Tyrosinemia type II: Mutation update, 11 novel mutations and description of 5 independent subjects with a novel founder mutation.

BACKGROUND: Tyrosinemia type II, also known as Richner-Hanhart Syndrome, is an extremely rare autosomal recessive disorder, caused by mutations in the gene encoding hepatic cytosolic tyrosine aminotransferase, leading to the accumulation of tyrosine and its metabolites which cause ocular and skin lesions, that may be accompanied by neurological manifestations, mostly intellectual disability. AIMS: To update disease-causing mutations and current clinical knowledge of the disease. MATERIALS AND METHODS: Genetic and clinical information were obtained from a collection of both unreported and previously reported cases. RESULTS: We report 106 families, represented by 143 individuals, carrying a total of 36 genetic variants, 11 of them not previously known to be associated with the disease. Variants include 3 large deletions, 21 non-synonymous and 5 nonsense amino-acid changes, 5 frameshifts and 2 splice variants. We also report 5 patients from Gran Canaria, representing the largest known group of unrelated families sharing the same P406L mutation. CONCLUSIONS: Data analysis did not reveal a genotype-phenotype correlation, but stressed the need of early diagnosis: All patients improved the oculocutaneous lesions after dietary treatment but neurological symptoms prevailed. The discovery of founder mutations in isolated populations, and the benefits of early intervention, should increase diagnostic awareness in newborns.

Screening for pre-eclampsia early in pregnancy: performance of a multivariable model combining clinical characteristics and biochemical markers.

OBJECTIVE: To investigate the performance of a multivariable model combining a priori clinical characteristics and biomarkers to detect, early in pregnancy, women at higher risk of developing pre-eclampsia (PE). DESIGN: Nested case-control study. SETTING: University medical centre, Quebec, Canada (CHU de Québec). POPULATION: A total of 7929 pregnant women recruited between 10 and 18 weeks of gestation. In all, 350 developed hypertensive disorders of pregnancy (HDP)-of which 139 had PE, comprising 68 with severe PE and 47 with preterm PE-and were matched with two women with a normal pregnancy. METHODS: We selected a priori clinical characteristics and promising markers to create multivariable logistic regression models: body mass index (BMI), mean arterial pressure (MAP), placental growth factor, soluble Fms-like tyrosine kinase-1, pregnancy-associated plasma protein A and inhibin A. MAIN OUTCOME MEASURES: PE, severe PE, preterm PE, HDP. RESULTS: At false-positive rates of 5 and 10%, the estimated detection rates were between 15% (5-29%) and 32% (25-39%), and between 39% (19-59%) and 50% (34-66%), respectively. Considering the low prevalence of PE in this population, the positive predictive values were 7% (5-9%) to 10% (7-13%) for PE and 2% (1-4%) to 4% (3-6%) in the preterm and severe PE subgroups. The multivariable model yielded areas under the receiver operating characteristics curves (AUC) between 0.72 (0.61-0.81) and 0.78 (0.68-0.88). When only BMI and MAP were included in the model, the AUC were similar to those of the a priori model. CONCLUSIONS: In a population with a low prevalence of preterm PE, a multivariable risk algorithm using an a priori combination of clinical characteristics and biochemical markers did not reach a performance justifying clinical implementation as screening test early in pregnancy.

Evolution of an influenza pandemic in 13 countries from 5 continents monitored by protein microarray from neonatal screening bloodspots.

BACKGROUND: Because of lack of worldwide standardization of influenza virus surveillance, comparison between countries of impact of a pandemic is challenging. For that, other approaches to allow internationally comparative serosurveys are welcome. OBJECTIVES: Here we explore the use of neonatal screening dried blood spots to monitor the trends of the 2009 influenza A (H1N1) pdm virus by the use of a protein microarray. STUDY DESIGN: We contacted colleagues from neonatal screening laboratories and asked for their willingness to participate in a study by testing anonymized neonatal screening bloodspots collected during the course of the pandemic. In total, 7749 dried blood spots from 13 countries in 5 continents where analyzed by using a protein microarray containing HA1 recombinant proteins derived from pandemic influenza A (H1N1) 2009 as well as seasonal influenza viruses. RESULTS: Results confirm the early start of the pandemic with extensive circulation in the US and Canada, when circulation of the new virus was limited in other parts of the world. The data collected from sites in Mexico suggested limited circulation of the virus during the early pandemic phase in this country. In contrast and to our surprise, an increase in seroprevalence early in 2009 was noted in the dataset from Argentina, suggestive of much more widespread circulation of the novel virus in this country than in Mexico. CONCLUSIONS: We conclude that this uniform serological testing of samples from a highly standardized screening system offers an interesting opportunity for monitoring population level attack rates of widespread diseases outbreaks and pandemics.

Cost effectiveness of newborn screening for cystic fibrosis: a simulation study.

BACKGROUND: Early detection of cystic fibrosis (CF) by newborn screening (NBS) reduces the rate of avoidable complications. NBS protocols vary by jurisdiction and the cost effectiveness of these different protocols is debated. OBJECTIVE: To compare the cost effectiveness of various CF NBS options. METHODS: A Markov model was built to simulate the cost effectiveness of various CF-NBS options for a hypothetical CF-NBS program over a 5-year time horizon assuming its integration into an existing universal NBS program. NBS simulated options were based on a combination of tests between the two commonly used immunoreactive trypsinogen (IRT) cutoffs (96th percentile and 99.5th percentile) as first tier tests, and, as a second tier test, either a second IRT, pancreatic-associated protein (PAP) or CFTR mutation panels. CFTR mutation panels were also considered as an eventual third tier test. Data input parameters used were retrieved from a thorough literature search. Outcomes considered were the direct costs borne by the Quebec public health care system and the number of cases of CF detected through each strategy, including the absence of screening option. RESULTS: IRT-PAP with an IRT cutoff at the 96th percentile is the most favorable option with a ratio of CAD$28,432 per CF case detected. The next most favorable alternative is the IRT1-IRT2 option with an IRT1 cutoff at the 96th percentile. The no-screening option is dominated by all NBS screening protocols considered. Results were robust in sensitivity analyses. CONCLUSION: This study suggests that NBS for cystic fibrosis is a cost-effective strategy compared to the absence of NBS. The IRT-PAP newborn screening algorithm with an IRT cutoff at the 96th percentile is the most cost effective NBS approach for Quebec.

Association between first-trimester placental volume and birth weight.

OBJECTIVE: To estimate the correlation between first-trimester placental volume, birth weight, small-for-gestational-age (SGA), and preeclampsia. METHODS: A prospective study of women with singleton pregnancy at 11-13 weeks of gestation was conducted. First-trimester placental volume was measured using three-dimensional ultrasound and reported as multiple of median (MoM) for gestational age. Participants were followed until delivery where birth weight, placental weight, and occurrence of preeclampsia were collected. Non-parametric analyses were performed. RESULTS: We reached a complete follow-up for 543 eligible women. First-trimester placental volume was significantly correlated with birth weight (correlation coefficient: 0.18; p < 0.0001) and placental weight (cc: 0.22; p < 0.0001) adjusted for gestational age. First-trimester placental volume was smaller in women who delivered SGA neonates (median MoM: 0.79; interquartile range: 0.62-1.00; p < 0.001) and greater in women who delivered large-for-gestational-age neonates (median MoM: 1.13; 0.95-1.49; p < 0.001) when compared to women with neonates between the 10th and 90th percentile (median MoM: 1.00; 0.81-1.25). First-trimester placental volume was not associated with the risk of preeclampsia (cc: 0.01; p = 0.87). CONCLUSION: First-trimester placental volume is strongly associated with fetal and placental growth. However, we did not observe a correlation between placental volume and the risk of preeclampsia.

OS090. Performance of candidate clinical and biochemical markers in screening early in pregnancy to detect women at high risk to develop preeclampsia.

INTRODUCTION: The advent of early preventive measures, such as low-dose aspirin targeting women at high risk of preeclampsia (PE), emphasizes the need for better detection. Despite the emergence of promising biochemical markers linked to the pathophysiological processes, systematic reviews have shown that, until now, no single tests fulfill the criteria set by WHO for biomarkers to screen for a disease. However, recent literature reveals that by combining various clinical, biophysical and biochemical markers into multivariate algorithms, one can envisage to estimate the risk of PE with a performance that would reach clinical utility and cost-effectiveness, but this remains to be demonstrated in various environments and health care settings. OBJECTIVES: To investigate, in a prospective study, the clinical utility of candidate biomarkers and clinical data to detect, early in pregnancy, women at risk to develop PE and to propose a multivariate prediction algorithm combining clinical parameters to biochemical markers. METHODS: 7929 pregnant women prospectively recruited at the first prenatal visit, provided blood samples, clinical and sociodemographic information. 214 pregnant women developed hypertensive disorders of pregnancy (HDP) of which 88 had PE (1.2%), including 44 with severe PE (0.6%). A nested case-control study was performed including for each case of HDP two normal pregnancies matched for maternal age, gestational age at recruitment, ethnicity, parity, and smoking status. Based on the literature we selected the most promising markers in a multivariate logistic regression model: mean arterial pressure (MAP), BMI, placental growth factor (PlGF), soluble Flt-1, inhibin A and PAPP-A. Biomarker results measured between 10-18 weeks gestation were expressed as multiples of the median. Medians were determined for each gestational week. RESULTS: When combined with MAP at the time of blood sampling and BMI at the beginning of pregnancy, the four biochemical markers discriminate normal pregnancies from those with HDP. At a 5% false positive rate, 37% of the affected pregnancies would have been detected. However, considering the prevalence of HDP in our population, the positive predictive value would have been only 15%. If all the predicted positive women would have been proposed a preventive intervention, only one out 6.7 women could have potentially benefited. In the case of severe PE, performance was not improved, sensitivity was the same, but the positive predictive value decreased to 3% (lower prevalence of severe PE). CONCLUSION: In our low-risk Caucasian population, neither individual candidate markers nor multivariate risk algorithm using an a priori combination of selected markers reached a performance justifying implementation. This also emphasizes the necessity to take into consideration characteristics of the population and environment influencing prevalence before promoting wide implementation of such screening strategies. In a perspective of personalized medicine, it appears more than ever mandatory to tailor recommendations for HDP screening according not only to individual but also to population characteristics.

PP054. Predicting preeclampsia at late mid-term pregnancy before occurrence of clinical symptoms: Clinical utility of biomarkers and clinical parameters in a low-risk population.

INTRODUCTION: Despite research efforts and healthcare improvement, preeclampsia (PE) continues to be a leading cause of maternal and fetal morbidity and mortality. Early identification of women at risk of developing PE is the most promising approach to implement preventive measures such as low-dose aspirin to reduce negative outcomes. However, it is still relevant to evaluate pregnant women to detect PE before the occurrence of clinical symptoms and/or to have better tools to assist in its differential diagnosis. Recently, measurements of biomarkers such as soluble fms-like tyrosine kinase-1 (SFLT-1) and placental growth factor (PlGF) have been proposed and some manufacturers are already marketing reagents for this purpose. OBJECTIVES: To examine in a prospective study the performance of selected clinical and biochemical markers for identifying late mid-term pregnancy women at risk of developing PE within a few weeks. METHODS: Seven thousand nine hundred and twenty nine pregnant women prospectively recruited at the first routine prenatal visit, provided blood samples, clinical and sociodemographic information. Two hundred and fourteen pregnant women developed hypertensive disorders of pregnancy (HDP) of which 88 had PE (1.2%), including 44 who presented with severe PE (0.6%). We performed a nested case-control study from the whole cohort including for each case of HDP two normal pregnancies after matching for maternal age, gestational age at recruitment, ethnicity, parity, and smoking status. Based on the literature, we selected the most promising clinical and biochemical markers to be included in a multivariate logistic regression model: mean arterial pressure and body mass index (BMI), PlGF, SFLT-1, inhibin A, and PAPP-A. All markers were measured between 20 and 32 weeks of gestation except for BMI (early pregnancy). All biological marker results were transformed in multiples of median. Medians were established for each gestational week. Multivariate logistic regression analyses were performed to develop prediction algorithm. RESULTS: The resulting regression model discriminated the affected from normal pregnancies as indicated by an area under the receiver operating characteristics (ROC) curve of 0.8. But at a 5% false positive rate, only 28% of the women who have developed HDP would have been detected. Even when the statistical analyses were limited to severe PE, the performance was poor: sensitivity 30%, positive predictive value 2.7%. CONCLUSION: In our low-risk Caucasian population, neither individual candidate markers nor multivariate risk algorithm using an a priori combination of selected clinical and biochemical markers reached a performance justifying implementation as a screening procedure. These results emphasize the necessity to take into consideration the environment, population and health care settings influencing prevalence and characteristics of HDP before promoting wide implementation of such screening strategies. It is imperative to tailor future recommendations for HDP screening not only according to the individual but also to the population characteristics if clinical utility has to be reached.

Alteration of calcium homeostasis in primary preeclamptic syncytiotrophoblasts: effect on calcium exchange in placenta.

Preeclampsia (PE) is characterized by maternal hypertension, proteinuria, oedema and, in 30% of cases, by intrauterine growth retardation. Causes are still unknown; however, epidemiological and clinical studies have suggested alterations in maternal calcium metabolism. We suggested that in PE, calcium transport by the syncytiotrophoblast (ST) is disturbed. From total placental tissues, we studied the expression of: calcium channels (TRPV5, TRPV6 [transient receptor potential vanilloid]), calcium binding proteins (CaBP-9K, CaBP-28K), plasma membrane calcium ATPase (PMCA)1,2,3,4 pumps, ATP synthase, genes implicated in Ca(2+) release [inositol-1,4,5-triphosphate receptor (IP3R)1,2,3; Ryanodine receptor (RyR)1,2,3] and replenishment (SERCA1,2,3 [sarcoendoplasmic reticulum Ca(2+) ATPases]) from endoplasmic reticulum, channels implicated in mitochondrial Ca(2+) accumulation (VDAC1,2,3 [voltage-dependent anion channels]) and a marker of oxidative stress (hOGG1 [Human 8-oxoguanine-DNA glycosylase 1]), as well as the influence of these variations on calcium transport in primary ST cultures. The mRNA and protein levels were thereby examined by real-time PCR and Western blot analysis, respectively, in two different groups of pregnant women with similar gestational age: a normal group (n= 16) and a PE group (n= 8), diagnosed by a clinician. Our study showed a significant decrease in calcium transport by the ST cultured from preeclamptic placentas. We found a significant (P < 0.05) decrease in mRNA levels of TRPV5, TRPV6, CaBP-9K, CaBP-28K, PMCA1, PMCA4, ATP synthase, IP3R1, IP3R2, RyR1, RyR2 and RyR3 in PE group compared to normal one. We also noted a significant decrease in protein levels of TRPV5, TRPV6, CaBP-9K, CaBP-28K and PMCA1/4 in PE group. In contrast, SERCA1, SERCA2, SERCA3, VDAC3 and hOGG1 mRNA expressions were significantly increased in PE placentas. Calcium homeostasis and transport through placenta is compromised in preeclamptic pregnancies and it appears to be affected by a lack of ATP and an excess of oxidative stress.

High dietary consumption of trans fatty acids decreases brain docosahexaenoic acid but does not alter amyloid-beta and tau pathologies in the 3xTg-AD model of Alzheimer's disease.

Dietary consumption of trans fatty acids (TFA) has increased during the 20th century and is a suspected risk factor for cardiovascular diseases. More recently, high TFA intake has been associated with a higher risk of developing Alzheimer's disease (AD). To investigate the impact of TFA on an animal model genetically programmed to express amyloid-beta (Abeta) and tau pathological markers of AD, we have fed 3xTg-AD mice with either control (0% TFA/total fatty acid), high TFA (16% TFA) or very high TFA (43% TFA) isocaloric diets from 2 to 16 months of age. Effects of TFA on plasma hepatic enzymes, glucose and lipid profile were minimal but very high TFA intake decreased visceral fat of non-transgenic mice. Importantly, dietary TFA increased brain TFA concentrations in a dose-related manner. Very high TFA consumption substantially modified the brain fatty acid profile by increasing mono-unsaturated fatty acids and decreasing polyunsaturated fatty acids (PUFA). Very high TFA intake induced a shift from docosahexaenoic acid (DHA, 22:6n-3) toward n-6 docosapentaenoic acid (DPA, 22:5n-6) without altering the n-3:n-6 PUFA ratio in the cortex of both control and 3xTg-AD mice. Changes in levels of Abeta(40), Abeta(42), tau protein, phosphorylated tau protein and synaptic markers were not statistically significant in the three groups of 3xTg-AD mice, despite a trend toward decreased insoluble tau in very high TFA-fed 3xTg-AD animals. In summary, TFA intake modulated brain fatty acid profiles but had no significant effect on major brain neuropathological hallmarks of AD in an animal model.

Modulation of placental protein expression of OLR1: implication in pregnancy-related disorders or pathologies.

The lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (OLR1) is a newly described receptor for oxidatively modified LDL. The human pregnancy is associated with hyperlipidemia and oxidative stress. It has been reported that modification in maternal lipid profile can induce disturbance during pregnancy. In this study, we have evaluated the expression protein level of OLR1 in human term placenta of women having plasma cholesterol level lower to 7 mM or higher to 8 mM and women of gestational diabetes mellitus (GDM) by western blot analysis. The present study demonstrates that the maternal lipid profile is associated with placental protein expression of OLR1. A significant increase in the protein expression of OLR1 was observed in placenta of women with elevated plasmatic total cholesterol level (>8 mM). In addition, the placental protein expression of OLR1 is increased in mothers having the highest pre-pregnancy body mass index (BMI) and low (<7 mM) plasmatic total cholesterol level at term. Interestingly, the placental protein expression of OLR1 is increased in the presence of GDM pregnancies compared with normal lipids level pregnancies, without the modification of mRNA expression. In conclusion, placental OLR1 protein expression is associated with maternal lipid profile, pre-pregnancy BMI, and pathology of GDM.

Assessment of the prevalence of the 985A>G MCAD mutation in the French-Canadian population using allele-specific PCR.

Inherited deficiency of medium-chain acyl-CoA dehydrogenase (MCAD) is a severe, sometimes fatal disorder. A single mutation in the MCAD gene, 985A>G, is involved in approximately 90% of cases. To evaluate the relevance of implementing a systematic population-based screening program in the province of Quebec using a biochemical test, we measured the prevalence of this mutation in a set of anonymous newborn samples from the Quebec City area, a region where the majority of its inhabitants are French-Canadians. An allele-specific polymerase chain reaction assay was designed and used to detect the mutation in 7143 DNA samples obtained from consecutive anonymous newborns. Pools of eight DNA samples were genotyped in parallel for the same mutation to validate this pooling strategy. The allelic frequency of the MCAD 985A>G mutation was found to be 0.71% and the carrier frequency 1:71 (95% confidence interval 1:55 to 1:98). This estimate predicts a homozygous frequency of 1:19,837. Ninety-nine heterozygous carriers and one homozygous individual were identified out of 7143 samples. There was 100% concordance between the individual and pooled analyses, and the pooling strategy reduced the total genotyping costs by approximately 70%. The carrier frequency estimated for this population is similar to other northwestern European populations and would support implementation of systematic newborn screening (such as tandem mass spectrometry screening) for this disease. Pooling DNA samples followed by genotyping appears to be cost-effective for estimating prevalence of rare mutations.

Influence of maternal lipid profile on placental protein expression of LDLr and SR-BI.

Maternal hyperlipidemia is a characteristic feature during pregnancy, it has been reported that modification of the maternal lipid profile can induce disturbance during pregnancy. In this study, we evaluated the impact of maternal lipid profile on the placental protein expression of two major receptors in cholesterol metabolism, the low density lipoprotein receptor (LDLr) and the scavenger receptor type B1 (SR-B1). We demonstrate an increase in the level of maternal total circulating cholesterol leads to a significant decrease in the level of the LDLr protein expression, while the level of the SR-BI expression remains unchanged. A similar change, for LDLr, is observed in association with the maternal pre-pregnancy body mass index and weight gain. Our data suggest that the LDLr plays a role in regulating cholesterol delivered to the baby from the placenta.

Leisure physical activity is associated with quantitative ultrasound measurements independently of bone mineral density in postmenopausal women.

The purpose of this study was to assess the magnitude of the relationship between leisure physical activity and bone status as measured either by an Achilles ultrasound bone densitometer (QUS) or dual-energy X-ray absorptiometry (DXA) in postmenopausal women. We studied 1162 French Canadian postmenopausal women, aged 33-84 years (mean age 58 years), for QUS parameters [broadband ultrasound attenuation (BUA), speed of sound (SOS), and stiffness index (SI)] measured at the right calcaneus, and bone mineral density (BMD) measured at the lumbar spine and femoral neck. Multivariate regression analyses revealed that leisure physical activity level was an independent predictor of the heel QUS parameters and of femoral neck BMD. No such association was observed for BMD of the lumbar spine. Heel QUS parameters (BUA, SOS, SI) and femoral neck BMD adjusted for interfering covariables showed a statistically significant difference between sedentary (less than three sessions/month) and active women (three or more sessions/week) (P < or = 0.001). Furthermore, after adjusting each heel QUS parameters for the mean lumbar spine BMD value, the association observed between leisure physical activity and QUS remained significant. These results suggest that regular leisure physical activity could influence QUS parameters, independently of BMD, and that quantitative ultrasound could be a suitable outcome measure in exercise studies in postmenopausal women.

Short polyglutamine tracts in the androgen receptor are protective against breast cancer in the general population.

We studied the association of breast cancer with the polymorphic polyglutamine repeat of the androgen receptor (AR) in 255 incident cases of breast cancer and 461 matched controls from the Quebec City metropolitan area. Women for whom the sum of both of the AR (CAG)n-repeats alleles is 39 or less (short-allele AR genotypes) have one-half the risk of breast cancer compared with women for whom the sum of AR (CAG)n-repeats is 40 or more [odds ratio (OR), 0.5; 95% confidence interval (CI), 0.3-0.83; P = 0.007]. This association is stronger in postmenopausal women (180 cases, 297 controls; OR, 0.36; 95% CI, 0.19-0.7; P = 0.003). We also observed an interaction between the type of menopause (natural versus surgical) and the AR genotype on breast cancer risk. Alternately, when subjects were grouped according to their (CAG)n-repeat genotype [homozygous for short alleles (CAG)n < or = 20; other genotypes ("long allele")], results were similar (OR. 0.5; 95% CI, 0.27-0.82; P = 0.007). Thus, women with short-alleles AR genotypes appear to be protected against breast cancer. Short-alleles AR genotypes were observed in 16% of the general population as represented by the control group. Short polyglutamine repeats in the AR protein have been reported to be associated with an increase in the capacity of the receptor to activate transcription of reporter genes in vitro. Furthermore, androgens have been previously shown to inhibit in vitro the growth of breast cancer cell lines. This suggests that differences in the number of polyglutamines in the AR protein may influence individual risk of breast cancer, especially in postmenopausal women, and that this apparent protection could be the consequence of an increased response/sensitivity to androgens.

The distribution of major lymphocyte subsets in cord blood is associated with its pH.

OBJECTIVES: To assess in venous cord blood the distribution of major lymphocyte subsets according to pH and medications used during labor. DESIGN AND METHODS: Venous cord blood was sampled immediately after labor from 70 newborns (35 males and 35 females) delivered vaginally. Lymphocytes were immunophenotyped by flow cytometry and pH was measured using the AVL 900 automated blood gas analysis system. Data on birth weight, gestational age at delivery, length of labor, presence of stained amniotic fluid, medications used during labor, maternal risk factors, age and parity were collected. RESULTS: The percentage of T lymphocytes decreased while the percentage of NK lymphocytes increased with decreasing pH over the whole range of pH values. The proportions of T and NK lymphocytes were associated with the administration of neuroplegics, spasmolytics or dihydroergotoxin in the first stage of labor. CONCLUSIONS: Cord blood pH and labor-associated variables should be taken into account to adequately interpret the profile of major lymphocyte subsets as a marker of the effect of different prenatal factors on the immune system of neonates.

The effect of dibenzo[a,1]pyrene and benzo[a]pyrene on human diploid lung fibroblasts: the induction of DNA adducts, expression of p53 and p21(WAF1) proteins and cell cycle distribution.

Polycyclic aromatic hydrocarbons (PAHs) present in ambient air are considered as potential human carcinogens, but the detailed mechanism of action is still unknown. Our aim was to study the in vitro effect of exposure to dibenzo[a,l]pyrene (DB[a,l]P), the most potent carcinogenic PAH ever tested, and benzo[a]pyrene (B[a]P) in a normal human diploid lung fibroblast cells (HEL) using multiple endpoints. DNA adduct levels were measured by 32P-postlabelling, the expression of p53 and p21(WAF1) proteins by western blotting and the cell cycle distribution by flow cytometry. For both PAHs, the DNA adduct formation was proportional to the time of exposure and dependent on the stage of cell growth in culture. DNA binding was detectable even at the lowest concentration used (24h exposure, 0.01 microM for both PAHs). The highest DNA adduct levels were observed after 24h of exposure in near-confluent cells (>90% of cells at G0/G1 phase), but DNA damage induced by DB[a,l]P was approximately 8-10 times higher at a concentration one order of magnitude lower as compared with B[a]P (for B[a]P at 1 microM and for DB[a,l]P at 0.1 microM: 237+/-107 and 2360+/-798 adducts/10(8) nucleotides, respectively). The induction of p53 and p21(WAF1) protein occurred subsequent to the induction of DNA adducts. The DNA adduct levels correlated with both p53 (R=0.832, P<0.001 and R=0.859, P<0.001, for DB[a,l]P and B[a]P, respectively) and p21(WAF1) levels (R=0.808, P<0.001 and R=0.797, P=0.001, for DB[a,l]P and B[a]P, respectively), regardless of the PAH exposure and the phase of cell growth. The results showed that a detectable increase of p53 and p21(WAF1) proteins (> or = 1.5-fold as compared with controls) requires a minimal DNA adduct level of approximately 200-250 adducts/10(8) nucleotides for both PAHs tested and suggest that the level of adducts rather than their structure triggers the p53 and p21(WAF1) responses. The cell cycle was altered after 12-16h of treatment, and after 24h of exposure to 0.1 microM DB[a,l]P in growing cells, there was approximately 24% increase in S phase cells accompanied by a decrease in G1 and G2/mitosis (G2/M) cells. Cell treatment with 1.0 microM B[a]P resulted in more subtle alterations. We conclude that DB[a,l]P, and to a lesser degree B[a]P, are able to induce DNA adducts as well as p53 and p21(WAF1) without eliciting G1 or G2/M arrests but rather an S phase delay/arrest. Whether the S phase delay observed in our study is beneficial for the survival of the cells remains to be further established.

The association between heel ultrasound and hormone replacement therapy is modulated by a two-locus vitamin D and estrogen receptor genotype.

Evidence supports the role of estrogen deprivation in the process of bone remodeling and increased risk of fracture in postmenopausal women but little is known about the genetic basis of individual differences in response to therapy. In a cross-sectional study, 425 ambulatory postmenopausal French-Canadian women from Quebec (age range, 42-85 years old) were genotyped for a common Bsm I polymorphism at the vitamin D receptor (VDR) gene as well as a Pvu II polymorphism in the estrogen receptor (ESR1) gene. Heel ultrasound was determined by right calcaneal quantitative ultrasound (QUS) and results were expressed as an age-and-weight-adjusted stiffness index (heel SI z score). Our aim was to investigate the interaction between hormone-replacement therapy (HRT) and receptor genotypes in an effect on heel SI. Notably, a two-locus genotype (VDR-bb/ESR-PP) present in 9.5% of women was responsible for over 30% of the total HRT-related heel SI difference in the whole sample. Women bearing this combined VDR/ESR1 genotype who received HRT for more than 5 years had a 21% (1.25 SD) greater heel SI (p = 0.002) than those bearing the same genotype but who received HRT for <5 years. This may translate into a 2- to 3-fold difference in the risk of fracture. Although follow-up studies are needed, our findings suggest that QUS of the heel in postmenopausal women taking HRT is affected by variation in VDR and ESR1 loci, jointly.

The genetics of osteoporosis: 'complexities and difficulties'.

Osteoporosis is characterized by a decrease in bone mass as well as a deterioration of the bone architecture resulting in an increased risk of fracture. Although the disease is multifactorial, twin studies have shown that genetic factors account for up to 80% of the variance in bone mineral density, the best known predictor of the risk of osteoporosis. Some loci, such as the vitamin D and estrogen receptor genes, as well as the collagen type Ialpha1 locus, are promising genetic determinants of bone mass, and possibly other bone phenotypes, but this is controversial and the molecular basis of osteoporosis remains largely undefined. Considering that the effect of each candidate gene is expected to be modest, discrepancies between allelic association studies may have arisen because different populations carry different genetic backgrounds and exposure to environmental factors. Also, we realize the importance of gene-gene as well as gene-environment interactions as significant determinants of bone density and risk of osteoporosis. The use of new tools such as small nucleotide polymorphism maps now allows the possibility to perform allelic association studies in the context of whole-genome search. However, specific study design strategies in large epidemiological studies as well as the best statistical approach will need to be established. We may expect the development of population-specific at-risk profiles for osteoporosis that would include genetic and environmental factors, as well as their interactions. This should eventually lead to better prevention strategies and more adapted therapies against osteoporosis.

Bone mineral density in French Canadian women.

This cross-sectional study investigated bone mineral density (BMD) at the lumbar spine (L2-4) and femoral neck in French Canadian women residing in the Quebec city area. Data collection was initiated in 1988 and completed in 1994. A total of 747 French Canadian Caucasian women (16-79 years of age) with no metabolic bone disease were evaluated. BMD measurements were obtained using dual-photon absorptiometry (DPA) or dual-energy X-ray absorptiometry (DXA). Anthropometric measures such as weight, height and body mass index (BMI) were recorded. Medical files provided information on demographic characteristics, hormonal profile and lifestyle habits. Results show a curvilinear trend of BMD with aging. Furthermore, the peak BMD at the lumbar spine (L2-4) was reached at 29 years followed by a stable phase until 35 years, after which BMD started to decrease. The pattern of bone evolution at the femoral neck was different, peak BMD being achieved earlier, at 21 years, while after age 26 years a significant decrease was already observed. Women older than 60 years showed the lowest BMD. Regression analysis showed that age, weight and height are determinants of BMD at the lumbar spine and explained 33.9% of inter-individual variation. At the femoral neck, 29.1% of variation was explained by age and height only. In conclusion, our data suggest that French Canadian women have a different pattern of bone loss at the femoral neck compared with the lumbar spine, according to their mean BMD values.