Vanya Peltekova

Functional variants of OCTN cation transporter genes are associated with Crohn disease

Crohn disease is a chronic, inflammatory disease of the gastrointestinal tract. A locus of ∼250 kb at 5q31 (IBD5) was previously associated with susceptibility to Crohn disease, as indicated by increased prevalence of a risk haplotype of 11 single-nucleotide polymorphisms among individuals with Crohn disease, but the pathogenic lesion in the region has not yet been identified. We report here that two variants in the organic cation transporter cluster at 5q31 (a missense substitution in SLC22A4 and a G→C transversion in the SLC22A5 promoter) form a haplotype associated with susceptibility to Crohn disease. These variants alter transcription and transporter functions of the organic cation transporters and interact with variants in another gene associated with Crohn disease, CARD15, to increase risk of Crohn disease. These results suggest that SLC22A4, SLC22A5 and CARD15 act in a common pathogenic pathway to cause Crohn disease.

Determinants of Peak Bone Mass: Clinical and Genetic Analyses in a Young Female Canadian Cohort

Peak bone mass has been shown to be a significant predictor of risk for osteoporosis. Previous studies have demonstrated that skeletal mass accumulation is under strong genetic control, and efforts have been made to identify candidate loci. Determinants of peak bone mass also include diet, physical activity, hormonal status, and other clinical factors. The overall contribution of these factors, genetic and nongenetic, and their interaction in determining peak bone density status have not been delineated. Six hundred and seventy‐seven healthy unrelated Caucasian women ages 18–35 years were assessed. A detailed, standardized interview was conducted to evaluate lifestyle factors, menstrual and reproductive history, medical conditions, medication use, and family history of osteoporosis. Bone mineral density (BMD) was measured at the lumbar spine (L2–L4) and the femoral neck (hip) using dual‐energy X‐ray absorptiometry. Genotyping of the vitamin D receptor (VDR) locus at three polymorphic sites (BsmI, ApaI, and TaqI) was performed. In bivariate analyses, BMD at the lumbar spine and hip was positively correlated with weight, height, body mass index (BMI), and level of physical activity, both now and during adolescence, but negatively correlated with a family history of osteoporosis. Hip, but not spine BMD, correlated positively with dietary intake of calcium, and negatively with amenorrhea of more than 3 months, with caffeine intake, and with age. Spine, but not hip BMD, correlated positively with age and with number of pregnancies. VDR haplotype demonstrated significant associations with BMD at the hip, level of physical activity currently, and BMI. In multivariate analysis, independent predictors of greater BMD (at the hip or spine) were: age (younger for the hip, older for the spine), greater body weight, greater height (hip only), higher level of physical activity now and during adolescence, no family history of osteoporosis, and VDR genotype (hip only). Weight, age, level of physical activity, and family history are independent predictors of peak BMD. Of these factors, weight accounts for over half the explained variability in BMD. VDR alleles are significant independent predictors of peak femoral neck, but not lumbar spine BMD, even after adjusting for family history of osteoporosis, weight, age, and exercise. However, the overall contribution of this genetic determinant is modest. Taken together, these factors explained ∼17% and 21% of the variability in peak spine and hip BMD, respectively, in our cohort. Future research should be aimed at further evaluation of genetic determinants of BMD. Most importantly, understanding the critical interactive nature between genes and the environment will facilitate development of targeted strategies directed at modifying lifestyle factors as well as earlier intervention in the most susceptible individuals.

A986S polymorphism of the calcium-sensing receptor and circulating calcium concentrations

BACKGROUND The regulation of extracellular calcium concentration by parathyroid hormone is mediated by a calcium-sensing, G-protein-coupled cell-surface receptor (CASR). Mutations of the CASR gene alter the set-point for extracellular ionised calcium [Ca2+]o and cause familial hypercalcaemia or hypocalcaemia. The CASR missense polymorphism, A986S, is common in the general population and is, therefore, a prime candidate as a genetic determinant of extracellular calcium concentration. METHODS We genotyped the CASR A986S variant (S allele frequency of 16.3%) in 163 healthy adult women and tested samples of their serum for total calcium, albumin, total protein, creatinine, phosphate, pH, and parathyroid hormone. A prospectively generated, random subset of 84 of these women provided a whole blood sample for assay of [Ca2+]o. FINDINGS The A986S genotype showed no association with total serum concentration of calcium, until corrected for albumin. In a multivariate regression model, biochemical and genetic variables accounted for 74% of the total variation in calcium. The significant predictors of serum calcium were: albumin (p<0.001), phosphate (p=0.02), parathyroid hormone (p=0.007), pH (p=0.001), and A986S genotype (p=0.009). Fasting whole-blood [Ca2+]o also showed an independent positive association with the 986S variant (p=0.013). INTERPRETATION The CASR A986S variant has a significant effect on extracellular calcium. The CASR A986S polymorphism is a likely candidate locus for genetic predisposition to various bone and mineral disorders in which extracellular calcium concentrations have a prominent part.

Vitamin D-receptor genotypes as independent genetic predictors of decreased bone mineral density in primary biliary cirrhosis.

BACKGROUND & AIMS Hepatic osteodystrophy is a complication of primary biliary cirrhosis (PBC). Allelic polymorphisms of the vitamin D receptor (VDR) gene are related to bone mineral density (BMD) in normal cohorts and those with primary osteoporosis. We sought to establish the prevalence of reduced bone mass in PBC, correlate BMD with VDR gene polymorphisms, and identify risk factors for the development of hepatic osteodystrophy. METHODS Seventy-two female patients with PBC were evaluated prospectively. Clinical information, BMD assessment, disease severity, and osteoporosis risk factors were documented, and multivariate regression modeling was performed. RESULTS Twenty-four percent of the patients were osteoporotic at the lumbar spine and 32% at the femur. Severe bone loss (z score <-2.0) occurs 4 times more frequently in patients with PBC compared with controls. Body weight (P = 0.003) and postmenopausal status (P = 0.012) correlated independently with BMD. VDR genotype (P = 0.01) correlated with lower BMD at the spine only. CONCLUSIONS Osteoporosis is a common complication of PBC. VDR genotype predicts lower BMD in patients with PBC. Studies are warranted to investigate the mechanism(s) by which VDR as well as other candidate genes may contribute to the development of hepatic osteodystrophy in PBC.

Hypercapnic acidosis in ventilator-induced lung injury

RationalePermissive hypercapnia is established in lung injury management. Therapeutic hypercapnia causes benefit or harm, depending on the context. Ventilator-associated lung injury has a wide spectrum of candidate mechanisms, affording multiple opportunities for intervention such as hypercapnia to exert benefit or harm.ObjectivesTo confirm (1) that hypercapnia attenuates in vivo ventilator-induced lung injury (VILI); (2) biological plausibility of such protection (e.g., dose-response, time series, inflammatory profile); and (3) that the associated biochemical events are consistently beneficial.MethodsA mouse model of VILI was established in vivo. Injurious ventilation was established, hypercapnia applied and markers of inflammation measured.MeasurementsLung injury was quantified by gas exchange, elastance, microvascular leak, histology and levels of cytokines and eicosanoids, cyclooxygenase and tissue nitrotyrosine.Main resultsInjurious ventilation caused significant lung injury (mechanics, microvascular leak, histology) and release of inflammatory cytokines, chemokines and eicosanoids. Hypercapnia attenuated these responses, with dose-response and time-dependent effects. No adverse effects of hypercapnia were observed in controls. Hypercapnia suppressed the transcription (mRNA) and translation (protein) of the major inducible prostanoid-generating enzyme (COX-2), but the effects on the downstream eicosanoids were modest. However, hypercapnia significantly increased lung tissue nitrotyrosine—at PaCO2 levels that were protective.ConclusionsHypercapnia provided consistent and biologically plausible in vivo protection against VILI, but elevated lung tissue levels of nitro-tyrosine as previously described in sepsis. Clinicians and those designing clinical trials need to be aware of the potential for detrimental effects when using hypercapnia in order to balance benefits versus harm with this approach.

Early Growth Response-1 Worsens Ventilator-induced Lung Injury by Up-Regulating Prostanoid Synthesis

RATIONALE Ventilator-induced lung injury (VILI) is common and serious and may be mediated in part by prostanoids. We have demonstrated increased expression of the early growth response-1 (Egr1) gene by injurious ventilation, but whether-or how-such up-regulation contributes to injury is unknown. OBJECTIVES We sought to define the role of Egr1 in the pathogenesis of VILI. METHODS An in vivo murine model of VILI was used, and Egr1(+/+) (wild-type) and Egr1(-/-) mice were studied; the effects of prostaglandin E receptor subtype 1 (EP1) inhibition were assessed. MEASUREMENTS AND MAIN RESULTS Injurious ventilation caused lung injury in wild-type mice, but less so in Egr1(-/-) mice. The injury was associated with expression of EGR1 protein, which was localized to type II cells and macrophages and was concentrated in nuclear extracts. There was a concomitant increase in expression of phosphorylated p44/p42 mitogen-activated protein kinases. The prostaglandin E synthase (mPGES-1) gene has multiple EGR1 binding sites on its promoter, and induction of mPGES-1 mRNA (as well as the prostanoid product, PGE2) by injurious ventilation was highly dependent on the presence of the Egr1 gene. PGE2 mediates many lung effects via EP1 receptors, and EP1 blockade (with ONO-8713) lessened lung injury. CONCLUSIONS This is the first demonstration of a mechanism whereby expression of a novel gene (Egr1) can contribute to VILI via a prostanoid-mediated pathway.

Constraints on proper folding of the amino terminal domains of group iii metabotropic glutamate receptors

The glutamate binding site of the G-protein coupled metabotropic glutamate receptors (mGluRs) is contained within the large extracellular amino terminal domain (ATD) of the receptor. In this study, we examined the ligand binding properties and cellular dispositions of the membrane-bound mGluR4 and mGluR8 subtypes of mGluRs, and a series of truncated versions of these receptors. Truncation of the ATDs of mGluR4 and mGluR8 40 amino acids upstream of the first transmembrane domain produced soluble proteins that were secreted into the cell culture media of transfected human embryonic kidney cells. The soluble receptors retained ligand binding capabilities. Additional constructs of the ATDs of mGluR4 and mGluR8 were assessed for their ability to bind the agonist [(3)H]L-AP4 and for secretion from cells. A shorter mGluR4 construct truncated 98 amino acids upstream from the first transmembrane domain failed to bind [(3)H]L-AP4, while the analogous mGluR8 construct displayed a low level of binding. Unlike the full-length receptors, which were expressed on the cell surface, or the soluble constructs which were secreted, the shorter constructs were primarily associated with intracellular membranes. These observations suggest that the cysteine-rich region may be important for efficient secretion, but not absolutely obligatory for ligand binding. Surprisingly, longer constructs encoding the entire ATDs of mGluR4 and mGluR8 failed to bind ligand and were localized intracellularly. Together, these findings demonstrate that there are strict limitations on the proper folding of truncated versions of the ATDs of mGluR4 and mGluR8. Specifically, all of the leucine-isoleucine-valine binding protein homology region, and part of the cysteine-rich region is required for optimal secretion in a soluble form that retains ligand binding activity.

Calcium sensing receptor gene: analysis of polymorphism frequency

The role that the Calcium Sensing Receptor (CASR) plays in extracellular calcium regulation had been ascertained through studies of inactivating as well as activating mutations of CASR gene in a number of multiplex families. We have extended these observations to a polymorphism analysis of the intercellular domain of CASR in a cohort of healthy young women. The results demonstrate significant allelic polymorphism as a result of nonconservative changes at two specific sites. Further studies will be required to determine what, if any, relationship this may have to CASR phenotype.

Dissociation of inflammatory mediators and function: experimental lung injury in nonpulmonary sepsis.

Background:Sepsis is a common indication for mechanical ventilation, which, with higher tidal volume, can cause ventilator-associated lung injury. Inflammatory mediators in the plasma or bronchoalveolar fluid are sometimes proposed as biomarkers in ICU patients. Objective:To test the hypothesis that “priming” with subthreshold sepsis in a clinically relevant model would worsen lung function, increase ventilator-induced mediator production, and differentially impact systemic vs. pulmonary mediator levels. The model used was cecal ligation and perforation modified so that alone it caused lung inflammatory responses but not injury. Methods and Main Results:Anesthetized mice were randomized to cecal ligation and perforation (vs. sham) with or without dexamethasone and 6 hrs later further randomized to: 1) sham, nonventilated, saline; 2) cecal ligation and perforation, nonventilated, saline; 3) cecal ligation and perforation, nonventilated, dexamethasone; 4) sham, high tidal volume, saline; 5) sham, high tidal volume, dexamethasone; 6) cecal ligation and perforation, high tidal volume, saline; or 7) cecal ligation and perforation, high tidal volume, dexamethasone. Mediators associated with sepsis and lung injury (cytokines: interleukin-6, tumor necrosis factor-&agr;; chemokine: keratinocyte stimulating factor) were measured in the plasma and the bronchoalveolar lavage, and lung function (compliance, oxygenation, alveolar protein leak) assessed. High tidal volume and cecal ligation and perforation increased individual bronchoalveolar lavage and plasma mediators; high tidal volume but not cecal ligation and perforation impaired lung function. Priming of high tidal volume by cecal ligation and perforation intensified plasma and bronchoalveolar lavage mediators; the plasma (but not the bronchoalveolar lavage) mediators were inhibited by dexamethasone pretreatment. Conclusions:Mediator—but not functional—responses to high tidal volume are augmented by subthreshold sepsis priming. There is important discordance among systemic and pulmonary mediators, physiologic function, and response to corticosteroids; thus, mediator levels may be incomplete surrogates for measures of lung injury or response to therapy in the context of systemic sepsis.

Direct haplotyping at the vitamin D receptor locus improves genetic resolution.

In this journal, Tokita et al. recently reported a significant association between restriction fragment length polymorphisms (RFLPs) of the vitamin D receptor (VDR) gene and bone mineral density (BMD) in a group of Japanese women. They concluded that VDR RFLPs affect BMD regardless of race on the basis of haplotypes inferred from analysis of three adjacent RFLP loci (B/b at a BsmI site, A/a at an ApaI site, and T/t at a TaqI site) in intron 8 and exon 9 of the VDR gene. However, the argument for increased predictive power with a three-locus genotype assumes complete linkage disequilibrium between RFLPs, unless direct haplotyping is performed. A direct haplotyping procedure developed by Uitterlinden et al. permits determination of the phase of the alleles at the three RFLP sites. In the Asian population, Tokita et al. describe the prevalent Asian genotype, BbAATt, as haplotype BAt/bAT [2,3], based on the relative frequency of the alleles inferred from the three most frequent homozygous genotypes: bbaaTT [1,1], BBAAtt [2,2], and bbAATT [3,3]. We wish to report an important counterexample, illustrated in Fig. 1. While subjects I and II share the same BbAATt genotype, as determined by individual RFLPs, the direct method reveals a BAt/bAT [2,3] haplotype in subject I, but unambiguously reveals BAT/bAt in subject II, to whom we can assign the haplotype [4,5] on the basis of allelic frequencies in our European and North American populations. For the fully heterozygote [1,2], ambiguity in determining the phase of alleles at the separate RFLP sites is an even greater problem and direct haplotyping becomes even more pertinent. While the less common haplotype alleles we have observed to date (BAT [4], bAt [5], and Bat [6]) collectively account for a minority of subjects, they are found in a varying fraction of each population sample (n . 3000 subjects). This has two important consequences. First, the lack of association reported by some, and allelic variation in association reported by us and others can be expected, if the degree of disequilibrium between the RFLP loci varies with the population studied. Second, the accuracy of the direct haplotyping method is intrinsically higher than independent RFLP analysis and is preferred. Moreover, with direct haplotyping, the number of gels required is reduced by two-thirds, and the DNA polymerase required for PCR is reduced by half. Direct haplotyping thus beFIG. 1. RFLP analysis of the VDR genotype BbAATt. (A) Schematic representation of the VDR region encompassing BsmI, ApaI, and TaqI polymorphic sites. Position and orientation of the OP1, OP2, OP3, and OP4 primers is shown along the top. The lower panel represents the position of restriction sites and the predicted fragment sizes after digestion. The asterisk (*) shows the position of the invariant TaqI site. (B) I and II represent the genomic DNA of two different individuals. The region encompassing the intron 8 and exon 9 was PCR amplified using primers OP1 and OP2 (lanes 2–4), OP3 and OP4 (lanes 5–10) or OP1 and OP4 (lanes 11–13). Primer sequences are as previously described. PCR products were digested with restriction endonucleases (shown at the bottom) and resolved by 4–20% gradient polyacrylamide TBE gel electrophoresis. Restriction fragments were visualized by staining the gels with silver stain (Pharmacia, Uppsala, Sweden). Lane 1 shows the migration of a 100-bp DNA ladder. Lane 14 is the marker that was made by mixing all the possible haplotypes and represents all fragments arising from the BsmI, ApaI, and TaqI digestions. Predicted haplotype is shown along the top. Lanes 2, 5, 8, and 11 are undigested PCR products. JOURNAL OF BONE AND MINERAL RESEARCH Volume 12, Number 3, 1997 Blackwell Science, Inc. q 1997 American Society for Bone and Mineral Research