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.

Safety, tolerability, and efficacy of fixed combination therapy with dorzolamide hydrochloride 2% and timolol maleate 0.5% in glaucoma and ocular hypertension.

Glaucoma is a collection of diseases characterized by multifactorial progressive changes leading to visual field loss and optic neuropathy most frequently due to elevated intraocular pressure (IOP). The goal of treatment is the lowering of the IOP to prevent additional optic nerve damage. Treatment usually begins with topical pharmacological agents as monotherapy, progresses to combination therapy with agents from up to 4 different classes of IOP-lowering medications, and then proceeds to laser or incisional surgical modalities for refractory cases. The fixed combination therapy with the carbonic anhydrase inhibitor dorzolamide hydrochloride 2% and the beta blocker timolol maleate 0.5% is now available in a generic formulation for the treatment of patients who have not responded sufficiently to monotherapy with beta adrenergic blockers. In pre- and postmarketing clinical studies, the fixed combination dorzolamide-timolol has been shown to be safe and efficacious, and well tolerated by patients. The fixed combination dorzolamide-timolol is convenient for patients, reduces their dosing regimen with the goal of increasing their compliance, reduces the effects of "washout" when instilling multiple drops, and reduces the preservative burden by reducing the number of drops administered per day.

Altered transmission of HOX and apoptotic SNPs identify a potential common pathway for clubfoot.

Clubfoot is a common birth defect that affects 135,000 newborns each year worldwide. It is characterized by equinus deformity of one or both feet and hypoplastic calf muscles. Despite numerous study approaches, the cause(s) remains poorly understood although a multifactorial etiology is generally accepted. We considered the HOXA and HOXD gene clusters and insulin-like growth factor binding protein 3 (IGFBP3) as candidate genes because of their important roles in limb and muscle morphogenesis. Twenty SNPs from the HOXA and HOXD gene clusters and 12 SNPs in IGFBP3 were genotyped in a sample composed of non-Hispanic white and Hispanic multiplex and simplex families (discovery samples) and a second sample of non-Hispanic white simplex trios (validation sample). Four SNPs (rs6668, rs2428431, rs3801776, and rs3779456) in the HOXA cluster demonstrated altered transmission in the discovery sample, but only rs3801776, located in the HOXA basal promoter region, showed altered transmission in both the discovery and validation samples (P = 0.004 and 0.028). Interestingly, HOXA9 is expressed in muscle during development. An SNP in IGFBP3, rs13223993, also showed altered transmission (P = 0.003) in the discovery sample. Gene-gene interactions were identified between variants in HOXA, HOXD, and IGFBP3 and with previously associated SNPs in mitochondrial-mediated apoptotic genes. The most significant interactions were found between CASP3 SNPS and variants in HOXA, HOXD, and IGFBP3. These results suggest a biologic model for clubfoot in which perturbation of HOX and apoptotic genes together affect muscle and limb development, which may cause the downstream failure of limb rotation into a plantar grade position.

Dilution of candidates: the case of iron-related genes in restless legs syndrome

Restless legs syndrome (RLS) is a common multifactorial disease. Some genetic risk factors have been identified. RLS susceptibility also has been related to iron. We therefore asked whether known iron-related genes are candidates for association with RLS and, vice versa, whether known RLS-associated loci influence iron parameters in serum. RLS/control samples (n = 954/1814 in the discovery step, 735/736 in replication 1, and 736/735 in replication 2) were tested for association with SNPs located within 4 Mb intervals surrounding each gene from a list of 111 iron-related genes using a discovery threshold of P = 5 × 10(-4). Two population cohorts (KORA F3 and F4 with together n = 3447) were tested for association of six known RLS loci with iron, ferritin, transferrin, transferrin-saturation, and soluble transferrin receptor. Results were negative. None of the candidate SNPs at the iron-related gene loci was confirmed significantly. An intronic SNP, rs2576036, of KATNAL2 at 18q21.1 was significant in the first (P = 0.00085) but not in the second replication step (joint nominal P-value = 0.044). Especially, rs1800652 (C282Y) in the HFE gene did not associate with RLS. Moreover, SNPs at the known RLS loci did not significantly affect serum iron parameters in the KORA cohorts. In conclusion, the correlation between RLS and iron parameters in serum may be weaker than assumed. Moreover, in a general power analysis, we show that genetic effects are diluted if they are transmitted via an intermediate trait to an end-phenotype. Sample size formulas are provided for small effect sizes.

Amyotrophic lateral sclerosis and Parkinsonism-dementia complex of Guam: Descriptive epidemiology, secular trends, and birth cohort effects on incidence, 1950--1989

This study describes the patterns of occurrence of amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia complex (PDC) of Guam during 1950-1989. Both ALS and PDC occur with high frequency among the indigenous Chamorro population, first recognized in the early 1950's. Reports in the early 1980's indicated that both ALS and PDC were disappearing, due to a purported reduction in exposure to harmful environmental factors as a result of the dramatic changes in lifestyle that took place after World War II. However, this study provides compelling evidence that ALS and PDC have not disappeared on Guam and that rates for both are higher during 1980-1989 than previously reported.^ The patterns of occurrence for both ALS and PDC overlap in most respects: (1) incidence and mortality are decreasing; (2) median age at onset is increasing; (3) males are at increased risk for developing disease; (4) risk is higher for those residing in the south compared to the non-south; and (5) age-specific incidence is decreasing over time except in the oldest age groups.^ Age-specific incidence of ALS and PDC, separately and together, is generally higher for cohorts born before 1920 than for those born after 1920. A significant birth cohort effect on the incidence of PDC for the 1906-1915 birth cohort was found, but not for ALS and for ALS and PDC together. Whether or not a cohort effect, period effect, or both are associated with incidence of ALS and PDC cannot be determined from the data currently available and will require additional follow-up of individuals born after 1920.^ The epidemiological data amassed over this 40-year period provide evidence that supports an environmental exposure model for disease occurrence as opposed to a simple genetic or infectious disease model. Whether neurodegenerative disease in this population occurs as a consequence of a single exposure or is explained by a multifactorial model such as a genetic predisposition with some environmental interaction is yet to be determined. However, descriptive studies such as this can provide clues concerning timing and location of potential adverse exposures but cannot determine etiology, underscoring the urgent need for analytic studies of ALS and PDC to further investigate existing etiologic hypotheses and to test new hypotheses. ^

Genes to blood pressure: The effects of variation in genes of the renin-angiotensin system on the hormonal and renal control of blood pressure

Objective. Essential hypertension affects 25% of the US adult population and is a leading contributor to morbidity and mortality. Because BP is a multifactorial phenotype that resists simple genetic analysis, intermediate phenotypes within the complex network of BP regulatory systems may be more accessible to genetic dissection. The Renin-Angiotensin System (RAS) is known to influence intermediate and long-term blood pressure regulation through alterations in vascular tone and renal sodium and fluid resorption. This dissertation examines associations between renin (REN), angiotensinogen (AGT), angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor (AT1) gene variation and interindividual differences in plasma hormone levels, renal hemodynamics, and BP homeostasis.^ Methods. A total of 150 unrelated men and 150 unrelated women, between 20.0 and 49.9 years of age and free of acute or chronic illness except for a history of hypertension (11 men and 7 women, all off medications), were studied after one week on a controlled sodium diet. RAS plasma hormone levels, renal hemodynamics and BP were determined prior to and during angiotensin II (Ang II) infusion. Individuals were genotyped by PCR for a variable number tandem repeat (VNTR) polymorphism in REN, and for the following restriction fragment length polymorphisms (RFLP): AGT M235T, ACE I/D, and AT1 A1166C. Associations between clinical measurements and allelic variation were examined using multiple linear regression statistical models.^ Results. Women homozygous for the AT1 1166C allele demonstrated higher intracellular levels of sodium (p = 0.044). Men homozygous for the AGT T235 allele demonstrated a blunted decrement in renal plasma flow in response to Ang II infusion (p = 0.0002). There were no significant associations between RAS gene variation and interindividual variation in RAS plasma hormone levels or BP.^ Conclusions. Rather than identifying new BP controlling genes or alleles, the study paradigm employed in this thesis (i.e., measured genes, controlled environments and interventions) may provide mechanistic insight into how candidate genes affect BP homeostasis. ^

The long non-coding RNA NEAT1 is increased in IUGR placentas, leading to potential new hypotheses of IUGR origin/development.

INTRODUCTION Intrauterine Growth Restriction (IUGR) is a multifactorial disease defined by an inability of the fetus to reach its growth potential. IUGR not only increases the risk of neonatal mortality/morbidity, but also the risk of metabolic syndrome during adulthood. Certain placental proteins have been shown to be implicated in IUGR development, such as proteins from the GH/IGF axis and angiogenesis/apoptosis processes. METHODS Twelve patients with term IUGR pregnancy (birth weight < 10th percentile) and 12 CTRLs were included. mRNA was extracted from the fetal part of the placenta and submitted to a subtraction method (Clontech PCR-Select cDNA Subtraction). RESULTS One candidate gene identified was the long non-coding RNA NEAT1 (nuclear paraspeckle assembly transcript 1). NEAT1 is the core component of a subnuclear structure called paraspeckle. This structure is responsible for the retention of hyperedited mRNAs in the nucleus. Overall, NEAT1 mRNA expression was 4.14 (±1.16)-fold increased in IUGR vs. CTRL placentas (P = 0.009). NEAT1 was exclusively localized in the nuclei of the villous trophoblasts and was expressed in more nuclei and with greater intensity in IUGR placentas than in CTRLs. PSPC1, one of the three main proteins of the paraspeckle, co-localized with NEAT1 in the villous trophoblasts. The expression of NEAT1_2 mRNA, the long isoform of NEAT1, was only modestly increased in IUGR vs. CTRL placentas. DISCUSSION/CONCLUSION The increase in NEAT1 and its co-localization with PSPC1 suggests an increase in paraspeckles in IUGR villous trophoblasts. This could lead to an increased retention of important mRNAs in villous trophoblasts nuclei. Given that the villous trophoblasts are crucial for the barrier function of the placenta, this could in part explain placental dysfunction in idiopathic IUGR fetuses.

Apoptosis of hippocampal neurons in organotypic slice culture models: direct effect of bacteria revisited

Neurons of the hippocampal dentate gyrus selectively undergo programmed cell death in patients suffering from bacterial meningitis and in experimental models of pneumococcal meningitis in infant rats. In the present study, a membrane-based organotypic slice culture system of rat hippocampus was used to test whether this selective vulnerability of neurons of the dentate gyrus could be reproduced in vitro. Apoptosis was assessed by nuclear morphology (condensed and fragmented nuclei), by immunochemistry for active caspase-3 and deltaC-APP, and by proteolytic caspase-3 activity. Co-incubation of the cultures with live pneumococci did not induce neuronal apoptosis unless cultures were kept in partially nutrient-deprived medium. Complete nutrient deprivation alone and staurosporine independently induced significant apoptosis, the latter in a dose-response way. In all experimental settings, apoptosis occurred preferentially in the dentate gyrus. Our data demonstrate that factors released by pneumococci per se failed to induce significant apoptosis in vitro. Thus, these factors appear to contribute to a multifactorial pathway, which ultimately leads to neuronal apoptosis in bacterial meningitis.

NGS Nominated CELA1, HSPG2, and KCNK5 as Candidate Genes for Predisposition to Balkan Endemic Nephropathy

Balkan endemic nephropathy (BEN) is a familial chronic tubulointerstitial disease with insidious onset and slow progression leading to terminal renal failure. The results of molecular biological investigations propose that BEN is a multifactorial disease with genetic predisposition to environmental risk agents. Exome sequencing of 22 000 genes with Illumina Nextera Exome Enrichment Kit was performed on 22 DNA samples (11 Bulgarian patients and 11 Serbian patients). Software analysis was performed via NextGene, Provean, and PolyPhen. The frequency of all annotated genetic variants with deleterious/damaging effect was compared with those of European populations. Then we focused on nonannotated variants (with no data available about them and not found in healthy Bulgarian controls). There is no statistically significant difference between annotated variants in BEN patients and European populations. From nonannotated variants with more than 40% frequency in both patients' groups, we nominated 3 genes with possible deleterious/damaging variants-CELA1, HSPG2, and KCNK5. Mutant genes (CELA1, HSPG2, and KCNK5) in BEN patients encode proteins involved in basement membrane/extracellular matrix and vascular tone, tightly connected to process of angiogenesis. We suggest that an abnormal process of angiogenesis plays a key role in the molecular pathogenesis of BEN.

Avoidable hospitalizations in Switzerland: a small area analysis on regional variation, density of physicians, hospital supply and rurality.

BACKGROUND Avoidable hospitalizations (AH) are hospital admissions for diseases and conditions that could have been prevented by appropriate ambulatory care. We examine regional variation of AH in Switzerland and the factors that determine AH. METHODS We used hospital service areas, and data from 2008-2010 hospital discharges in Switzerland to examine regional variation in AH. Age and sex standardized AH were the outcome variable, and year of admission, primary care physician density, medical specialist density, rurality, hospital bed density and type of hospital reimbursement system were explanatory variables in our multilevel poisson regression. RESULTS Regional differences in AH were as high as 12-fold. Poisson regression showed significant increase of all AH over time. There was a significantly lower rate of all AH in areas with more primary care physicians. Rates increased in areas with more specialists. Rates of all AH also increased where the proportion of residences in rural communities increased. Regional hospital capacity and type of hospital reimbursement did not have significant associations. Inconsistent patterns of significant determinants were found for disease specific analyses. CONCLUSION The identification of regions with high and low AH rates is a starting point for future studies on unwarranted medical procedures, and may help to reduce their incidence. AH have complex multifactorial origins and this study demonstrates that rurality and physician density are relevant determinants. The results are helpful to improve the performance of the outpatient sector with emphasis on local context. Rural and urban differences in health care delivery remain a cause of concern in Switzerland.

Familial occurrence and heritable connective tissue disorders in cervical artery dissection

OBJECTIVE In a large series of patients with cervical artery dissection (CeAD), a major cause of ischemic stroke in young and middle-aged adults, we aimed to examine frequencies and correlates of family history of CeAD and of inherited connective tissue disorders. METHODS We combined data from 2 large international multicenter cohorts of consecutive patients with CeAD in 23 neurologic departments participating in the CADISP-plus consortium, following a standardized protocol. Frequency of reported family history of CeAD and of inherited connective tissue disorders was assessed. Putative risk factors, baseline features, and 3-month outcome were compared between groups. RESULTS Among 1,934 consecutive patients with CeAD, 20 patients (1.0%, 95% confidence interval: 0.6%-1.5%) from 17 families (0.9%, 0.5%-1.3%) had a family history of CeAD. Family history of CeAD was significantly more frequent in patients with carotid location of the dissection and elevated cholesterol levels. Two patients without a family history of CeAD had vascular Ehlers-Danlos syndrome with a mutation in COL3A1. This diagnosis was suspected in 2 additional patients, but COL3A1 sequencing was negative. Two patients were diagnosed with classic and hypermobile Ehlers-Danlos syndrome, one patient with Marfan syndrome, and one with osteogenesis imperfecta, based on clinical criteria only. CONCLUSIONS In this largest series of patients with CeAD to date, family history of symptomatic CeAD was rare and inherited connective tissue disorders seemed exceptional. This finding supports the notion that CeAD is a multifactorial disease in the vast majority of cases.

When the systems fail to communicate. Cerebral resting state networks in schizophrenia spectrum disorders.

There have been numerous attempts to reveal the neurobiological basis of schizophrenia spectrum disorders. Results however, remain as heterogeneous as the schizophrenia spectrum disorders itself. Therefore, one aim of this thesis was to divide patients affected by this disorder into subgroups in order to homogenize the results of future studies. In a first study it is suggested that psychopathological rating scales should focus on symptoms-clusters that may have a common neurophysiological background. The here presented Bern Psychopathology Scale (BPS) proposes that alterations in three wellknown brain systems (motor, language, and affective) are largely leading to the communication failures observable on a behavioral level, but also - as repeatedly hypothesized - to dysconnectivity within and between brain systems in schizophrenia spectrum disorders. The external validity of the motor domain in the BPS was tested against the objective measure of 24 hours wrist actigraphy, in a second study. The subjective, the quantitative, as well as the global rating of the degree of motor disorders in this patient group showed significant correlations to the acquired motor activity. This result confirmed in a first step the practicability of the motor domain of the BPS, but needs further validation regarding pathological brain alterations. Finally, in a third study (independent from the two other studies), two cerebral Resting State Networks frequently altered in schizophrenia were investigated for the first time using simultaneous EEG/fMRI: The well-known default mode network and the left working memory network. Besides the changes in these fMRI-based networks, there are well-documented findings that patients exhibit alterations in EEG spectra compared to healthy controls. However, only through the multimodal approach it was possible to discover that patients with schizophrenia spectrum disorders have a slower driving frequency of the Resting State Networks compared to the matched healthy controls. Such a dysfunctional coupling between neuronal frequency and functional brain organization could explain in a uni- or multifactorial way (dysfunctional cross-frequency coupling, maturational effects, vigilance fluctuations, task-related suppression), how the typical psychotic symptoms might occur. To conclude, the major contributions presented in this thesis were on one hand the development of a psychopathology rating scale that is based on the assumption of dysfunctional brain networks, as well as the new evidence of a dysfunctional triggering frequency of Resting State Networks from the simultaneous EEG/fMRI study in patients affected by a schizophrenia spectrum disorder.

Climatic conditions as a risk factor in canine gastric dilatation-volvulus

Canine acute gastric dilatation-volvulus (GDV) is a life-threatening condition of multifactorial origin. The risk of developing GDV is influenced by a variety of factors, including breed, age, gender, temperament, diet and management. A relationship between seasonal variations and the frequency of GDV has been previously documented although no association was found with any specific climatic event. Variables in weather conditions within a defined geographic region were investigated in a retrospective study of 287 client-owned dogs diagnosed with GDV between 1992 and 1999. Monthly incidences were evaluated and differences in atmospheric temperature, humidity and pressure between days in which GDV cases were observed and days in which no case was presented were examined. Although temperature was significantly associated with the occurrence of GDV, the difference in temperatures between days with and days without GDV cases was so small that it is unlikely to be of clinical relevance. Moreover, no significant association was found between GDV occurrence and atmospheric pressure or humidity, and a seasonal variation in GDV incidence was not observed.

Canine mast cell tumours: a review of the pathogenesis, clinical features, pathology and treatment

Mast cells (MCs) are well known for their neoplastic transformation in solitary and multiple cutaneous mast cell tumours (MCTs), as well as visceral and systemic mastocytosis. Dogs have a unique risk of developing cutaneous MCTs, and they account for 7% to 21% of all canine skin tumours. The aetiology of canine MCTs is unknown but is probably multifactorial. This article reviews up-to-date knowledge on the pathogenesis, the clinical presentation, the clinical prognostic factors, the diagnostic workup including clinical staging, cytological findings, histological findings and the various grading systems which have been evaluated based on morphology, the assessment of proliferation markers and other factors such as vessel density. Furthermore, detailed information about current treatment protocols for canine cutaneous MCTs is provided.

BRCA2-Deficient Sarcomatoid Mammary Tumors Exhibit Multidrug Resistance.

Pan- or multidrug resistance is a central problem in clinical oncology. Here, we use a genetically engineered mouse model of BRCA2-associated hereditary breast cancer to study drug resistance to several types of chemotherapy and PARP inhibition. We found that multidrug resistance was strongly associated with an EMT-like sarcomatoid phenotype and high expression of the Abcb1b gene, which encodes the drug efflux transporter P-glycoprotein. Inhibition of P-glycoprotein could partly resensitize sarcomatoid tumors to the PARP inhibitor olaparib, docetaxel, and doxorubicin. We propose that multidrug resistance is a multifactorial process and that mouse models are useful to unravel this. Cancer Res; 75(4); 732-41. ©2014 AACR.