Intrauterine Smoke Exposure, microRNA Expression during Human Lung Development, and Childhood Asthma.

Intrauterine smoke (IUS) exposure during early childhood has been associated with a number of negative health consequences, including reduced lung function and asthma susceptibility. The biological mechanisms underlying these associations have not been established. MicroRNAs regulate the expression of numerous genes involved in lung development. Thus, investigation of the impact of IUS on miRNA expression during human lung development may elucidate the impact of IUS on post-natal respiratory outcomes. We sought to investigate the effect of IUS exposure on miRNA expression during early lung development. We hypothesized that miRNA-mRNA networks are dysregulated by IUS during human lung development and that these miRNAs may be associated with future risk of asthma and allergy. Human fetal lung samples from a prenatal tissue retrieval program were tested for differential miRNA expression with IUS exposure (measured using placental cotinine concentration). RNA was extracted and miRNA-sequencing was performed. We performed differential expression using IUS exposure, with covariate adjustment. We also considered the above model with an additional sex-by-IUS interaction term, allowing IUS effects to differ by male and female samples. Using paired gene expression profiles, we created sex-stratified miRNA-mRNA correlation networks predictive of IUS using DIABLO. We additionally evaluated whether miRNAs were associated with asthma and allergy outcomes in a cohort of childhood asthma. We profiled pseudoglandular lung miRNA in n = 298 samples, 139 (47%) of which had evidence of IUS exposure. Of 515 miRNAs, 25 were significantly associated with intrauterine smoke exposure (q-value < 0.10). The IUS associated miRNAs were correlated with well-known asthma genes (e.g., ORM1-Like Protein 3, ORDML3) and enriched in disease-relevant pathways (oxidative stress). Eleven IUS-miRNAs were also correlated with clinical measures (e.g., Immunoglobulin E andlungfunction) in children with asthma, further supporting their likely disease relevance. Lastly, we found substantial differences in IUS effects by sex, finding 95 significant IUS-miRNAs in male samples, but only four miRNAs in female samples. The miRNA-mRNA correlation networks were predictive of IUS (AUC = 0.78 in males and 0.86 in females) and suggested that IUS-miRNAs are involved in regulation of disease-relevant genes (e.g., A disintegrin and metalloproteinase domain 19 (ADAM19), LBH regulator of WNT signaling (LBH)) and sex hormone signaling (Coactivator associated methyltransferase 1(CARM1)). Our study demonstrated differential expression of miRNAs by IUS during early prenatal human lung development, which may be modified by sex. Based on their gene targets and correlation to clinical asthma and atopy outcomes, these IUS-miRNAs may be relevant for subsequent allergy and asthma risk. Our study provides insight into the impact of IUS in human fetal lung transcriptional networks and on the developmental origins of asthma and allergic disorders.

Co-methylation analysis in lung tissue identifies pathways for fetal origins of COPD.

COPD likely has developmental origins; however, the underlying molecular mechanisms are not fully identified. Investigation of lung tissue-specific epigenetic modifications such as DNA methylation using network approaches might facilitate insights linking in utero smoke (IUS) exposure and risk for COPD in adulthood.We performed genome-wide methylation profiling for adult lung DNA from 160 surgical samples and 78 fetal lung DNA samples isolated from discarded tissue at 8-18 weeks of gestation. Co-methylation networks were constructed to identify preserved modules that shared methylation patterns in fetal and adult lung tissues and associations with fetal IUS exposure, gestational age and COPD.Weighted correlation networks highlighted preserved and co-methylated modules for both fetal and adult lung data associated with fetal IUS exposure, COPD and lower adult lung function. These modules were significantly enriched for genes involved in embryonic organ development and specific inflammation-related pathways, including Hippo, phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), Wnt, mitogen-activated protein kinase and transforming growth factor-ß signalling. Gestational age-associated modules were remarkably preserved for COPD and lung function, and were also annotated to genes enriched for the Wnt and PI3K/AKT pathways.Epigenetic network perturbations in fetal lung tissue exposed to IUS and of early lung development recapitulated in adult lung tissue from ex-smokers with COPD. Overlapping fetal and adult lung tissue network modules highlighted putative disease pathways supportive of exposure-related and age-associated developmental origins of COPD.

Developmental Expression of SULT1C4 Transcript Variants in Human Liver: Implications for Discordance Between SULT1C4 mRNA and Protein Levels.

The cytosolic sulfotransferases (SULTs) metabolize a variety of xenobiotic and endogenous substrates. Several SULTs are expressed in the fetus, implying that these enzymes have important functions during human development. We recently reported that while SULT1C4 mRNA is abundant in prenatal human liver specimens, SULT1C4 protein is barely detectable. Two coding transcript variants (TVs) of SULT1C4 are indexed in GenBank, TV1 (full-length) and TV2 (lacking exons 3 and 4). The purpose of this study was to evaluate expression of the individual TVs as a clue for understanding the discordance between mRNA and protein levels. Reverse-transcription polymerase chain reaction was initially performed to identify TVs expressed in intestinal and hepatic cell lines. This analysis generated fragments corresponding to TV1, TV2, and a third variant that lacked exon 3 (E3DEL). Using reverse-transcription quantitative polymerase chain reaction assays designed to quantify TV1, TV2, or E3DEL individually, all three TVs were more highly expressed in prenatal than postnatal specimens. TV2 levels were ∼fivefold greater than TV1, while E3DEL levels were minimal. RNA sequencing (RNA-seq) analysis of another set of liver specimens confirmed that TV1 and TV2 levels were highest in prenatal liver, with TV2 higher than TV1. RNA-seq also detected a noncoding RNA, which was also more abundant in prenatal liver. Transfection of HEK293T cells with plasmids expressing individual Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-tagged SULT1C4 isoforms demonstrated that TV1 produced much more protein than did TV2. These data suggest that the lack of correspondence between SULT1C4 mRNA and protein levels in human liver is likely attributable to the inability of the more abundant TV2 to produce stable protein. SIGNIFICANCE STATEMENT: Cytosolic sulfotransferases (SULTs) metabolize a variety of xenobiotic and endogenous substrates, and several SULTs are highly expressed in the fetus, implying that they have important functions during human development. SULT1C4 is highly expressed in prenatal liver at the mRNA level but not the protein level. This study provides an explanation for this discordance by demonstrating that the predominant SULT1C4 transcript is a variant that produces relatively little protein.

Developmental Expression of the Cytosolic Sulfotransferases in Human Liver.

The liver is the predominant organ of metabolism for many endogenous and foreign chemicals. Cytosolic sulfotransferases (SULTs) catalyze the sulfonation of drugs and other xenobiotics, as well as hormones, neurotransmitters, and sterols, with consequences that include enhanced drug elimination, hormone inactivation, and procarcinogen bioactivation. SULTs are classified into six gene families, but only SULT1 and SULT2 enzymes are expressed in human liver. We characterized the developmental expression patterns of SULT1 and SULT2 mRNAs and proteins in human liver samples using reverse transcription quantitative polymerase chain reaction (RT-qPCR), RNA sequencing, and targeted quantitative proteomics. Using a set of prenatal, infant, and adult liver specimens, RT-qPCR analysis demonstrated that SULT1A1 (transcript variant 1) expression did not vary appreciably during development; SULT1C2, 1C4, and 1E1 mRNA levels were highest in prenatal and/or infant liver, and 1A2, 1B1, and 2A1 mRNA levels were highest in infant and/or adult. Hepatic SULT1A1 (transcript variant 5), 1C3, and 2B1 mRNA levels were low regardless of developmental stage. Results obtained with RNA sequencing of a different set of liver specimens (prenatal and pediatric) were generally comparable results to those of the RT-qPCR analysis, with the additional finding that SULT1A3 expression was highest during gestation. Analysis of SULT protein content in a library of human liver cytosols demonstrated that protein levels generally corresponded to the mRNAs, with the major exception that SULT1C4 protein levels were much lower than expected based on mRNA levels. These findings further support the concept that hepatic SULTs play important metabolic roles throughout the human life course, including early development.

Evaluating metronidazole as a novel, safe CYP2A6 phenotyping probe in healthy adults.

AIMS: CYP2A6 is a genetically polymorphic enzyme resulting in differential substrate metabolism and health behaviours. Current phenotyping probes for CYP2A6 exhibit limitations related to procurement (deuterated cotinine), toxicity (coumarin), specificity (caffeine) and age-appropriate administration (nicotine, NIC). In vitro, CYP2A6 selectively forms 2-hydroxymetronidazole (2HM) from metronidazole (MTZ). The purpose of this study was to evaluate MTZ as a CYP2A6 phenotyping probe drug in healthy adults against the well-established method of measuring trans-3-hydroxycotinine (3HC)/cotinine (COT). METHODS: A randomized, cross-over, pharmacokinetic study was completed in 16 healthy, nonsmoking adults. Separated by a washout period of at least 2 weeks, MTZ 500 mg and NIC gum 2 mg were administered and plasma was sampled over 48 hours and 8 hours, respectively. Correlations of plasma metabolite/parent ratios (2HM/MTZ; 3HC/COT) were assessed by Pearson coefficient. CYP2A6 genotyping was conducted and incorporated as a variable of plasma ratio response. RESULTS: Correlations between the plasma ratio 2HM/MTZ and 3HC/COT were ≥ 0.9 at multiple time points (P < 0.001), demonstrating a wide window during which 2HM/MTZ can be queried post-MTZ dose. CYP2A6 genotype had significant impacts on both MTZ and NIC phenotyping ratios with decreased activity predicted phenotypes demonstrating 2HM/MTZ ratios ≤58% and 3HC/COT ratios ≤56% compared with extensive activity predicted phenotypes at all time points examined in the study (P < 0.05). No adverse events were reported in the MTZ arm while 38% (n = 6) of participants reported mild adverse events in the NIC arm. CONCLUSIONS: Metronidazole via 2HM/MTZ performed well as a novel, safe phenotyping probe for CYP2A6 in healthy adults.

Age, Sexual Dimorphism, and Disease Associations in the Developing Human Fetal Lung Transcriptome.

The fetal origins of disease hypothesis suggests that variations in the course of prenatal lung development may affect life-long pulmonary function growth, decline, and pathobiology. Many studies support the existence of differences in the developing lung trajectory in males and females, and sex-specific differences in the prevalence of chronic lung diseases, such as asthma and bronchopulmonary dysplasia. The objectives of this study were to investigate the early developing fetal lung for transcriptomic correlates of postconception age (maturity) and sex, and their associations with chronic lung diseases. We analyzed whole-lung transcriptome profiles of 61 females and 78 males at 54-127 days postconception (dpc) from nonsmoking mothers using unsupervised principal component analysis and supervised linear regression models. We identified dominant transcriptomic correlates for postconception age and sex with corresponding gene sets that were enriched for developing lung structural and functional ontologies. We observed that the transcriptomic sex difference was not a uniform global time shift/lag, rather, lungs of males appear to be more mature than those of females before 96 dpc, and females appear to be more mature than males after 96 dpc. The age correlate gene set was consistently enriched for asthma and bronchopulmonary dysplasia genes, but the sex correlate gene sets were not. Despite sex differences in the developing fetal lung transcriptome, postconception age appears to be more dominant than sex in the effect of early fetal lung developments on disease risk during this early pseudoglandular phase of development.

Dynamics of Cytosine Methylation in the Proximal Promoters of CYP3A4 and CYP3A7 in Pediatric and Prenatal Livers.

Members of the human CYP3A family of metabolizing enzymes exhibit developmental changes in expression whereby CYP3A7 is expressed in fetal tissues, followed by a transition to expression of CYP3A4 in the first months of life. Despite knowledge about the general pattern of CYP3A activity in human development, the mechanisms that regulate developmental expression remain poorly understood. Epigenetic changes, including cytosine methylation, have been suggested to play a role in the regulation of CYP3A expression. The objective of this study was to investigate changes in cytosine methylation of the CYP3A4 and CYP3A7 genes in human pediatric and prenatal livers. The methylation status of cytosine-phospho-guanine dinucleotides was determined in 16 pediatric liver samples using methyl-seq and confirmed by bisulfite sequencing of 48 pediatric and 34 prenatal liver samples. Samples were separated by age into five groups (prenatal, < 1 year of age, 1.8-6 years, 7-11 years, and 12-17 years). Methyl-seq anaylsis revealed that cytosines in the proximal promoter of CYP3A7 are hypomethylated in neonates compared with adolescents (P < 0.001). In contrast, a cytosine 383 base pair upstream of CYP3A4 is hypermethylated in liver samples from neonates compared with adolescents (P = 0.00001). Developmental changes in methylation of cytosines in the proximal promoters of CYP3A4 and CYP3A7 in pediatric livers were confirmed by bisulfite sequencing. In addition, the methylation status of cytosine in the CYP3A4 and CYP3A7 proximal promoters correlated with changes in developmental expression of mRNA for the two enzymes.

Decreased Pregnane X Receptor Expression in Children with Active Crohn's Disease.

Expression of the pregnane X receptor (PXR) has been reported to be decreased in animal models of inflammatory bowel disease (IBD). To investigate the differential expression of PXR in children with Crohn's disease, a type of IBD, RNA was extracted from archived intestinal biopsies from 18 children with Crohn's disease (CD) and 12 age- and sex-matched controls (aged 7-17yrs). The aim of this investigation was to compare the relative mRNA expression of PXR, cytochrome p450 3A4 (CYP3A4), and villin 1 (VIL1) (a marker of epithelial cell integrity) in the inflamed terminal ileum (TI) versus noninflamed duodenum of children with CD. Relative expression was determined via reverse transcription real-time quantitative polymerase chain reaction, data normalized to glyceraldehyde 3-phosphate dehydrogenase, and differences in gene expression explored via paired t tests. PXR expression was decreased in the inflamed TI versus noninflamed duodenum (TI = 1.88 ± 0.89 versus duodenum = 2.5 ± 0.67; P < 0.001) in CD, but not controls (TI = 2.11 ± 0.41 versus duodenum = 2.26 ± 0.61; P = 0.52). CYP3A4 expression was decreased in CD (TI = -0.89 ± 3.11 versus duodenum = 1.90 ± 2.29; P < 0.05), but not controls (TI = 2.46 ± 0.51 versus duodenum = 2.60 ± 0.60; P = 0.61), as was VIL1 (CD TI = 3.80 ± 0.94 versus duodenum = 4.61 ± 0.52; P < 0.001; controls TI = 4.30 ± 0.35 versus duodenum = 4.47 ± 0.40; P = 0.29). PXR expression correlated with VIL1 (r = 0.78, P = 0.01) and CYP3A4 (r = 0.52, P = 0.01) expression. In conclusion, PXR, CYP3A4, and VIL1 expression was decreased only in the actively inflamed small intestinal tissue in children with CD. Our findings suggest that inflammation has the potential to influence expression of genes, and potentially intestinal proteins, important to drug disposition and response. The observed differential patterns of gene expression support further investigation of the role of PXR in the pathogenesis and/or treatment of pediatric Crohn's disease.

Genetic Variation along the Histamine Pathway in Children with Allergic versus Nonallergic Asthma.

Histamine is an important mediator in the pathogenesis of asthma. Variation in genes along the histamine production, response, and degradation pathway may be important in predicting response to antihistamines. We hypothesize that differences exist among single-nucleotide polymorphisms (SNPs) in genes of the histamine pathway between children with allergic versus nonallergic asthma. Children (7-18 yr of age; n = 202) with asthma were classified as allergic or nonallergic based on allergy skin testing. Genotyping was performed to detect known SNPs (n = 10) among genes (HDC, HNMT, ABP1, HRH1, and HRH4) within the histamine pathway. Chi square tests and Cochran-Armitage Trend were used to identify associations between genetic variants and allergic or nonallergic asthma. Significance was determined by P < 0.05 and false-positive report probability. After correction for race differences in genotype were observed, HRH1-17 TT (6% allergic versus 0% nonallergic; P = 0.04), HNMT-464 TT (41% allergic versus 29% nonallergic; P = 0.04), and HNMT-1639 TT (30% allergic versus 20% nonallergic; P = 0.04) were overrepresented among children with allergic asthma. Genotype differences specifically among the African-American children were also observed: HRH1-17 TT (13% allergic versus 0% nonallergic; P = 0.04) and HNMT-1639 TT (23% allergic versus 3% nonallergic; P = 0.03) genotypes were overrepresented among African-American children with allergic asthma. Our study suggests that genetic variation within the histamine pathway may be associated with an allergic versus nonallergic asthma phenotype. Further studies are needed to determine the functional significance of identified SNPs and their impact on antihistamine response in patients with asthma and allergic disease.

Glucocorticoid genes and the developmental origins of asthma susceptibility and treatment response.

Antenatal corticosteroids enhance lung maturation. However, the importance of glucocorticoid genes on early lung development, asthma susceptibility, and treatment response remains unknown. We investigated whether glucocorticoid genes are important during lung development and their role in asthma susceptibility and treatment response. We identified genes that were differentially expressed by corticosteroids in two of three genomic datasets: lymphoblastoid cell lines of participants in the Childhood Asthma Management Program, a glucocorticoid chromatin immunoprecipitation/RNA sequencing experiment, or a murine model; these genes made up the glucocorticoid gene set (GCGS). Using gene expression profiles from 38 human fetal lungs and C57BL/6J murine fetal lungs, we identified developmental genes that were in the top 5% of genes contributing to the top three principal components (PCs) most highly associated with post-conceptional age. Glucocorticoid genes that were enriched in this set of developmental genes were then included in the developmental glucocorticoid gene set (DGGS). We then investigated whether glucocorticoid genes are important during lung development, and their role in asthma susceptibility and treatment response. A total of 232 genes were included in the GCGS. Analysis of gene expression demonstrated that glucocorticoid genes were enriched in lung development (P = 7.02 × 10(-26)). The developmental GCGS was enriched for genes that were differentially expressed between subjects with asthma and control subjects (P = 4.26 × 10(-3)) and were enriched after treatment of subjects with asthma with inhaled corticosteroids (P < 2.72 × 10(-4)). Our results show that glucocorticoid genes are overrepresented among genes implicated in fetal lung development. These genes influence asthma susceptibility and treatment response, suggesting their involvement in the early ontogeny of asthma.

CYP3A isoforms in Ewing's sarcoma tumours: an immunohistochemical study with clinical correlation.

Ewing's sarcoma is an aggressive malignancy of bone and soft tissue with high incidence of metastasis and resistance to chemotherapy. Cytochrome P450 (CYP) monooxygenases are a family of enzymes that are involved in the metabolism of exogenous and endogenous compounds, including anti-cancer drugs, and have been implicated in the aggressive behaviour of various malignancies. Tumour samples and clinical information including age, sex, tumour site, tumour size, clinical stage and survival were collected from 36 adult and paediatric patients with Ewing's sarcoma family tumours. Tissue microarrays slides were processed for immunohistochemical labelling for CYP3A4, CYP3A5 and CYP3A7 using liver sections as positive control. The intensity of staining was scored as negative, low or high expression and was analysed statistically for any association with patients' clinical information. Four cases were later excluded due to inadequate viable tissue. CYP3A4 staining was present in 26 (81%) cases with high expression noted in 13 (40%) of 32 cases. High expression was significantly associated with distant metastases (P < 0.05). CYP3A5 and CYP3A7 were expressed in 5 and 13 cases respectively (15.6%, 40.6%). There was no association between the expression of CYP3A isoforms and age, sex, tumour size, or location (pelvic or extra-pelvic). None of the biomarkers showed any correlation with overall or disease-free survival. In conclusion, expression of CYP3A isoforms is noted in Ewing's sarcoma tumours and high CYP3A4 expression may be associated with metastasis. Additional studies are needed to further investigate the role of CYP3A4 in the prognosis of these tumours.

Variability in Expression of CYP3A5 in Human Fetal Liver.

Members of the cytochrome P450 3A (CYP3A) subfamily of drug metabolizing enzymes exhibit developmental changes in expression in human liver characterized by a transition between CYP3A7 and CYP3A4 over the first few years of life. In contrast, the developmental expression of CYP3A5 is less well understood due to polymorphic expression of the enzyme in human tissues as a result of the prevalence of the CYP3A5*3 allele, which leads to alternative splicing. We further explored the expression of CYP3A5 and the impact of alternative splicing on the variability of CYP3A5 functional activity in a large bank of human prenatal liver samples (7 to 32 weeks of age postconception). The expression of normally spliced CYP3A5 mRNA in all human fetal liver samples varied 235-fold whereas CYP3A5 SV1 mRNA was only detected in fetal liver samples with at least one CYP3A5*3 allele. Formation of 1'-OH midazolam (MDZ) varied 79-fold, and the ratio of 1'-OH MDZ to 4-OH MDZ varied 8-fold and depended on the presence or absence of the CYP3A5*3 allele. Formation of 4-OH MDZ was significantly associated with 1'-OH MDZ (r(2) = 0.76, P < 0.0001) but varied (36-fold) independently of CYP3A5 genotype or expression. The substantial interindividual variability that remains even after stratification for CYP3A5 genotype suggests that factors such as environmental exposure and epigenetic alterations act in addition to genetic variation to contribute to the variability of CYP3A5 expression in human prenatal liver.

Genetic variation within the histamine pathway among patients with asthma--a pilot study.

OBJECTIVE: Histamine is an important mediator in the pathophysiology of asthma. We have previously reported that HRH1 is differentially expressed among those with asthma compared to those without asthma. Single histamine-related genes have also been associated with asthma. We aimed to evaluate known single nucleotide polymorphisms (SNPs) in genes along the histamine biotransformation and response pathway, and determine their association with asthma and HRH1 mRNA expression. METHODS: We enrolled children and adults (n = 93) with/without asthma who met inclusion/exclusion criteria. Genotyping was performed for nine known SNPs in the HDC, HRH1, HRH4, HNMT and ABP1 genes. HRH1 mRNA expression was determined on RNA from buccal tissue. General linear model, Fisher's exact test and Chi-square test were used to determine differences in allele, genotype and haplotype frequency between subjects with and without asthma and differential HRH1 mRNA expression relative to genotype. Statistical significance was determined by p < 0.05. RESULTS: No difference was observed in genotype/allele frequency for the nine SNPs between subjects with and without asthma. The HNMT-1639C/-464C/314C/3'UTRA haplotype was more frequently observed in those without asthma than those with asthma (p = 0.03). We also observed genetic differences relative to race and gender. HNMT 314 genotype CT was more frequent in males with asthma compared to those without asthma (p = 0.04). CONCLUSIONS: Histamine pathway haplotype was associated with a diagnosis of asthma in our cohort but allele and genotype were not. Subgroup evaluations may also be important. Further studies are needed to determine the potential biological/clinical significance of our findings.

Genomic insights into pediatric intestinal inflammatory and eosinophilic disorders using single-cell RNA-sequencing.

Introduction: Chronic inflammation of the gastrointestinal tissues underlies gastrointestinal inflammatory disorders, leading to tissue damage and a constellation of painful and debilitating symptoms. These disorders include inflammatory bowel diseases (Crohn's disease and ulcerative colitis), and eosinophilic disorders (eosinophilic esophagitis and eosinophilic duodenitis). Gastrointestinal inflammatory disorders can often present with overlapping symptoms necessitating the use of invasive procedures to give an accurate diagnosis. Methods: This study used peripheral blood mononuclear cells from individuals with Crohn's disease, ulcerative colitis, eosinophilic esophagitis, and eosinophilic duodenitis to better understand the alterations to the transcriptome of individuals with these diseases and identify potential markers of active inflammation within the peripheral blood of patients that may be useful in diagnosis. Single-cell RNA-sequencing was performed on peripheral blood mononuclear cells isolated from the blood samples of pediatric patients diagnosed with gastrointestinal disorders, including Crohn's disease, ulcerative colitis, eosinophilic esophagitis, eosinophilic duodenitis, and controls with histologically healthy gastrointestinal tracts. Results: We identified 730 (FDR < 0.05) differentially expressed genes between individuals with gastrointestinal disorders and controls across eight immune cell types. Discussion: There were common patterns among GI disorders, such as the widespread upregulation of MTRNR2L8 across cell types, and many differentially expressed genes showed distinct patterns of dysregulation among the different gastrointestinal diseases compared to controls, including upregulation of XIST across cell types among individuals with ulcerative colitis and upregulation of Th2-associated genes in eosinophilic disorders. These findings indicate both overlapping and distinct alterations to the transcriptome of individuals with gastrointestinal disorders compared to controls, which provide insight as to which genes may be useful as markers for disease in the peripheral blood of patients.

Recommendations for Method Development and Validation of qPCR and dPCR Assays in Support of Cell and Gene Therapy Drug Development.

The emerging use of qPCR and dPCR in regulated bioanalysis and absence of regulatory guidance on assay validations for these platforms has resulted in discussions on lack of harmonization on assay design and appropriate acceptance criteria for these assays. Both qPCR and dPCR are extensively used to answer bioanalytical questions for novel modalities such as cell and gene therapies. Following cross-industry conversations on the lack of information and guidelines for these assays, an American Association of Pharmaceutical Scientists working group was formed to address these gaps by bringing together 37 industry experts from 24 organizations to discuss best practices to gain a better understanding in the industry and facilitate filings to health authorities. Herein, this team provides considerations on assay design, development, and validation testing for PCR assays that are used in cell and gene therapies including (1) biodistribution; (2) transgene expression; (3) viral shedding; (4) and persistence or cellular kinetics of cell therapies.

Cotinine in human placenta predicts induction of gene expression in fetal tissues.

Maternal cigarette smoking during pregnancy is associated with increased risk of perinatal morbidity and mortality. However, the mechanisms underlying adverse birth outcomes following prenatal exposure to cigarette smoke remain unknown due, in part, to the absence or unreliability of information regarding maternal cigarette smoke exposure during pregnancy. Our goal was to determine if placental cotinine could be a reliable biomarker of fetal cigarette smoke exposure during pregnancy. Cotinine levels were determined in placentas from 47 women who reported smoking during pregnancy and from 10 women who denied cigarette smoke exposure. Cotinine levels were significantly higher in placentas from women reporting cigarette smoking (median = 27.2 ng/g) versus women who reported no smoke exposure (2.3 ng/g, P < 0.001). Receiver operating characteristic curve analysis identified an optimal cut point of 7.5 ng/g (sensitivity = 78.7%, specificity = 100%) to classify placenta samples from mothers who smoked versus those from mothers who did not. Among 415 placentas for which maternal cigarette smoking status was unavailable, 167 had cotinine levels > 7.5 ng/g and would be considered positive for cigarette smoke exposure. Data from quantitative reverse-transcription polymerase chain reaction analyses demonstrated that in utero cigarette smoke exposure predicted by cotinine in placenta is associated with changes in the expression of xenobiotic-metabolizing enzymes in fetal tissues. CYP1A1 mRNA in fetal lung and liver tissue and CYP1B1 mRNA in fetal lung tissue were significantly induced when cotinine was detected in placenta. These findings indicate that cotinine in placenta is a reliable biomarker for fetal exposure and response to maternal cigarette smoking during pregnancy.

Detecting gene expression in buccal mucosa in subjects with asthma versus subjects without asthma.

BACKGROUND: Differences in mRNA expression for inflammatory markers have been observed between subjects with asthma vs. controls and in relation to corticosteroid response. However, these studies utilized methods (e.g., bronchoscopy) that are too invasive to be used routinely in children and in the clinic. The primary purpose of this study was to determine the feasibility of obtaining RNA of adequate quantity and quality from buccal mucosa of children and adults for gene expression studies. Secondly, this study aimed to determine whether gene expression patterns in buccal mucosa are similar to those that have been observed in respiratory epithelium. METHODS: We enrolled 94 subjects with and without asthma between 5 and 54 years of age. Relative gene expression in buccal mucosa was determined with quantitative RT-PCR for the following genes: CCL2, EDN1, FKBP5, IL8, IFNAR2, NFKB1, RELA, SERPINB2, DENND1B, HRH1, ICAM1, ORMDL3, NR3C1, CLCA1, CRHR1, MUC5B, FCER2, POSTN, GAPDH, PPIA. RESULTS: mRNA Expression of the following genes was detected in buccal mucosa: CCL2, EDN1, FKBP5, IL8, IFNAR2, NFKB1, RELA, SERPINB2, DENND1B, HRH1, ICAM1, ORMDL3, NR3C1, GAPDH, PPIA. HRH1 was differentially expressed in adults with asthma vs. controls (p = 0.04), and EDN1 was differentially expressed in children with asthma vs. controls 12-18 years old (p = 0.03). A similar trend for HRH1 was observed in children 12-18 years old. CONCLUSIONS: Buccal mucosa sampling is a reliable method for detecting changes in gene expression in patients with asthma. This non-invasive technique may serve as a valuable tool for diagnosing asthma and evaluating therapeutic response.

Mixed modeling and sample size calculations for identifying housekeeping genes.

Normalization of gene expression data using internal control genes that have biologically stable expression levels is an important process for analyzing reverse transcription polymerase chain reaction data. We propose a three-way linear mixed-effects model to select optimal housekeeping genes. The mixed-effects model can accommodate multiple continuous and/or categorical variables with sample random effects, gene fixed effects, systematic effects, and gene by systematic effect interactions. We propose using the intraclass correlation coefficient among gene expression levels as the stability measure to select housekeeping genes that have low within-sample variation. Global hypothesis testing is proposed to ensure that selected housekeeping genes are free of systematic effects or gene by systematic effect interactions. A gene combination with the highest lower bound of 95% confidence interval for intraclass correlation coefficient and no significant systematic effects is selected for normalization. Sample size calculation based on the estimation accuracy of the stability measure is offered to help practitioners design experiments to identify housekeeping genes. We compare our methods with geNorm and NormFinder by using three case studies. A free software package written in SAS (Cary, NC, U.S.A.) is available at http://d.web.umkc.edu/daih under software tab.

Genetic variation in the TNFA promoter region and TNFA gene expression in subjects with asthma.

OBJECTIVE: Asthma is a chronic disease that affects millions of people. Messenger RNA (mRNA) expression of specific inflammatory markers has been associated with asthma and corticosteroid response. Tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine, has been shown to have increased expression in airways of asthmatics and may be related to corticosteroid sensitivity. The purpose of this study was to determine how genetic variants within the promoter region of the TNFA gene differ between subjects with asthma and controls. We also investigated how genetic variation affects gene expression. METHODS: We enrolled 94 subjects between 5 to 54 years of age who met the inclusion and exclusion criteria. TNFA mRNA expression was determined by qRT-PCR on total RNA isolated from the buccal mucosa. Genotyping was performed for TNFA-1031T/C, -857C/T, and -308G/A on genomic DNA isolated from blood with commercially available assays. Gene expression was log-2 transformed and corrected with 2 normalization genes. General linear model, Chi-square test, Fisher's exact test, and Cochran-Mantel-Haenszel test were performed with p < .05. RESULTS: The TNFA-857C/T polymorphism is associated with asthma in this cohort. The TNFA-857 T allele is underrepresented in pediatric subjects with asthma relative to those without asthma (3% and 29% of individuals, respectively, p = .01). Furthermore, a TNFA haplotype combination containing -1031T/-857C/-308G and -1031T/-857T/-308G is associated with lower expression of TNF-α mRNA (p = .01) in pediatric subjects. CONCLUSIONS: Presence of the TNFA-857T allele may be protective in the development of asthma and a haplotype combination that contains the TNFA-857T allele is associated with TNFA expression.

A Validatable Droplet Digital Polymerase Chain Reaction Assay for the Detection of Adeno-Associated Viral Vectors in Bioshedding Studies of Tears.

The use of viral vectors to treat genetic diseases has increased substantially in recent years, with over 2,000 studies registered to date. Adeno-associated viral (AAV) vectors have found particular success in the treatment of eye related diseases, as exemplified by the approval of voretigene neparvovec-rzyl. To bring new therapies to market, regulatory agencies typically request qualified or validated bioshedding studies to evaluate release of the vector into the environment. However, no official guidelines for the development of molecular based assays to support such shedding studies have been released by the United States Food and Drug Administration, leaving developers to determine best practices for themselves. The purpose of this protocol is to present a validatable protocol for the detection of AAV vectors in human tears by droplet digital polymerase chain reaction (ddPCR) in support of clinical bioshedding studies. This manuscript discusses current industry approaches to molecular assay validation and demonstrates that the method exceeds the target assay acceptance criteria currently proposed in white papers. Finally, steps critical in the performance of any ddPCR assay, regardless of application, are discussed.