Scott R. Diehl

Application of automated DNA sizing technology for genotyping microsatellite loci

Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, we report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. We capitalize on the availability of three distinct fluorescent dyes to label uniquely loci that overlap in size, and this innovation increases by threefold the number of loci that can be analyzed simultaneously. We label size standards with a fourth dye and combine these with the microsatellite PCR products in each gel lane. Computer programs provide very rapid and accurate sizing of microsatellite alleles and efficient data management. In addition, fluorescence signals are linear over a much greater range of intensity than conventional autoradiography. This facilitates multiplexing of loci (since signal intensities often vary greatly) and helps distinguish major peaks from artifacts, thereby improving genotyping accuracy.

Association Between Changes in Air Pollution Levels During the Beijing Olympics and Biomarkers of Inflammation and Thrombosis in Healthy Young Adults

CONTEXT Air pollution is a risk factor for cardiovascular diseases (CVD), but the underlying biological mechanisms are not well understood. OBJECTIVE To determine whether markers related to CVD pathophysiological pathways (biomarkers for systemic inflammation and thrombosis, heart rate, and blood pressure) are sensitive to changes in air pollution. DESIGN, SETTING, AND PARTICIPANTS Using a quasi-experimental opportunity offered by greatly restricted air pollution emissions during the Beijing Olympics, we measured pollutants daily and the outcomes listed below in 125 healthy young adults before, during, and after the 2008 Olympics (June 2-October 30). We used linear mixed-effects models to estimate the improvement in outcome levels during the Olympics and the anticipated reversal of outcome levels after pollution controls ended to determine whether changes in outcome levels were associated with changes in pollutant concentrations. MAIN OUTCOME MEASURES C-reactive protein (CRP), fibrinogen, von Willebrand factor, soluble CD40 ligand (sCD40L), soluble P-selectin (sCD62P) concentrations; white blood cell count (WBC); heart rate; and blood pressure. RESULTS Concentrations of particulate and gaseous pollutants decreased substantially (-13% to -60%) from the pre-Olympic period to the during-Olympic period. Using 2-sided tests conducted at the .003 level, we observed statistically significant improvements in sCD62P levels by -34.0% (95% CI, -38.4% to -29.2%; P < .001) from a pre-Olympic mean of 6.29 ng/mL to a during-Olympic mean of 4.16 ng/mL and von Willebrand factor by -13.1% (95% CI, -18.6% to -7.5%; P < .001) from 106.4% to 92.6%. After adjustments for multiple comparisons, changes in the other outcomes were not statistically significant. In the post-Olympic period when pollutant concentrations increased, most outcomes approximated pre-Olympic levels, but only sCD62P and systolic blood pressure were significantly worsened from the during-Olympic period. The fraction of above-detection-limit values for CRP (percentage ≥ 0.3 mg/L) was reduced from 55% in the pre-Olympic period to 46% in the during-Olympic period and reduced further to 36% in the post-Olympic period. Interquartile range increases in pollutant concentrations were consistently associated with statistically significant increases in fibrinogen, von Willebrand factor, heart rate, sCD62P, and sCD40L concentrations. CONCLUSIONS Changes in air pollution levels during the Beijing Olympics were associated with acute changes in biomarkers of inflammation and thrombosis and measures of cardiovascular physiology in healthy young persons. These findings are of uncertain clinical significance.

Behavioral evidence for host races in Rhagoletis pomonella flies

SummaryOne of the most controversial putative cases of host race formation in insects is that of the apple maggot fly, Rhagoletis pomonella (Diptera: Tephritidae). A principal cause of the controversy is lack of relevant data. In laboratory and field enclosure experiments, we compared the host acceptance behavior of sympatric populations of flies originating from naturally infested hawthorn (the native host) and apple (an introduced host) in Amherst, Massachusetts or East Lansing, Michigan. In general, hawthorn fruit were accepted for ovipositional attempts nearly equally by apple and hawthorn origin females, whereas apples were accepted much more often by apple than hawthorn origin females. Similarly, males of apple and hawthorn origin exhibited about equal duration of residence on hawthorn fruits as sites at which to acquire potential mates, while males of apple origin tended to reside substantially longer than males of hawthorn origin on apples. Irrespective of fly origin, both sexes always responded more positively to hawthorn fruit than to apples. Because all flies assayed were naive (ruling out effects of prior host experience of adults) and because tests revealed no influence of pre-imaginal fruit exposure on pattern of host fruit acceptance by females, the combined evidence suggests the phenotypic differences we observed in host response pattern between hawthorn and apple origin flies may have an underlying genetic basis. Further tests showed that while larval progeny of flies of each origin survived better in naturally growing hawthorn fruit than in naturally growing apples, there was no differential effect of fly origin on larval survival ability in either host. We discuss our findings in relation to restriction in gene flow between sympatric populations of R. pomonella and in relation to current models of host shifts in insects.

Inflammatory and Oxidative Stress Responses of Healthy Young Adults to Changes in Air Quality during the Beijing Olympics

RATIONALE Unprecedented pollution control actions during the Beijing Olympics provided a quasi-experimental opportunity to examine biologic responses to drastic changes in air pollution levels. OBJECTIVES To determine whether changes in levels of biomarkers reflecting pulmonary inflammation and pulmonary and systemic oxidative stress were associated with changes in air pollution levels in healthy young adults. METHODS We measured fractional exhaled nitric oxide, a number of exhaled breath condensate markers (H(+), nitrite, nitrate, and 8-isoprostane), and urinary 8-hydroxy-2-deoxyguanosine in 125 participants twice in each of the pre- (high pollution), during- (low pollution), and post-Olympic (high pollution) periods. We measured concentrations of air pollutants near where the participants lived and worked. We used mixed-effects models to estimate changes in biomarker levels across the three periods and to examine whether changes in biomarker levels were associated with changes in pollutant concentrations, adjusting for meteorologic parameters. MEASUREMENTS AND MAIN RESULTS From the pre- to the during-Olympic period, we observed significant and often large decreases (ranging from -4.5% to -72.5%) in levels of all the biomarkers. From the during-Olympic to the post-Olympic period, we observed significant and larger increases (48-360%) in levels of these same biomarkers. Moreover, increased pollutant concentrations were consistently associated with statistically significant increases in biomarker levels. CONCLUSIONS These findings support the important role of oxidative stress and that of pulmonary inflammation in mediating air pollution health effects. The findings demonstrate the utility of novel and noninvasive biomarkers in the general population consisting largely of healthy individuals.

Factors Associated with Delay in the Diagnosis of Oral Cancer

Early detection and treatment improve the prognosis for oral cancer. Delays from the onset of symptoms to clinical diagnosis are common. Our aim is to identify factors associated with this delay. Between 1995 and 1998, we interviewed 105 consecutive patients with histologically confirmed oral cancer in Greece. If 21 or more days elapsed from the time the patient noticed major symptoms to a definitive diagnosis, we called it a delay (52% of cases). We used logistic and linear regression to estimate odds ratios of delayed diagnosis and to identify correlates of length of delay, respectively. Former smokers had a 4.3 times greater risk of delayed diagnosis compared with current smokers (95% confidence interval: 1.1-17.1). The length of delay was greater among single patients, non-smokers, or those with stage IV tumors. Clinicians should be advised that delay in the diagnosis of oral cancer occurs frequently, even in individuals who do not smoke heavily.

Mapping a gene for neuropathic pain-related behavior following peripheral neurectomy in the mouse

&NA; Total hindpaw denervation in rodents elicits an abnormal behavior of licking, scratching and self‐injury of the anesthetic limb (‘autotomy’). Since the same denervation produces phantom limb pain and anesthesia dolorosa in humans, autotomy has been used as a model of human neuropathic pain. Autotomy is an inherited trait in rodents, attributable to a few genes of major effect. Here we used recombinant inbred (RI) mouse lines of the AXB‐BXA RI set to map a gene for autotomy. Autotomy levels following unilateral sciatic and saphenous nerve section were scored daily for 36 days, using a standardized scale, in all 23 RI lines available for this set. We used a genetic map of 395 marker loci and a permutation‐based statistical method for categorical data to assess the statistical significance of mapping results. We identified a marker on chromosome 15 with statistical support (P=0.0003) in the range considered significant for genome‐wide scans in the mouse. Several genes located in this chromosomal region encode for neural functions related to neuropathic pain and may indicate targets for development of novel analgesics.

Smoking and alcohol in the etiology of oral cancer: gender-specific risk profiles in the south of Greece

Oral and pharyngeal cancer (OC) mortality is very low in Greece, especially among men, compared to other European countries. We conducted a case-control study of OC in Athens, and obtained information on tobacco, alcohol use and other potential risk factors and confounding variables for 110 incident cases and 115 hospital-based controls. We used multivariate logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Tobacco smoking (pack years, P(trend)=0.01) and alcohol use (drinks/week, P(trend)=0.07) were independent risk factors, with a multiplicative effect for combined exposures (OR, 8.3; 95% CI, 2.4-29.1, for >28 alcohol drinks/week and >50 pack years of cigarette smoking). The type of alcoholic beverage also seemed important: drinking ouzo and tsipouro (liquors of high ethanol concentration) was associated with greater increased OC risk than drinking comparable amounts of wine, beer or dark spirits. While alcohol drinking is more common for male cases versus controls, few men reported regularly consuming large quantities of ethanol associated with highest risk of OC in other studies. This may partially explain the low rates of male OC mortality in Greece. Among the 38% of our cases who were women, however, neither smoking nor alcohol drinking frequencies were significantly elevated compared to controls, and so the etiology of OC risk in females requires further investigation.

Mouthwash in the etiology of oral cancer in Puerto Rico

AbstractObjectives: To determine if the risk of cancers of the mouth and pharynx is associated with mouthwash use in Puerto Rico, an area of relatively high risk. Methods: Interviews were conducted with 342 cases of oral and pharyngeal cancer registered in Puerto Rico and diagnosed between 1992 and 1995 and with 521 population-based controls regarding mouthwash use and other factors. Mouthwash-related risks were estimated using unconditional logistic regression controlling for potential confounders. Results: The adjusted odds ratio associated with using mouthwash with an alcohol content of 25% or greater was 1.0. Risks were not higher with greater frequency, years of use, or lifetime mouthwash exposure. Among tobacco and alcohol abstainers the odds ratio associated with mouthwash use was 2.8 (CI = 0.8–9.9), in contrast to 0.8 (CI = 0.4–1.7) and 0.9 (CI = 0.6–1.3) among those with light and heavy cigarette smoking/alcohol drinking behaviors, respectively. Conclusions: There was no overall increased risk of oral cancer associated with mouthwash use. An elevated, but not statistically significant, risk was observed among the small number of subjects who neither smoked cigarettes nor drank alcohol, among whom an effect of alcohol-containing mouthwash would be most likely evident. Our findings indicate the need to clarify the mechanisms of oral carcinogenesis, including the possible role of alcohol-containing mouthwash.

Reevaluation of the chromosome 4q candidate region for early onset periodontitis

Evidence of linkage (lod=3.1, θ=0.05) was reported previously in one large kindred (the Brandywine genetic isolate) for an autosomal dominant form of early onset periodontitis (EOP) with a protein polymorphism in the vitamin D binding protein (GC) located on chromosome 4q12-q13. To evaluate the generality of this finding, 19 unrelated families (228 individuals), each with two or more EOP affected individuals, were ascertained and sampled. A restriction fragment length polymorphism (RFLP) at the GC locus and eight other polymorphic DNA markers and two red blood cell antigens located on proximal chromosome 4q in the vicinity of the GC locus were typed. Twelve genetic models of EOP were evaluated, which varied in diagnostic classification, penetrance, and mode of disease transmission. Results for all models strongly exclude linkage between an EOP susceptibility gene and this chromosomal region assuming locus homogeneity. Our data statistically exclude (lod≤ -2.0) the possibility that more than 40% of our families are linked to this candidate region for one model tested. Linkage under heterogeneity was excluded less strongly for other models, but no significant evidence in support of linkage was obtained for any model. Our results indicate that either the previous report of linkage was a false positive, or that there are two or more unlinked forms of EOP, with the form located in 4q12-q13 being less common.

Interaction between a single nucleotide polymorphism in the alcohol dehydrogenase 3 gene, alcohol consumption and oral cancer risk†

We investigated effects on oral cancer (OC) risk of an interaction between a single nucleotide polymorphism (SNP) in the alcohol dehydrogenase 3 (ADH3) gene and alcohol consumption levels using a hospital‐based study of 93 cases and 99 controls conducted in Athens, Greece. This SNP affects ethanol metabolism in vitro and appeared to interact with alcohol consumption in a previous OC study. We also evaluated a SNP in CYP2E1, another gene involved in ethanol metabolism, reported to be associated with OC risk in a European population. Data on genotypes and risk factors obtained from interviews were analyzed using multivariate logistic regression, accounting for potential confounders. No overall (marginal) association was found between OC risk and ADH3 genotypes. An interaction between ADH3 genotypes and alcohol consumption levels, however, was suggested. In non‐drinkers, the ADH31‐1 genotype has higher risk than ADH31‐2 or ADH32‐2 genotypes, but for subjects consuming alcohol, lower risk was observed for ADH3 We fit a logistic regression model to estimate the increase in OC risk associated with each alcohol drink consumed per week. We estimated that OC risk increased by 31.5% per drink/week for the ADH32‐2 genotype, 4.1% for the ADH31‐2 genotype and 1.6% for the ADH31‐1 genotype. Evidence of genotype‐environment interaction was suggestive (p = 0.048, Wald χ p = 0.145, likelihood ratio). This finding is opposite to that reported for a population in Puerto Rico, where the ADH31‐1 genotype seemed more sensitive to ethanol exposure. In Greece, genetic variation at the CYP2E1 SNP is almost entirely absent, with only 1 case and 1 control heterozygous for the variant. By contrast, in a population in France where an OC association was reported, the frequency of CYP2E1 heterozygotes was 5% in controls and 9% in OC cases. These findings illustrate the importance of replicating SNP associations both within and between different racial and ethnic groups and geographic regions. Published 2001 Wiley‐Liss, Inc.