Lizhi Yu

Developmentally restricted genetic determinants of human arsenic metabolism: Association between urinary methylated arsenic and CYT19 polymorphisms in children

We report the results of a screen for genetic association with urinary arsenic metabolite levels in three arsenic metabolism candidate genes, PNP, GSTO, and CYT19, in 135 arsenic-exposed subjects from the Yaqui Valley in Sonora, Mexico, who were exposed to drinking water concentrations ranging from 5.5 to 43.3 ppb. We chose 23 polymorphic sites to test in the arsenic-exposed population. Initial phenotypes evaluated included the ratio of urinary inorganic arsenic(III) to inorganic arsenic(V) and the ratio of urinary dimethylarsenic(V) to monomethylarsenic(V) (D:M). In the initial association screening, three polymorphic sites in the CYT19 gene were significantly associated with D:M ratios in the total population. Subsequent analysis of this association revealed that the association signal for the entire population was actually caused by an extremely strong association in only the children (7–11 years of age) between CYT19 genotype and D:M levels. With children removed from the analysis, no significant genetic association was observed in adults (18–79 years). The existence of a strong, developmentally regulated genetic association between CYT19 and arsenic metabolism carries import for both arsenic pharmacogenetics and arsenic toxicology, as well as for public health and governmental regulatory officials.

Association between exposure to farming, allergies and genetic variation in CARD4/NOD1

Background:  Caspase recruitment domain protein (CARD) 4 has been recently identified as an intracellular pattern recognition receptor that interacts with muropeptides found in common Gram‐negative bacteria. We therefore aimed to explore whether the previously observed inverse association between exposure to microbial products and asthma and allergies in childhood is modified by genetic variation in CARD4.

Genetic regulation of rejection and survival following human lung transplantation by the innate immune receptor CD14.

We have developed the hypothesis that genetic polymorphisms which alter the expression or function of innate immune receptors contribute to the marked interindividual differences in the onset and severity of lung transplant rejection. In this analysis, we considered the effects of a common promotor polymorphism of the lipopolysaccharide receptor CD14 associated with increased transcriptional activity upon the development of posttransplant rejection and graft survival. Genotyping was performed in 226 lung transplant recipients well characterized with regards to clinical outcomes. An earlier onset of acute rejection, bronchiolitis obliterans syndrome (BOS) and worse posttransplant graft survival due to greater BOS related deaths was evident in patients with the CD14 –159 TT genotype (TT). The adverse effect upon graft survival of the TT genotype remained significant in a multivariate Cox model (Hazard Ratio 1.65, 95% CI, 1.03–2.64, p‐value = 0.04) after adjusting for other important covariates. Furthermore, TT patients have significantly greater sCD14, TNF‐α and IFN‐γ in the peripheral blood implying a heightened state of innate immune activation drives the development of increased post‐transplant rejection. Inhibition of innate immune activation through CD14 represents a novel and potentially important therapeutic target to prevent post‐transplant rejection and improve outcomes after human lung transplantation.

Automated high-throughput sex-typing assay

Determining the sex of the donor of a human DNA sample in a rapid and accurate manner is of importance in several settings, including basic research, clinical practice, and forensics (1,2). Our group routinely performs high-throughput ge-notyping within large epidemiological studies. It is not uncommon to receive thousands of DNA samples, with the tube identification as the only accompanying information. These samples will then be distributed through a chain of multi-well plates, ultimately ending in 384-well PCR plates in which there is no individual identification for a given sample, only a plate identifier. In such cases, a basic test of incoming sample identification integrity and of sample processing/tracking integrity is to sex type both the original DNA sample tubes as well as the final 384-well reaction plates. These data are compared to the sex of the study participant , which is recorded in the database storing the epidemiological information, providing basic surveillance for significant sample tracking and identification problems. Recently, Gold et al. (3) published a multiplex PCR-based assay designed to produce a 475-bp fragment from the ABCD1 gene (X-chromosome) and a 231-bp fragment from the SRY gene (Y-chromosome). As the authors point out, this assay design has significant advantages, including the lack of nested primers and genomic targeting to unique, reliable sequences. In this report, we describe a significant refinement of this assay, in which these genetic regions are targeted in an end-point-based 5′-exonuclease (TaqMan ®) assay. This assay takes advantage of the 5′-exonuclease activity of Taq DNA polymerase (Applied Biosystems, Foster City, CA, USA) to cleave the reporter fluorescent molecule from the 5′ end of a probe that is hybridized 3′ to an extending PCR primer on a template strand of DNA. A key strength of this assay is that it is a homogenous reaction , requiring only one reaction setup and no subsequent reaction processing. Notably, this assay does not rely on a cumbersome electrophoretic analysis. This improvement, coupled with a 384-well reaction plate format, allows the assay to be performed in a high-throughput automated setting. Oligonucleotides were designed according to default parameters using Primer Express ® software (Applied Biosystems). PCR primer and probe sequences are described in Table 1 and were synthesized by Integrated DNA Technologies (Coralville, IA, USA). Both probes contained the Black Hole Quencher-1™ (BHQ-1) fluorescence quencher at their 3′ ends. TaqMan reactions were performed in 5 μL volumes, consisting of the following final …

Genetic Variation in Genes Associated with Arsenic Metabolism

Individual variability in human arsenic metabolism has been reported frequently in the literature. This variability could be an underlying determinant of individual susceptibility to arsenic-induced disease in humans. Recent analysis revealing familial aggregation of arsenic metabolic profiles suggests that genetic factors could underlie interindividual variation in arsenic metabolism. We screened two genes responsible for arsenic metabolism, human purine nucleoside phosphorylase (hNP), which functions as an arsenate reductase converting arsenate to arsenite, and human glutathione S-transferase omega 1-1 (hGSTO1-1), which functions as a monomethylarsonic acid (MMA) reductase, converting MMA(V) to MMA(III), to develop a comprehensive catalog of commonly occurring genetic polymorphisms in these genes. This catalog was generated by DNA sequencing of 22 individuals of European ancestry (EA) and 24 individuals of indigenous American (IA) ancestry. In (Italic)hNP(/Italic), 48 polymorphic sites were observed, including 6 that occurred in exons, of which 1 was nonsynonymous (G51S). One intronic polymorphism occurred in a known enhancer region. In hGSTO1-1, 33 polymorphisms were observed. Six polymorphisms occurred in exons, of which 4 were nonsynonymous. In contrast to hNP, in which the IA group was more polymorphic than the EA group, in hGSTO1-1 the EA group was more polymorphic than the IA group, which had only 1 polymorphism with a frequency > 10%. Populations representing genetic admixture between the EA and IA groups, such as Mexican Hispanics, could vary in the extent of polymorphism in these genes based upon the extent of admixture. These data provide a framework in which to conduct genetic association studies of these two genes in relevant populations, thereby allowing hNP and hGSTO1-1 to be evaluated as potential susceptibility genes in human arsenicism.