Xenobiotic Metabolism Genes and Clubfoot

Idiopathic or isolated clubfoot is a common orthopedic birth defect that affects approximately 135,000 children worldwide. It is characterized by equinus, varus and adductus deformities of the ankle and foot. Correction of clubfoot involves months of serial manipulations, castings and bracing, with surgical correction needed in forty percent of cases. Multifactorial etiology has been suggested in numerous studies with both environmental and genetic factors playing an etiologic role. Maternal smoking during pregnancy is the only common environmental factor that has consistently been shown to increase the risk for clubfoot. Moreover, a positive family history of clubfoot and maternal smoking increases the risk of clubfoot twenty fold. These findings suggest that genetic variation in smoking metabolism genes may increase susceptibility to clubfoot. Based on this reasoning, we interrogated eight candidate genes, chosen based on their involvement in phase 1 and 2 cigarette smoke metabolism. Twenty-two SNPs and two null alleles in eight genes (CYP1A1, CYP1A2, CYP1B1, CYP2A6, EPHX1, NAT2, GSTM1 and GSTT1) were genotyped in a dataset composed of nonHispanic white and Hispanic multiplex and simplex families. Only one SNP in CYP1A1, rs1048943, had significantly altered transmission in the aggregate and multiplex NHW datasets (p=0.003 and p=0.009). Perturbation of CYP1A1 by rs1048943 polymorphism causes an increase in the amount of harmful, adduct forming metabolic intermediates. A significant gene interaction between EPHX1 and NAT2 was also found (p=0.007). This interaction may affect the metabolism of harmful metabolic intermediates. Additionally, marginal interactions were found for other xenobiotic genes and these interactions may play a contributory role in clubfoot. Importantly, for CYP1A2, significant maternal (p=0.03; RR=1.24; 95% CI: 1.04-1.44) and fetal (p=0.01; RR=1.33; 95% CI: 1.13-1.54) genotypic effects were identified suggesting that both maternal and fetal genotypes impact normal limb development. No association was found for maternal smoking status and tobacco metabolism genes. Together, these results suggest that xenobiotic metabolism genes may play a contributory role in the etiology of clubfoot regardless of maternal smoking status and may impact foot development through perturbation of tobacco metabolic pathways.

EPSTEIN-BARR VIRUS TRANSFORMATION OF B LYMPHOCYTE SUBPOPULATIONS

Epstein-Barr virus is a herpes virus distinguished by its remarkable specificity for the B lymphocyte of humans and certain other primates. Although the transformation process is very efficient, is has become clear that only a fraction of B lymphocytes is susceptible. Therefore the question may be raised if transformation is related to B cell stage of activation. B cells were purified from peripheral blood mononuclear cells by the removal of monocytes using elutriation and sheep red blood cell rosetting to remove T cells. Retesting B cells were purified using discontinuous Percoll gradients. Activation of resting cells for 24 hours with anti-mu or Staphylococcus aureus Cowan I (SAC) resulted in transition of susceptible cells into the G(,1) phase of the cell cycle as shown by an increase in cell size, an increase in uridine incorporation and an increase in sensitivity to B cell growth factor (BCGF). Entry into S phase was achieved by extending the period of activation to 48-96 hr as shown by an increase in thymidine incorporation. By this criterion, SAC activated cells entered S phase on day 2 and anti-mu treated cells on day 3. Control (G(,0)) cells and cells activated for varying lengths of time (G(,1), G(,1) plus S) were exposed to EBV and plated in a limiting dilution assay to determine the frequency of EBV-transformable cells. Control cells and cells activated for 24 hr had a precursor frequency of 1% to 2%. With continued activation, however, precursor frequency decreased as a function of the duration of activation. The decrease in frequency of transformable cells correlated with the entry of the population into S phase. The transformation frequency in the SAC-treated population was reduced twenty-fold on day 4, whereas in the anti-mu treated population it was reduced ten-fold. Treating cells with BCGF in conjunction with low concentrations of anti-mu decreased the transformation frequency to levels lower than anti-mu alone, further suggesting that entry into S phase is accompanied by a reduction in transformability. These results indicate that resting B cells are highly susceptible to transformation and that with in vitro activation into the cell cycle B cells become progressively insensitive to EBV. ^

Proactive aggression: A two part study

Aggressive behavior can be divided into the subtypes: reactive and proactive. Reactive aggressive acts occur in response to a stimulus or provocation. Proactive aggressive acts occur without provocation and are goal-directed. A number of findings have suggested that individuals displaying proactive aggression may be discerned from individuals not displaying proactive aggression on measures of personality, psychopathology and psychopathy, as well as on aggressive histories and type and severity of aggressive behaviors committed. The current study was conducted in two phases; phase 1 and 2. This was because phase 1 compared proactive aggressive, reactive aggressive and non-aggressive subjects on questionnaire measures, while phase 2 observed the acute effects of the benzodiazepine alprazolam on only proactive aggressive subjects. The phase 1 hypotheses were that proactive aggressive subjects would show greater numbers of personality disorders and have greater psychopathy relative to reactive and non-aggressive subjects. To verify these hypotheses subjects were recruited from the community and classified as proactive (n = 20), reactive (n = 20) or non-aggressive (n = 10) via laboratory behavioral testing. Classified subjects were administered a battery of questionnaires pertaining to personality disorders (SCID-II, OMNI-IV), psychopathy (PCL-R) and aggression history. The results of these questionnaire measures were subjected to statistical analyses, which confirmed the hypotheses. In the second phase, the acute effects of three doses of the benzodiazepine alprazolam were evaluated in proactive aggressive subjects on proactive aggressive responding in the computer-based Point Subtraction Aggression Paradigm (PSAP). In phase 2 it was hypothesized that alprazolam would produce dose dependent decreases in aggressive responding. Subjects were never provoked in this phase, and aggressive responding was classified as proactive. Studies of drugs acting on the GABA system have frequently found decreases in aggression in animals and humans, although there have also been findings of increased (paradoxical) aggression. The hypothesis was tested by statistical analysis of proactive aggressive responding under placebo vs. under alprazolam. The hypothesis was supported by six of seven subjects. Aggressive responding was significantly, decreased under alprazolam relative to placebo in six subjects. One subject showed increases in aggressive responding.^