Exome sequencing-driven discovery of coding polymorphisms associated with common metabolic phenotypes.

AIMS/HYPOTHESIS: Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes. METHODS: The study comprised three stages. We performed medium-depth (8×) whole exome sequencing in 1,000 cases with type 2 diabetes, BMI >27.5 kg/m(2) and hypertension and in 1,000 controls (stage 1). We selected 16,192 polymorphisms nominally associated (p < 0.05) with case-control status, from four selected annotation categories or from loci reported to associate with metabolic traits. These variants were genotyped in 15,989 Danes to search for association with 12 metabolic phenotypes (stage 2). In stage 3, polymorphisms showing potential associations were genotyped in a further 63,896 Europeans. RESULTS: Exome sequencing identified 70,182 polymorphisms with MAF >1%. In stage 2 we identified 51 potential associations with one or more of eight metabolic phenotypes covered by 45 unique polymorphisms. In meta-analyses of stage 2 and stage 3 results, we demonstrated robust associations for coding polymorphisms in CD300LG (fasting HDL-cholesterol: MAF 3.5%, p = 8.5 × 10(-14)), COBLL1 (type 2 diabetes: MAF 12.5%, OR 0.88, p = 1.2 × 10(-11)) and MACF1 (type 2 diabetes: MAF 23.4%, OR 1.10, p = 8.2 × 10(-10)). CONCLUSIONS/INTERPRETATION: We applied exome sequencing as a basis for finding genetic determinants of metabolic traits and show the existence of low-frequency and common coding polymorphisms with impact on common metabolic traits. Based on our study, coding polymorphisms with MAF above 1% do not seem to have particularly high effect sizes on the measured metabolic traits.

Chromosome-wide distribution of haplotype blocks and the role of recombination hot spots.

Recent studies of human populations suggest that the genome consists of chromosome segments that are ancestrally conserved ('haplotype blocks'; refs. 1-3) and have discrete boundaries defined by recombination hot spots. Using publicly available genetic markers, we have constructed a first-generation haplotype map of chromosome 19. As expected for this marker density, approximately one-third of the chromosome is encompassed within haplotype blocks. Evolutionary modeling of the data indicates that recombination hot spots are not required to explain most of the observed blocks, providing that marker ascertainment and the observed marker spacing are considered. In contrast, several long blocks are inconsistent with our evolutionary models, and different mechanisms could explain their origins.

A role for coding functional variants in HNF4A in type 2 diabetes susceptibility.

AIMS/HYPOTHESIS: Rare mutations in the gene HNF4A, encoding the transcription factor hepatocyte nuclear factor 4α (HNF-4A), account for ~5% of cases of MODY and more frequent variants in this gene may be involved in multifactorial forms of diabetes. Two low-frequency, non-synonymous variants in HNF4A (V255M, minor allele frequency [MAF] ~0.1%; T130I, MAF ~3.0%)-known to influence downstream HNF-4A target gene expression-are of interest, but previous type 2 diabetes association reports were inconclusive. We aimed to evaluate the contribution of these variants to type 2 diabetes susceptibility through large-scale association analysis. METHODS: We genotyped both variants in at least 5,745 cases and 14,756 population controls from the UK and Denmark. We also undertook an expanded association analysis that included previously reported and novel genotype data obtained in Danish, Finnish, Canadian and Swedish samples. A meta-analysis incorporating all published association studies of the T130I variant was subsequently carried out in a maximum sample size of 14,279 cases and 26,835 controls. RESULTS: We found no association between V255M and type 2 diabetes in either the initial (p = 0.28) or the expanded analysis (p = 0.44). However, T130I demonstrated a modest association with type 2 diabetes in the UK and Danish samples (additive per allele OR 1.17 [95% CI 1.08-1.28]; p = 1.5 × 10⁻4), which was strengthened in the meta-analysis (OR 1.20 [95% CI 1.10-1.30]; p = 2.1 × 10⁻5). CONCLUSIONS/INTERPRETATION: Our data are consistent with T130I as a low-frequency variant influencing type 2 diabetes risk, but are not conclusive when judged against stringent standards for genome-wide significance. This study exemplifies the difficulties encountered in association testing of low-frequency variants.

High-density SNP association study and copy number variation analysis of the AUTS1 and AUTS5 loci implicate the IMMP2L-DOCK4 gene region in autism susceptibility.

Autism spectrum disorders are a group of highly heritable neurodevelopmental disorders with a complex genetic etiology. The International Molecular Genetic Study of Autism Consortium previously identified linkage loci on chromosomes 7 and 2, termed AUTS1 and AUTS5, respectively. In this study, we performed a high-density association analysis in AUTS1 and AUTS5, testing more than 3000 single nucleotide polymorphisms (SNPs) in all known genes in each region, as well as SNPs in non-genic highly conserved sequences. SNP genotype data were also used to investigate copy number variation within these regions. The study sample consisted of 127 and 126 families, showing linkage to the AUTS1 and AUTS5 regions, respectively, and 188 gender-matched controls. Further investigation of the strongest association results was conducted in an independent European family sample containing 390 affected individuals. Association and copy number variant analysis highlighted several genes that warrant further investigation, including IMMP2L and DOCK4 on chromosome 7. Evidence for the involvement of DOCK4 in autism susceptibility was supported by independent replication of association at rs2217262 and the finding of a deletion segregating in a sib-pair family.

Decoding of standard and non-standard visuomotor associations from parietal cortex.

OBJECTIVE: Neural signals can be decoded and used to move neural prostheses with the purpose of restoring motor function in patients with mobility impairments. Such patients typically have intact eye movement control and visual function, suggesting that cortical visuospatial signals could be used to guide external devices. Neurons in parietal cortex mediate sensory-motor transformations, encode the spatial coordinates for reaching goals, hand position and movements, and other spatial variables. We studied how spatial information is represented at the population level, and the possibility to decode not only the position of visual targets and the plans to reach them, but also conditional, non-spatial motor responses. APPROACH: The animals first fixated one of nine targets in 3D space and then, after the target changed color, either reached toward it, or performed a non-spatial motor response (lift hand from a button). Spiking activity of parietal neurons was recorded in monkeys during two tasks. We then decoded different task related parameters. MAIN RESULTS: We first show that a maximum-likelihood estimation (MLE) algorithm trained separately in each task transformed neural activity into accurate metric predictions of target location. Furthermore, by combining MLE with a Naïve Bayes classifier, we decoded the monkey's motor intention (reach or hand lift) and the different phases of the tasks. These results show that, although V6A encodes the spatial location of a target during a delay period, the signals they carry are updated around the movement execution in an intention/motor specific way. SIGNIFICANCE: These findings show the presence of multiple levels of information in parietal cortex that could be decoded and used in brain machine interfaces to control both goal-directed movements and more cognitive visuomotor associations.

Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein.

The rotavirus nonstructural glycoprotein NSP4 is an intracellular receptor that mediates the acquisition of a transient membrane envelope as subviral particles bud into the endoplasmic reticulum. NSP4 also causes an increase in intracellular calcium in insect cells. Purified NSP4 or a peptide corresponding to NSP4 residues 114 to 135 induced diarrhea in young (6 to 10 days old) CD1 mice. This disease response was age-dependent, dose-dependent, and specific. Electrophysiologic data from intestinal mucosa showed that the NSP4 114-135 peptide potentiates chloride secretion by a calcium-dependent signaling pathway. Diarrhea is induced when NSP4, acting as a viral enterotoxin, triggers a signal transduction pathway.

Genome-wide association and epidemiological analyses reveal common genetic origins between uterine leiomyomata and endometriosis.

Uterine leiomyomata (UL) are the most common neoplasms of the female reproductive tract and primary cause for hysterectomy, leading to considerable morbidity and high economic burden. Here we conduct a GWAS meta-analysis in 35,474 cases and 267,505 female controls of European ancestry, identifying eight novel genome-wide significant (P < 5 × 10-8) loci, in addition to confirming 21 previously reported loci, including multiple independent signals at 10 loci. Phenotypic stratification of UL by heavy menstrual bleeding in 3409 cases and 199,171 female controls reveals genome-wide significant associations at three of the 29 UL loci: 5p15.33 (TERT), 5q35.2 (FGFR4) and 11q22.3 (ATM). Four loci identified in the meta-analysis are also associated with endometriosis risk; an epidemiological meta-analysis across 402,868 women suggests at least a doubling of risk for UL diagnosis among those with a history of endometriosis. These findings increase our understanding of genetic contribution and biology underlying UL development, and suggest overlapping genetic origins with endometriosis.

Increased beta-catenin expression and nuclear translocation accompany cellular hyperproliferation in vivo.

Beta-catenin performs critical roles in development and cellular adhesion. More recently, an oncogenic role has been described. In colon cancer, decreased E-cadherin/beta-catenin association is causally linked to increased beta-catenin-regulated gene expression and increased cellular division. Whether the same pathway is active in native epithelia remains unknown. To address this question, we used the transmissible murine colonic hyperplasia model to measure changes in beta-catenin abundance, nuclear partitioning, target gene (c-myc and cyclin D1) expression, and subcellular distribution. Colonocyte hyperproliferation was associated with a 4.3 +/- 0.56 (SD)-fold increase in total cellular beta-catenin protein content, whereas modest changes in gamma-catenin and E-cadherin expression were recorded. The beta-catenin signal increased before changes in mucosal crypt length, a gross index of cellular proliferation/apoptosis. Beta-catenin detected in Triton X-100-soluble (cytosolic) cellular fractions was enriched 4.3 +/- 0.9 (SD)-fold, whereas a modest decrease of 0.9 +/- 0.09 (SD)-fold was recorded in Triton X-100-insoluble (cytoskeletal) fractions. After these changes, nuclear beta-catenin partitioning increased 2.4 +/- 0.4 (SD)-fold, accompanied by 2.5 +/- 0.4- and 4.0 +/- 0.8-fold (SD) increases in cellular c-myc and cyclin D1 levels, respectively. Thus, increased cellular cytosolic and nuclear beta-catenin levels were associated with increased beta-catenin target protein expression. Significant alterations in beta-catenin subcellular distribution were also recorded immunohistochemically. Apical/lateral junctional labeling was observed in normal crypts with increased lateral membrane staining within the upper regions. During transmissible murine colonic hyperplasia, these gradients were dissipated, and basilar plaques were formed within a subset of basal crypt cells. These findings predict that an oncogenic signaling mechanism related to non-E-cadherin-bound beta-catenin is active in hyperproliferating native colonocytes and is similar to that recorded during the early stages of colon carcinogenesis.

Plate failure by bending following tibial fracture stabilisation in 10 cats.

OBJECTIVE: To describe the clinical findings and management of tibial fractures in cats in which osteosynthesis failed due to plate bending. METHODS: Case records and radiographs of cat tibial fracture repairs from five referral centres were reviewed for signalment and to assess incidence of plate failure by bending. Cats that sustained plate bending following plate or plate-rod fixation were reviewed for fracture configuration, repair method, initial postoperative and postfailure tibial alignment, revision treatment and outcome. RESULTS: The incidence of plate bending in cat fractures managed with plate and plate-rod fixation in the four referral centres where the overall number could be established was 13% (8/60). In the 10 cats in which plates bent, initial fractures were generally oblique or spiral with mild comminution and located in the middle or distal third of the tibia. Mean time to implant failure was 24 days (range 2 to 56 days). Mean tibial valgus angle increased from 12·9° to 30·9° following bending of the plate. Short-term outcome following revision surgery using orthogonal plating or stacked medial plates was favourable with improvement in tibial valgus in all five fractures with follow-up data. CLINICAL SIGNIFICANCE: Plate bending following tibial fracture stabilisation in these 10 cats resulted in tibial valgus deformation. Consideration of plate and/or intramedullary rod selection and application should be given to avoid a plate strain environment that exceeds the yield point of the plate.

Cytoplasmic calcium measurement in rotavirus enterotoxin-enhanced green fluorescent protein (NSP4-EGFP) expressing cells loaded with Fura-2.

The green fluorescent protein (GFP) and its analogs are standard markers of protein expression and intracellular localization of proteins. The fluorescent properties of GFP complicate accurate measurement of intracellular calcium using calcium sensitive fluorophores, which show a great degree of spectral overlap with GFP, or their K(d) values are too high for accurate measurement of subtle changes in cytoplasmic calcium concentrations. Here we describe a simple modification of the standard microscope-based Fura-2 calcium-imaging technique which permits the quantitative measurement of intracellular calcium levels in cells expressing enhanced green fluorescent protein (EGFP) fusion proteins. Longpass emission filtering of the Fura-2 signal in cells expressing an EGFP fusion protein is sufficient to eliminate the EGFP-Fura-2 emission spectra overlap and allows quantitative calibration of intracellular calcium. To validate this technique, we investigated the ability of rotavirus enterotoxin NSP4-EGFP to elevate intracellular calcium levels in mammalian HEK 293 cells. We show here that inducible intracellular expression of NSP4-EGFP fusion protein elevates basal intracellular calcium more than two-fold by a phospholipase C (PLC) independent mechanism.

Dietary pectin and calcium inhibit colonic proliferation in vivo by differing mechanisms.

Diet plays an important role in promoting and/or preventing colon cancer; however, the effects of specific nutrients remain uncertain because of the difficulties in correlating epidemiological and basic observations. Transmissible murine colonic hyperplasia (TMCH) induced by Citrobacter rodentium, causes significant hyperproliferation and hyperplasia in the mouse distal colon and increases the risk of subsequent neoplasia. We have recently shown that TMCH is associated with an increased abundance of cellular beta-catenin and its nuclear translocation coupled with up-regulation of its downstream targets, c-myc and cyclin D1. In this study, we examined the effects of two putatively protective nutrients, calcium and soluble fibre pectin, on molecular events linked to proliferation in the colonic epithelium during TMCH. Dietary intervention incorporating changes in calcium [high (1.0%) and low (0.1%)] and alterations in fibre content (6% pectin and fibre-free) were compared with the standard AIN-93 diet (0.5% calcium, 5% cellulose), followed by histomorphometry and immunochemical assessment of potential oncogenes. Dietary interventions did not alter the time course of Citrobacter infection. Both 1.0% calcium and 6% pectin diet inhibited increases in proliferation and crypt length typically seen in TMCH. Neither the low calcium nor fibre-free diets had significant effect. Pectin diet blocked increases in cellular beta-catenin, cyclin D1 and c-myc levels associated with TMCH by 70%, whereas neither high nor low calcium diet had significant effect on these molecules. Diets supplemented with either calcium or pectin therefore, exert anti-proliferative effects in mouse distal colon involving different molecular pathways. TMCH is thus a diet-sensitive model for examining the effect of specific nutrients on molecular characteristics of the pre-neoplastic colonic epithelium.

Cholinergic receptors regulate a voltage-insensitive but Na+-dependent calcium influx pathway in salivary acinar cells.

The calcium-sensitive fluorescent probe, quin 2, was employed to investigate the cholinergic receptor regulation of cytosolic free calcium concentration [( Ca2+]i) in isolated rat submandibular acinar cells. Cholinergic receptor stimulation results in a marked and sustained elevation [Ca2+]i. The major component of this response is shown to be due to activation of a calcium influx pathway. There is no evidence in K+-depolarized cells of any voltage-gated calcium influx. Acetylcholine failed to activate the calcium influx pathway in cells bathed in Na+-free solutions. The cholinergic receptors thus regulate a voltage-insensitive but Na+-dependent transport system for calcium entry in submandibular acinar cells.

A large conductance, voltage- and calcium-activated K+ channel in the basolateral membrane of rat enterocytes.

The patch-clamp technique was employed to record single channel currents in patches of basolateral membrane from enterocytes isolated from rat small intestine. We demonstrate the presence of a large conductance (250 pS), voltage- and calcium-activated, the maxi, K+ channel in this membrane. Currents in this K+ channel were blocked by the application of barium (5 mM) to the extracellular membrane face.

DNA variation in a 13-Mb region including the F9 gene: inferring the genealogical history and causal role of a hemophilia B mutation (IVS 5+13 A-->G).

About 5.5% of all UK hemophilia B patients have the base substitution IVS 5+13 A-->G as the only change in their factor (F)IX gene (F9). This generates a novel donor splice site which fits the consensus better than the normal intron 5 donor splice. Use of the novel splice site should result in a missense mutation followed by the abnormal addition of four amino acids to the patients' FIX. In order to explain the prevalence of this mutation, its genealogical history is examined. Analysis of restriction fragment length polymorphism in the 21 reference UK individuals (from different families) with the above mutation showed identical haplotypes in 19 while two differed from the rest and from each other. In order to investigate the history of the mutation and to verify that it had occurred independently more than once, the sequence variation in 1.5-kb segments scattered over a 13-Mb region including F9 was examined in 18 patients and 15 controls. This variation was then analyzed with a recently developed Bayesian approach that reconstructs the genealogy of the gene investigated while providing evidence of independent mutations that contribute disconnected branches to the genealogical tree. The method also provides minimum estimates of the age of the mutation inherited by the members of coherent trees. This revealed that 17 or 18 mutant genes descend from a founder who probably lived 450 years ago, while one patient carries an independent mutation. The independent recurrence of the IVS5+13 A-->G mutation strongly supports the conclusion that it is the cause of these patients' mild hemophilia.

Cholinergic receptor-regulation of potassium channels and potassium transport in human submandibular acinar cells.

The cholinergic receptor-regulation of K+ transport was studied in human submandibular glands. Acetylcholine stimulation 10 mumol/L results in an increase in membrane permeability (86Rb+ efflux) for, and a net efflux of, K+ ions from the glandular tissue. In the post-stimulus period, there is a net re-uptake of K+ ions into the tissue. Patch-clamp electrophysiological techniques were employed to demonstrate the presence of a large conductance K+ selective ion channel in the basolateral membranes of isolated human submandibular acinar cells. The patch-clamp results indicate that this voltage- and calcium-activated K+ channel operates to regulate the K+ permeability in both the resting and acetylcholine-stimulated acinar cells. We discuss the role of the K+ channel, K+ efflux, and K+ re-uptake in relation to stimulus-secretion coupling.

Chest and abdominal injuries caused by seat belt loading.

Injuries will inevitably occur when restraining loads are exerted on a car occupant by a seat belt during a crash. This study investigated the nature, frequency, and severity of such injuries to the chest and abdomen. Vehicle, occupant, and injury details were obtained from accidents occurring in the Midlands of England. The sample was chosen with emphasis on fatal and serious injuries while also representing slight injuries as rated by the British government scheme. All causes of injuries to 3,276 front-seat restrained occupants were considered. 29.6% had a minor (Abbreviated Injury Scale [AIS] 1) injury caused by seat belt loading, more than from any other cause. The study went on to focus on 1,025 occupants sustaining injuries caused solely by seat belt loading. Of those, 19.4% sustained chest/abdominal injuries rated at Maximum AIS > or = 2 with sternum fractures predominating, and 4.5% were rated at Maximum AIS > or = 3. Occupants were not excluded if they had injuries at other body regions enabling the frequency and severity of head injuries to be considered also. The role played by impact type, speed change at impact, seat belt usage problems, and some aspects of occupant characteristics were investigated. While females were at much greater risk of serious injury (AIS > or = 3) when aged > or = 70 years, the effects of aging were more obvious in the cases with chest injuries rated at AIS 2. Serious chest injuries were predominantly a function of higher speed changes at impact. Occupant height and weight were shown to influence injury outcome, and the study concluded that work is required to further define occupants most at risk. Comparisons were made with two studies into other types of injury at other body regions, and injuries rated AIS > or = 2 caused by seat belt loading were seen to be relatively unlikely. It must also be stressed that casualties sustained seat belt injuries would most likely have received more severe injuries had a seat belt not been worn.

Unexpected benefit of sorbitol placebo in Mg intervention study of premenstrual symptoms: implications for choice of placebo in RCTs.

We carried out a randomized, double-blind, crossover study of 85 women, designed to investigate the dose-response of daily Mg supplementation on premenstrual symptoms. Each woman took one of four treatments: Mg (200, 350 or 500 mg/day) or sorbitol (placebo) for 2 months. This was followed by a washout of 1 month, and then each woman received one of the three remaining treatments for a further 2 months. Unexpectedly, sorbitol (1305 mg) reduced anxiety-related and total premenstrual symptoms after 2 months compared with Mg treatments (P<0.001 and P<0.001, respectively). We conclude that low-dose sorbitol reduces premenstrual symptoms beyond that expected of a placebo. After 2 months of treatment, sorbitol also reduced urinary Mg excretion compared to baseline (no intervention) and Mg treatments (P=0.005). A follow-up study on 17 healthy volunteers confirmed lack of effect on urinary Mg output of a similar sorbitol intervention regime compared with either baseline or cellulose placebo. It appears that sorbitol may influence Mg homeostasis in women suffering premenstrual symptoms, but not in healthy individuals. Implications for placebo choice in RCTs are discussed.

A viral enterotoxin. A new mechanism of virus-induced pathogenesis.

Acute infectious gastroenteritis is a major cause of infant morbidity in developed countries and of infant mortality in developing areas of the world. Rotavirus is recognized as the most important etiologic agent of infantile gastroenteritis, and studies of rotavirus serve as models to understand the complex interactions between enteric viruses and the multifunctional cells of the gastrointestinal tract. Understanding such interactions is significant for microbial pathogenesis because most (> 80%) infections are initiated at mucosal surfaces. Rotaviruses are pathogens that infect the mature enterocytes of the villi in the intestine and infection appears to be limited to these highly differentiated cells in immunologically competent hosts. In such hosts, infections are generally acute yet diarrheal disease can be severe and life-threatening. Disease generally is resolved within 2-5 days after infection if affected hosts receive adequate rehydration. In immunocompromised hosts, virus infections persist, virus can be detected extraintestinally and virus excretion may be detected for extended periods of time (many months). Rotaviruses infect almost all mammalian and some avian species and much of our understanding of rotavirus pathogenesis has come from studies in animal models, particularly in small animal models (mice and rabbits), but also in larger animals (cows and piglets). Studies in children are limited due to the difficulty and lack of clinical need of obtaining biopsies from infants and the inability to determine the precise time of natural infections. In all animal species where naïve animals can be infected, disease is age-dependent; for example, in mice and rabbits, diarrheal disease is the outcome of infections that occur only during the first two weeks of life (Ciarlet et al., 1998; Starkey et al., 1986; Ramig 1988; Ward et al., 1990; Burns et al., 1995), while animals remain susceptible to viral infection into adulthood. Rotavirus infections have been reported to occur repeatedly in humans from birth to old age, but the majority of infections after the first 2 years of life are asymptomatic or associated with mild gastrointestinal symptoms. The age-related resistance to rotavirus-induced diarrhea in humans is thought to be mediated primarily by acquired immunity, but it is not possible to directly test if humans also exhibit an age-dependent resistance to disease based on other factors such as intestinal development and maturation. Currently, our best understanding of the mechanisms of rotavirus pathogenesis rely on results obtained in animal models.

The regulation of epithelial cell cAMP- and calcium-dependent chloride channels.

This chapter has focused on two types of chloride conductance found in epithelial cells. The leap from the Ussing chamber to patch-clamp studies has identified yet other conductances present which have also been electrophysiologically characterized. In the case of the swelling activated wholecell chloride current, a physiological function is apparent and a single-channel basis found, but its genetic identity remains unknown (see reviews by Frizzell and Morris, 1994; and Strange et al., 1996). The outwardly rectified chloride channel has been the subject of considerable electrophysiological interest over the past 10 years and is well characterized at the single-channel level, but its physiological function remains controversial (reviewed by Frizzell and Morris, 1994; Devidas and Guggino, 1997). Yet other conductances related to the CLC gene family also appear to be present in epithelial cells of the kidney (reviewed by Jentsch, 1996; Jentsch and Gunter, 1997) where physiological functions for some isoforms are emerging. Clearly, there remain many unknowns. Chief among these is the molecular basis of GCa2+Cl and many of other the conductances. As sequences become available it is expected that the wealth of information gained by investigation into CFTR function will provide a conceptual blueprint for similar studies in these later channel clones.