Ankyrin-B p.S646F undergoes increased proteasome degradation and reduces cell viability in the H9c2 rat ventricular cardiomyoblast cell line.

Ankyrin-B (AnkB) is scaffolding protein that anchors integral membrane proteins to the cardiomyocyte cytoskeleton. We recently identified an AnkB variant, AnkB p.S646F (ANK2 c.1937 C>T) associated with a phenotype ranging from predisposition for cardiac arrhythmia to cardiomyopathy. AnkB p.S646F exhibited reduced expression levels in the H9c2 rat ventricular-derived cardiomyoblast cell line relative to wildtype AnkB. Here, we demonstrate that AnkB is regulated by proteasomal degradation and proteasome inhibition rescues AnkB p.S646F expression levels in H9c2 cells, although this effect is not conserved with differentiation. We also compared the impact of wildtype AnkB and AnkB p.S646F on cell viability and proliferation. AnkB p.S646F expression resulted in decreased cell viability at 30 h after transfection, whereas we observed a greater proportion of cycling, Ki67-positive cells at 48 h after transfection. Notably, the number of GFP-positive cells was low and was consistent between wildtype AnkB and AnkB p.S646F expressing cells, suggesting that AnkB and AnkB p.S646F affected paracrine communication between H9c2 cells differentially. This work reveals that AnkB levels are regulated by the proteasome and that AnkB p.S646F compromises cell viability. Together, these findings provide key new insights into the putative cellular and molecular mechanisms of AnkB-related cardiac disease.

Heavy smoking during pregnancy as a marker for other risk factors of adverse birth outcomes: a population-based study in British Columbia, Canada.

BACKGROUND: Smoking during pregnancy is associated with known adverse perinatal and obstetrical outcomes as well as with socio-economic, demographic and other behavioural risk factors that independently influence outcomes. Using a large population-based perinatal registry, we assess the quantity of cigarettes smoked for the magnitude of adverse birth outcomes and also the association of other socio-economic and behavioural risk factors documented within the registry that influence pregnancy outcomes. Our goal was to determine whether number of cigarettes smoked could identify those in greatest need for comprehensive intervention programs to improve outcomes. METHODS: Our population-based retrospective study of singleton births from 2001 to 2006 (N = 237,470) utilized data obtained from the BC Perinatal Database Registry. Smoking data, self reported at the earliest prenatal visit, was categorized as: never, former, light (1 to 4), moderate (5 to 9), or heavy smoker (10 or more per day). Crude and adjusted odds ratios (AOR) with 95% confidence intervals (95% CI) were calculated using logistic regression models for smoking frequency and adverse birth outcomes. A partial proportional odds (pp-odds) model was used to determine the association between smoking status and other risk factors. RESULTS: There were 233,891 singleton births with available smoking status data. A significant dose-dependent increase in risk was observed for the adverse birth outcomes small-for-gestational age, term low birth weight and intra-uterine growth restriction. Results from the pp-odds model indicate heavy smokers were more likely to have not graduated high school: AOR (95% CI) = 3.80 (3.41-4.25); be a single parent: 2.27 (2.14-2.42); have indication of drug or alcohol use: 7.65 (6.99-8.39) and 2.20 (1.88-2.59) respectively, attend fewer than 4 prenatal care visits: 1.39 (1.23-1.58), and be multiparous: 1.59 (1.51-1.68) compared to light, moderate and non-smokers combined. CONCLUSION: Our data suggests that self reports of heavy smoking early in pregnancy could be used as a marker for lifestyle risk factors that in combination with smoking influence birth outcomes. This information may be used for planning targeted intervention programs for not only smoking cessation, but potentially other support services such as nutrition and healthy pregnancy education.

Mechanisms underlying the role of ankyrin-B in cardiac and neurological health and disease.

The ANK2 gene encodes for ankyrin-B (ANKB), one of 3 members of the ankyrin family of proteins, whose name is derived from the Greek word for anchor. ANKB was originally identified in the brain (B denotes "brain") but has become most widely known for its role in cardiomyocytes as a scaffolding protein for ion channels and transporters, as well as an interacting protein for structural and signaling proteins. Certain loss-of-function ANK2 variants are associated with a primarily cardiac-presenting autosomal-dominant condition with incomplete penetrance and variable expressivity characterized by a predisposition to supraventricular and ventricular arrhythmias, arrhythmogenic cardiomyopathy, congenital and adult-onset structural heart disease, and sudden death. Another independent group of ANK2 variants are associated with increased risk for distinct neurological phenotypes, including epilepsy and autism spectrum disorders. The mechanisms underlying ANKB's roles in cells in health and disease are not fully understood; however, several clues from a range of molecular and cell biological studies have emerged. Notably, ANKB exhibits several isoforms that have different cell-type-, tissue-, and developmental stage- expression profiles. Given the conservation within ankyrins across evolution, model organism studies have enabled the discovery of several ankyrin roles that could shed important light on ANKB protein-protein interactions in heart and brain cells related to the regulation of cellular polarity, organization, calcium homeostasis, and glucose and fat metabolism. Along with this accumulation of evidence suggesting a diversity of important ANKB cellular functions, there is an on-going debate on the role of ANKB in disease. We currently have limited understanding of how these cellular functions link to disease risk. To this end, this review will examine evidence for the cellular roles of ANKB and the potential contribution of ANKB functional variants to disease risk and presentation. This contribution will highlight the impact of ANKB dysfunction on cardiac and neuronal cells and the significance of understanding the role of ANKB variants in disease.

Rates of congenital anomalies and other adverse birth outcomes in an offspring cohort of registered nurses from British Columbia, Canada.

OBJECTIVES: Although there is concern that occupational exposures in some nursing professions may confer reproductive risks, there are few relevant studies. Our objective is to determine if a cohort of female registered nurses (RN) from British Columbia (BC), in comparison to BC women in general, have elevated rates of congenital anomalies, stillbirths, low birth weight, or prematurity in their offspring. METHODS: A cohort of RNs from BC was linked to Vital Statistics birth records and the BC Health Status Registry (HSR) between the years 1986 and 2000. The RN offspring cohort included 23,222 births. For each outcome, odds ratios (OR) with 95% confidence intervals (CI) were determined by comparing rates in the nurses' cohort with the rates in the general population standardized for the year of birth. RESULTS: Apart from chromosomal anomalies, for each of the ICD-9 congenital anomaly categories there were fewer congenital anomalies than expected, resulting in an overall prevalence of congenital anomaly cases (with at least one anomaly) that was significantly lower in the RN cohort (1,567 observed vs. 1,846 expected; OR 0.84, 95% CI 0.78-0.90) in comparison to the general population. Low birth weight (1,138 observed vs. 1,260 expected; OR 0.90, 95% CI 0.83-0.98) was also significantly lower, while prevalence of prematurity and stillbirths were not significantly different than among the general population. CONCLUSION: Offspring of RNs in BC had a lower prevalence of congenital anomalies and low birth weight compared to the rates found among the general population. Further studies are underway to determine if subpopulations within the RN cohort are at risk.

Maternal smoking at first prenatal visit as a marker of risk for adverse pregnancy outcomes in the Qikiqtaaluk (Baffin) Region.

INTRODUCTION: In Nunavut, 60-80% of pregnant women report smoking in pregnancy, a rate five times the Canadian average. Nunavut also has the highest rates of preterm birth and low birth weight infants in Canada. The present study assessed whether the number of cigarettes smoked per day, as recorded in the first trimester, influenced birth outcomes. METHODS: Maternal-newborn charts were reviewed for infants born between 1 January 2003 and 31 December 2005 to at least one Inuit parent in the Qikiqtaaluk (Baffin) region of Nunavut. Smoking data, as reported by the mother at an early prenatal visit, were extracted from the prenatal record. Birth outcomes including birth weights (for term births), low birth weight, small for gestational age births and rates of preterm birth, were analysed according to category of reported number of cigarettes smoked (0, 1-5, 6-10, and >10 per day). Maternal age, alcohol and street drug use were also assessed for each category of smokers. Statistical analysis among groups was carried out. RESULTS: Of 918 births meeting the study criteria, more than 80% of women reported smoking. For 80% of those, the amount smoked per day was available. Non-smokers and women smoking less than 5 cigarettes daily had perinatal outcomes equal to or better than Canadian averages for low birth weight, small for gestational age, and preterm birth. Furthermore, average birth weights at term significantly decreased from 3681 g for non smokers to 3310 g for those smoking more than 10 cigarettes per day. Compared with non-smokers, women in the highest smoking category (>10 cigarettes daily) had a six-fold increase in low birth weight infants (OR 6.7, 95% CI 2.3-19.6), almost a four-fold increase for small for gestational age births (OR 3.7, 95% CI 1.6-8.8) and twice the chance of a premature birth compared with non-smokers (OR 2.14, 95% CI 1.1-4.2). Those in the highest smoking category were also most likely to report alcohol and other substance use. CONCLUSIONS: Inuit women who reported not smoking, or smoking less than 5 cigarettes per day had birth outcomes equal or superior to average Canadian outcomes in each category evaluated. Those women reporting smoking more than 10 cigarettes daily had significantly increased risk for preterm birth, low birth weight and small for gestational age infants. Because those in the highest smoking category also reported the highest rates of alcohol and other substance use, it is likely that a combination of factors assessed in this study influenced the outcomes. Along with public health preventative measures to reduce smoking directed to this high risk group, other contributing factors for adverse birth outcomes need to be assessed more fully to understand the complex interactions that lead to increased smoking, substance use and, therefore, adverse birth outcomes. Furthermore, evidence from this study suggests that smoking more than 10 cigarettes per day, reported at first prenatal visit, may provide a marker for those women at highest risk of poor outcomes, which could provide direction for focused public health efforts.

Ankyrin B and Ankyrin B variants differentially modulate intracellular and surface Cav2.1 levels.

Ankyrin B (AnkB) is an adaptor and scaffold for motor proteins and various ion channels that is ubiquitously expressed, including in the brain. AnkB has been associated with neurological disorders such as epilepsy and autism spectrum disorder, but understanding of the underlying mechanisms is limited. Cav2.1, the pore-forming subunit of P/Q type voltage gated calcium channels, is a known interactor of AnkB and plays a crucial role in neuronal function. Here we report that wildtype AnkB increased overall Cav2.1 levels without impacting surface Cav2.1 levels in HEK293T cells. An AnkB variant, p.S646F, which we recently discovered to be associated with seizures, further increased overall Cav2.1 levels, again with no impact on surface Cav2.1 levels. AnkB p.Q879R, on the other hand, increased surface Cav2.1 levels in the presence of accessory subunits α2δ1 and ß4. Additionally, AnkB p.E1458G decreased surface Cav2.1 irrespective of the presence of accessory subunits. In addition, we found that partial deletion of AnkB in cortex resulted in a decrease in overall Cav2.1 levels, with no change to the levels of Cav2.1 detected in synaptosome fractions. Our work suggests that depending on the particular variant, AnkB regulates intracellular and surface Cav2.1. Notably, expression of the AnkB variant associated with seizure (AnkB p.S646F) caused further increase in intracellular Cav2.1 levels above that of even wildtype AnkB. These novel findings have important implications for understanding the role of AnkB and Cav2.1 in the regulation of neuronal function in health and disease.

Red blood cell folate levels in Canadian Inuit women of childbearing years: influence of food security, body mass index, smoking, education, and vitamin use.

BACKGROUND: The benefits of folic acid for prevention of congenital anomalies are well known. For the Inuit of Canada, where vitamin use is low and access to folate-rich foods limited, fortification is likely a major source of intake. We sought to determine whether red blood cell folate (RBCF) levels of Inuit women reached accepted target levels. METHODS: The Inuit Health Survey, 2007-2008, included evaluation of RBCF levels among 249 randomly selected non-pregnant women of reproductive age. Using descriptive statistics and linear regression analyses, RBCF levels were assessed and compared across several socio-demographic variables to evaluate the characteristics associated with RBCF status. RESULTS: Mean (SD) RBCF levels of 935.5 nmol/L (± 192) reached proposed target levels (> 906 nmol/L); however, 47% of women had lower than target levels. In bivariate analysis, non-smoking, higher education, higher income, food security, increased body mass index, and vitamin use were each significantly associated with higher RBCF. Increased levels of smoking had a negative association with RBCF levels (- 5.8 nmol/L per cigarette smoked per day (p = 0.001)). A total of 6.8% of women reported taking vitamin supplements, resulting in a 226 nmol/L higher RBCF level on average compared to non-users (p < 0.001). CONCLUSION: While mean levels of folate reached target levels, this was largely driven by the small number of women taking vitamin supplements. Our results suggest that folate status is often too low in Inuit women of childbearing years. Initiatives to improve food security, culturally relevant education on folate-rich traditional foods, vitamin supplements, and smoking cessation/reduction programs may benefit Inuit women and improve birth outcomes.

Retrospective review of pediatric and adult autoimmune hepatitis in two quaternary care centres in British Columbia: increased prevalence seen in British Columbia's First Nations community.

BACKGROUND: It has been previously reported that British Columbia's (BC's) First Nations (Aboriginal) community has an increased risk of autoimmune diseases, including rheumatological conditions (rheumatoid arthritis, systemic lupus) and primary biliary cirrhosis. The researchers hypothesized that this community may also be at increased risk for autoimmune hepatitis (AIH). METHODS: Independent, retrospective reviews of the databases of two separate tertiary/quaternary British Columbia university-affiliated health care institutions, the Adult Liver Transplant Program of the BC Transplant Society and the Division of Pediatric Gastroenterology, BC Children's Hospital (Vancouver, BC), were performed. All patients referred with a diagnosis of probable or definite AIH who identified themselves as being of First Nations descent from 1988 to 2004 were reviewed. The liver transplant database records all adult patients in the province referred for transplant assessment. The pediatric database records all children referred to the BC Children's Hospital. RESULTS: A total of 68 adult patients with a definite or probable diagnosis of AIH were referred to the liver transplant program. Twelve patients (17.6%) were Aboriginal, 11 of which were female. Similarly, a total of 30 children with probable or definite AIH were identified from the pediatric database. Six of these cases (20%) were identified in Aboriginal children. CONCLUSIONS: The findings suggest an increased prevalence of AIH among BC's First Nations community. A disproportionate First Nations representation was found on independent review of two databases. Future studies are needed to determine the true prevalence of AIH in this community, and to uncover the genetic predisposition and the environmental triggers explaining this phenomenon.

Novel Variant in the ANK2 Membrane-Binding Domain Is Associated With Ankyrin-B Syndrome and Structural Heart Disease in a First Nations Population With a High Rate of Long QT Syndrome.

BACKGROUND: Long QT syndrome confers susceptibility to ventricular arrhythmia, predisposing to syncope, seizures, and sudden death. While rare globally, long QT syndrome is ≈15× more common in First Nations of Northern British Columbia largely because of a known mutation in KCNQ1. However, 2 large multigenerational families were affected, but negative for the known mutation. METHODS AND RESULTS: Long QT syndrome panel testing was carried out in the index case of each family, and clinical information was collected. Cascade genotyping was performed. Biochemical and myocyte-based assays were performed to evaluate the identified gene variant for loss-of-function activity. Index cases in these 2 families harbored a novel ANK2 c.1937C>T variant (p.S646F). An additional 16 carriers were identified, including 2 with structural heart disease: one with cardiomyopathy resulting in sudden death and the other with congenital heart disease. For all carriers of this variant, the average QTc was 475 ms (±40). Although ankyrin-B p.S646F is appropriately folded and expressed in bacteria, the mutant polypeptide displays reduced expression in cultured H9c2 cells and aberrant localization in primary cardiomyocytes. Furthermore, myocytes expressing ankyrin-B p.S646F lack normal membrane targeting of the ankyrin-binding partner, the Na/Ca exchanger. Thus, ankyrin-B p.S646F is a loss-of-function variant. CONCLUSIONS: We identify the first disease-causing ANK2 variant localized to the membrane-binding domain resulting in reduced ankyrin-B expression and abnormal localization. Further study is warranted on the potential association of this variant with structural heart disease given the role of ANK2 in targeting and stabilization of key structural and signaling molecules in cardiac cells.

Autoimmune liver disease and the Canadian First Nations Aboriginal Communities of British Columbia's Pacific Northwest.

Primary biliary cirrhosis (PBC) is a well-known but uncommon chronic liver disease that is presumed to be of autoimmune etiology. Recently, investigations in British Columbia (BC), a province of Canada situated along the Pacific North-West of North America, have suggested that PBC is not a rare disease amongst BC's Aboriginal (i.e. First Nations) communities. Geographically, BC is adjacent to South East Alaska, an American state that has also reported an increased prevalence of PBC amongst its Aboriginal communities. In this article, the medical evidence supporting a hypothesis of increased risk of PBC amongst BC's First Nations communities is reviewed. Evidence suggesting that autoimmune hepatitis is also more likely amongst BC's First Nations communities is also presented.

Age differences in vitamin A intake among Canadian Inuit.

BACKGROUND: Inuit traditional food provides ample amounts of preformed vitamin A. However, the dietary transition away from traditional food raises concerns regarding dietary adequacy. Vitamin A is an essential nutrient with inadequate and excessive exposures having adverse effects. OBJECTIVE: To evaluate total dietary vitamin A intake for Canadian Inuit from market food and traditional food sources and to evaluate retinol concentrations in liver and blubber. METHODS: Dietary surveys were conducted in 18 communities representing 5 Inuit regions, and traditional food items were evaluated for nutrient content. RESULTS: Among those 15-40 years of age, 68% of men and 60% of women had a dietary vitamin A intake below the estimated average requirement (EAR) for retinol activity equivalents (RAE)/day. Among those over 40 years of age, only 11 % of men and 15% of women had a dietary vitamin A intake below the EAR. Young Inuit men had a relative risk of 6.2 (95% CI= 4.5-8.4), and young Inuit women had a relative risk of 4.0 (95% CI= 3.1-5.0) for dietary inadequacy compared to the older Inuit men and women, respectively. The median retinol content of liver of ringed seal, caribou, and fish were comparable to levels observed in market food liver. Liver was less frequently consumed by those 15-40 years of age than among older Inuit. DISCUSSION: Sub-optimal vitamin A intake is the predominant nutritional concern rather than excessive exposures. Public health education campaigns are needed to improve vitamin A intake among the younger generations of Inuit men and women.