Maternal smoking, xenobiotic metabolizing enzyme gene variants, and gastroschisis risk.

Maternal smoking during pregnancy is one proposed risk factor for gastroschisis, but reported associations have been modest, suggesting that differences in genetic susceptibility might play a role. We included 108 non-Hispanic white and 62 Hispanic families who had infants with gastroschisis, and 1,147 non-Hispanic white and 337 Hispanic families who had liveborn infants with no major structural birth defects (controls) in these analyses. DNA was extracted from buccal cells collected from infants and mothers, and information on periconceptional smoking history was obtained from maternal interviews, as part of the National Birth Defects Prevention Study. We analyzed five polymorphisms in three genes that code for enzymes involved in metabolism of some cigarette smoke constituents (CYP1A1, CYP1A2, and NAT2). Logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) independently for maternal smoking and maternal and infant gene variants, and to assess joint associations of maternal smoking and maternal or infant gene variants with gastroschisis. In analyses adjusted for maternal age at delivery and stratified by maternal race-ethnicity, we identified three suggestive associations among 30 potential associations with sufficient numbers to calculate ORs: CYP1A1*2A for non-Hispanic white mothers who smoked periconceptionally (aOR = 0.38, 95% CI 0.15-0.98), and NAT2*6 for Hispanic non-smoking mothers (aOR = 2.17, 95% CI 1.12-4.19) and their infants (aOR = 2.11, 95% CI 1.00-4.48). This analysis does not support the occurrence of effect modification between periconceptional maternal smoking and most of the xenobiotic metabolizing enzyme gene variants assessed.

Evaluation of mailed pediatric buccal cytobrushes for use in a case-control study of birth defects.

BACKGROUND: Buccal cell collection is a convenient DNA collection method; however, little attention has been given to the quality of DNA obtained from pediatric populations. The purpose of this study was to determine the effect of a modified cytobrush collection method on the yield and quality of infant buccal DNA collected as part of a population-based case-control study of birth defects. METHODS Cytobrushes were collected from infants, mothers, and fathers using a standard collection method in 1997 to 2003 and a modified protocol that allows air-drying of the cytobrushes after collection from 2003 to the present. Yield and quality of DNA from 1057 cytobrushes was assessed by quantitative PCR and short tandem repeat (STR) genotyping, respectively. RESULTS Air-dried cytobrushes from infants had higher median DNA yields (1300 ng) and STR completion rates (99.5%) than standard collection method cytobrushes (60 ng and 59.5%, respectively). A subset of DNA aliquots was genotyped for six single nucleotide polymorphisms (SNPs). Aliquots from both collection methods that passed the quality protocol (DNA concentration >1 ng/µl, and successful amplification of ≥1 STR) had high genotype completion rates (99-100%). The median DNA yield following whole genome amplification was more than twofold higher for air-dried than standard collection specimens (p < 0.001). CONCLUSION Yield and quality of buccal DNA collected from infants are improved by using a method that incorporates air-drying; however, DNA collected by both methods is suitable for genotyping if stringent quality control procedures are instituted. These findings may be helpful for future epidemiologic studies of birth defects and other adverse pediatric outcomes.

Clinical evaluation of two methods for genotyping hepatitis C virus based on analysis of the 5' noncoding region.

We compared the performance characteristics of a standardized direct sequencing method (TRUGENE HCV 5'NC; Visible Genetics Inc., Toronto, Ontario, Canada) and a reverse hybridization line probe assay (INNO-LiPA HCV II; Bayer Corp., Tarrytown, N.Y.) for genotyping of hepatitis C virus (HCV). Both methods are based on detection of sequence heterogeneity in the 5' noncoding (5'NC) region. Concordance between the genotyping methods was assessed by testing 172 samples representing the six major genotypes. Sequence analysis of the more phylogenetically informative nonstructural 5B (NS5B) region was also done with 148 (86%) samples to confirm the accuracy of and resolve discrepancies between the 5'NC genotyping results. The sensitivities of the methods were assessed by using the 5'NC amplicon from both the qualitative and quantitative AMPLICOR HCV tests (Roche Diagnostics Corp., Indianapolis, Ind.). The ability of the methods to detect mixed-genotype infections was determined with mixtures of two different genotypes at relative concentrations ranging from 1 to 50%. Both 5'NC methods were able to genotype 99.4% of the samples with type agreement for 99.5% and subtype agreement for 68.2% of the samples. No or ambiguous subtype results were found by the line probe assay for 16.5% and by the TRUGENE 5'NC test for 17.1% of the samples. Discrepancies occurred between the line probe assay and NS5B results at the type level for 1.4% of the samples and at the subtype level for 14.2% of the samples. Discrepancies also occurred between the TRUGENE 5'NC and NS5B results at the type level for 2% of the samples and at the subtype level for 8.1% of the samples. We also found two distinct strains of HCV classified as type 2 by analysis of the 5'NC region that were type 1 by analysis of the NS5B region. The sensitivities of the two 5'NC genotyping methods were comparable and dependent on the amplification test used ( approximately 10(3) IU/ml with the qualitative HCV RNA tests and approximately 10(5) IU/ml with the quantitative HCV RNA tests). Genotype mixtures were successfully identified at a relative concentration of 5% by the line probe assay and 10% by the TRUGENE 5'NC test. In conclusion, the performance characteristics of the 5'NC methods were similar and both methods produced accurate results at the genotype level but neither method should be used for subtyping.