5, RORA and HLA-G. Among the asthma genes identified in genome wide association studies, ROBO1, RORA, HLA-DQB1, IL2RB and PDE10A were differentially expressed during human lung development.
Our data provide insight about the role of asthma susceptibility genes during lung development and suggest common mechanisms underlying lung morphogenesis and pathogenesis of respiratory diseases.
PMCID: PMC3141421  PMID: 21699702
Asthma; Development; Expression; Genetics; Lung
8.  A Role for Wnt Signaling Genes in the Pathogenesis of Impaired Lung Function in Asthma 
Rationale: Animal models demonstrate that aberrant gene expression in utero can result in abnormal pulmonary phenotypes.
Objectives: We sought to identify genes that are differentially expressed during in utero airway development and test the hypothesis that variants in these genes influence lung function in patients with asthma.
Methods: Stage 1 (Gene Expression): Differential gene expression analysis across the pseudoglandular (n = 27) and canalicular (n = 9) stages of human lung development was performed using regularized t tests with multiple comparison adjustments. Stage 2 (Genetic Association): Genetic association analyses of lung function (FEV1, FVC, and FEV1/FVC) for variants in five differentially expressed genes were conducted in 403 parent-child trios from the Childhood Asthma Management Program (CAMP). Associations were replicated in 583 parent-child trios from the Genetics of Asthma in Costa Rica study.
Measurements and Main Results: Of the 1,776 differentially expressed genes between the pseudoglandular (gestational age: 7–16 wk) and the canalicular (gestational age: 17–26 wk) stages, we selected 5 genes in the Wnt pathway for association testing. Thirteen single nucleotide polymorphisms in three genes demonstrated association with lung function in CAMP (P < 0.05), and associations for two of these genes were replicated in the Costa Ricans: Wnt1-inducible signaling pathway protein 1 with FEV1 (combined P = 0.0005) and FVC (combined P = 0.0004), and Wnt inhibitory factor 1 with FVC (combined P = 0.003) and FEV1/FVC (combined P = 0.003).
Conclusions: Wnt signaling genes are associated with impaired lung function in two childhood asthma cohorts. Furthermore, gene expression profiling of human fetal lung development can be used to identify genes implicated in the pathogenesis of lung function impairment in individuals with asthma.
PMCID: PMC2822972  PMID: 19926868
asthma; lung development; lung function; genetic variation; gene expression
9.  Transcriptomic Analysis of Human Lung Development 
Rationale: Current understanding of the molecular regulation of lung development is limited and derives mostly from animal studies.
Objectives: To define global patterns of gene expression during human lung development.
Methods: Genome-wide expression profiling was used to measure the developing lung transcriptome in RNA samples derived from 38 normal human lung tissues at 53 to 154 days post conception. Principal component analysis was used to characterize global expression variation and to identify genes and bioontologic attributes contributing to these variations. Individual gene expression patterns were verified by quantitative reverse transcriptase–polymerase chain reaction analysis.
Measurements and Main Results: Gene expression analysis identified attributes not previously associated with lung development, such as chemokine-immunologic processes. Lung characteristics attributes (e.g., surfactant function) were observed at an earlier-than-anticipated age. We defined a 3,223 gene developing lung characteristic subtranscriptome capable of describing a majority of the process. In gene expression space, the samples formed a time-contiguous trajectory with transition points correlating with histological stages and suggesting the existence of novel molecular substages. Induction of surfactant gene expression characterized a pseudoglandular “molecular phase” transition. Individual gene expression patterns were independently validated. We predicted the age of independent human lung transcriptome profiles with a median absolute error of 5 days, supporting the validity of the data and modeling approach.
Conclusions: This study extends our knowledge of key gene expression patterns and bioontologic attributes underlying early human lung developmental processes. The data also suggest the existence of molecular phases of lung development.
PMCID: PMC2797628  PMID: 19815808
microarrays; surfactant; principal component analysis
10.  Human Carboxylesterases HCE1 and HCE2: Ontogenic Expression, Inter-Individual Variability and Differential Hydrolysis of Oseltamivir, Aspirin, Deltamethrin and Permethrin13 
Biochemical pharmacology  2008;77(2):238-247.
Carboxylesterases hydrolyze chemicals containing such functional groups as a carboxylic acid ester, amide and thioester. The liver contains the highest carboxylesterase activity and expresses two major carboxylesterases: HCE1 and HCE2. In this study, we analyzed 104 individual liver samples for the expression patterns of both carboxylesterases. These samples were divided into three age groups: adults (≥ 18 years of age), children (0 days-10 years) and fetuses (82-224 gestation days). In general, the adult group expressed significantly higher HCE1 and HCE2 than the child group, which expressed significantly higher than the fetal group. The age-related expression was confirmed by RT-qPCR and Western immunoblotting. To determine whether the expression patterns reflected the hydrolytic activity, liver microsomes were pooled from each group and tested for the hydrolysis of drugs such as oseltamivir and insecticides such as deltamethrin. Consistent with the expression patterns, adult microsomes were ∼4 times as active as child microsomes and 10 times as active as fetal microsomes in hydrolyzing these chemicals. Within the same age group, particularly in the fetal and child groups, a large inter-individual variability was detected in mRNA (430 fold), protein (100 fold) and hydrolytic activity (127 fold). Carboxylesterases are recognized to play critical roles in drug metabolism and insecticide detoxication. The findings on the large variability among different age groups or even within the same age group have important pharmacological and toxicological implications, particularly in relation to pharmacokinetic alterations of ester drugs in children and vulnerability of fetuses and children to pyrethroid insecticides.
PMCID: PMC2671154  PMID: 18983829
11.  Platform dependence of inference on gene-wise and gene-set involvement in human lung development 
BMC Bioinformatics  2009;10:189.
With the recent development of microarray technologies, the comparability of gene expression data obtained from different platforms poses an important problem. We evaluated two widely used platforms, Affymetrix U133 Plus 2.0 and the Illumina HumanRef-8 v2 Expression Bead Chips, for comparability in a biological system in which changes may be subtle, namely fetal lung tissue as a function of gestational age.
We performed the comparison via sequence-based probe matching between the two platforms. "Significance grouping" was defined as a measure of comparability. Using both expression correlation and significance grouping as measures of comparability, we demonstrated that despite overall cross-platform differences at the single gene level, increased correlation between the two platforms was found in genes with higher expression level, higher probe overlap, and lower p-value. We also demonstrated that biological function as determined via KEGG pathways or GO categories is more consistent across platforms than single gene analysis.
We conclude that while the comparability of the platforms at the single gene level may be increased by increasing sample size, they are highly comparable ontologically even for subtle differences in a relatively small sample size. Biologically relevant inference should therefore be reproducible across laboratories using different platforms.
PMCID: PMC2711081  PMID: 19545372
12.  Variability of CYP2J2 expression in human fetal tissues 
CYP2J2 metabolizes arachidonic acid to 20-HETE and EETs which play a critical role in the regulation of renal, pulmonary, cardiac and vascular function. However, the contribution of CYP2J2 to EET formation in the liver remains poorly characterized. Similarly, information is sparse regarding the extent and variability of CYP2J2 expression during human development. This investigation was undertaken to characterize the variability of CYP2J2 expression in fetal liver, heart, kidney, lung, intestine and brain and postnatal liver samples. CYP2J2 mRNA expression was measured using quantitative PCR, and immunoreactive CYP2J2 was examined using two anti-CYP2J2 antibodies. CYP2J2 mRNA was ubiquitously expressed in pre- and postnatal samples. Fetal hepatic mRNA expression varied 127-fold (1351 ± 717 transcripts/ng total RNA), but this variation was reduced to 8-fold after exclusion of four samples with extremely low levels of mRNA. Amounts of immunoreactive protein also varied substantially among samples without an apparent relationship with transcript number or genotype. Western blot analysis revealed a different protein pattern between prenatal and postnatal liver samples. DNA resequencing of selected subjects identified a single novel SNP (CYP2J2*10), which was found in only one subject and therefore did not explain the large variability in CYP2J2 protein content. In vitro expression suggests that the protein product of CYP2J2*10 confers reduced enzymatic activity. Aberrant splicing produces three minor transcripts which were present in all samples tested. Due to premature termination codons none encodes functional protein. The mechanisms leading to variable amounts of immunoreactive protein and distinct pre- and postnatal CYP2J2 protein patterns warrant further investigation.
PMCID: PMC1876721  PMID: 16868033
13.  Genetic Heterogeneity in the rRNA Gene Locus of Trichophyton tonsurans 
Journal of Clinical Microbiology  2003;41(12):5478-5487.
Trichophyton tonsurans is the major pediatric pathogen in tinea capitis, causing disparate disease presentations. Little is known about genetic variation, which may ultimately be linked to divergent disease status. This investigation was aimed at identifying genetic variants of T. tonsurans by methods that can facilitate strain discrimination in population-based studies. Ninety-two isolates were acquired from six U.S. microbiology laboratories, and genomic DNA was isolated from mature colonies. The nontranscribed spacer (NTS) was amplified by PCR, and products from isolates with various amplicon sizes were fully sequenced. Nested amplification, targeting a variable internal repeat (VIR) region, allowed assignment of variant type by fragment size. Subvariant type was assigned by a combination of PCR-restriction fragment length polymorphism-based assays. Five variants differing in size (348 to 700 bp) and sequence were identified within the VIR region comprised of several large repeats (104, 140, and 194 bp) arranged in tandem. Seven single-nucleotide polymorphisms (SNPs) were detected across the NTS, with five occurring in the constant regions flanking the VIR region and two occurring in the VIR region. Additionally, a 10-bp insertion and a 14-bp deletion were identified upstream of the VIR region. The combination of SNPs revealed seven haplotype patterns which were stable upon serial passage over 1 year. No sequence variations were identified within the internal transcribed spacer regions. Unique NTS sequences were utilized to develop a duplex PCR assay that discriminated T. tonsurans from other dermatophytes. Of the 92 isolates evaluated, this genotyping scheme distinguished 12 distinct strains, providing evidence of genetic heterogeneity in T. tonsurans.
PMCID: PMC309017  PMID: 14662928
14.  Characterization of Mayven, a Novel Actin-binding Protein Predominantly Expressed in Brain 
Molecular Biology of the Cell  1999;10(7):2361-2375.
The cytoskeleton plays an important role in neuronal morphogenesis. We have identified and characterized a novel actin-binding protein, termed Mayven, predominantly expressed in brain. Mayven contains a BTB (broad complex, tramtrack, bric-a-brac)/POZ (poxvirus, zinc finger) domain-like structure in the predicted N terminus and “kelch repeats” in the predicted C-terminal domain. Mayven shares 63% identity (77% similarity) with the Drosophila ring canal (“kelch”) protein. Somatic cell-hybrid analysis indicated that the human Mayven gene is located on chromosome 4q21.2, whereas the murine homolog gene is located on chromosome 8. The BTB/POZ domain of Mayven can self-dimerize in vitro, which might be important for its interaction with other BTB/POZ-containing proteins. Confocal microscopic studies of endogenous Mayven protein revealed a highly dynamic localization pattern of the protein. In U373-MG astrocytoma/glioblastoma cells, Mayven colocalized with actin filaments in stress fibers and in patchy cortical actin-rich regions of the cell margins. In primary rat hippocampal neurons, Mayven is highly expressed in the cell body and in neurite processes. Binding assays and far Western blotting analysis demonstrated association of Mayven with actin. This association is mediated through the “kelch repeats” within the C terminus of Mayven. Depolarization of primary hippocampal neurons with KCl enhanced the association of Mayven with actin. This increased association resulted in dynamic changes in Mayven distribution from uniform to punctate localization along neuronal processes. These results suggest that Mayven functions as an actin-binding protein that may be translocated along axonal processes and might be involved in the dynamic organization of the actin cytoskeleton in brain cells.
PMCID: PMC25454  PMID: 10397770

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