Population genetic screening efficiently identifies carriers of autosomal dominant diseases.

Three inherited autosomal dominant conditions-BRCA-related hereditary breast and ovarian cancer (HBOC), Lynch syndrome (LS) and familial hypercholesterolemia (FH)-have been termed the Centers for Disease Control and Prevention Tier 1 (CDCT1) genetic conditions, for which early identification and intervention have a meaningful potential for clinical actionability and a positive impact on public health1. In typical medical practice, genetic testing for these conditions is based on personal or family history, ethnic background or other demographic characteristics2. In this study of a cohort of 26,906 participants in the Healthy Nevada Project (HNP), we first evaluated whether population screening could efficiently identify carriers of these genetic conditions and, second, we evaluated the impact of genetic risk on health outcomes for these participants. We found a 1.33% combined carrier rate for pathogenic and likely pathogenic (P/LP) genetic variants for HBOC, LS and FH. Of these carriers, 21.9% of participants had clinically relevant disease, among whom 70% had been diagnosed with relevant disease before age 65. Moreover, 90% of the risk carriers had not been previously identified, and less than 19.8% of these had documentation in their medical records of inherited genetic disease risk, including family history. In a direct follow-up survey with all carriers, only 25.2% of individuals reported a family history of relevant disease. Our experience with the HNP suggests that genetic screening in patients could identify at-risk carriers, who would not be otherwise identified in routine care.

Fine-mapping the MHC locus in juvenile idiopathic arthritis (JIA) reveals genetic heterogeneity corresponding to distinct adult inflammatory arthritic diseases.

OBJECTIVES: Juvenile idiopathic arthritis (JIA) is a heterogeneous group of diseases, comprising seven categories. Genetic data could potentially be used to help redefine JIA categories and improve the current classification system. The human leucocyte antigen (HLA) region is strongly associated with JIA. Fine-mapping of the region was performed to look for similarities and differences in HLA associations between the JIA categories and define correspondences with adult inflammatory arthritides. METHODS: Dense genotype data from the HLA region, from the Immunochip array for 5043 JIA cases and 14 390 controls, were used to impute single-nucleotide polymorphisms, HLA classical alleles and amino acids. Bivariate analysis was performed to investigate genetic correlation between the JIA categories. Conditional analysis was used to identify additional effects within the region. Comparison of the findings with those in adult inflammatory arthritic diseases was performed. RESULTS: We identified category-specific associations and have demonstrated for the first time that rheumatoid factor (RF)-negative polyarticular JIA and oligoarticular JIA are genetically similar in their HLA associations. We also observe that each JIA category potentially has an adult counterpart. The RF-positive polyarthritis association at HLA-DRB1 amino acid at position 13 mirrors the association in adult seropositive rheumatoid arthritis (RA). Interestingly, the combined oligoarthritis and RF-negative polyarthritis dataset shares the same association with adult seronegative RA. CONCLUSIONS: The findings suggest the value of using genetic data in helping to classify the categories of this heterogeneous disease. Mapping JIA categories to adult counterparts could enable shared knowledge of disease pathogenesis and aetiology and facilitate transition from paediatric to adult services.

Association of Toll-like receptor 4 (TLR4) with chronic plaque type psoriasis and psoriatic arthritis.

Family studies have provided overwhelming evidence for an underlying genetic component to psoriasis. Toll-like receptors (TLRs) are key transmembrane proteins in both the innate and adaptive immune responses which are known to be integral processes in psoriasis. Recent functional studies support this notion having suggested a role for TLR4 in the pathogenesis of psoriasis. Furthermore a missense polymorphism in the TLR4 gene has been associated with a number of autoimmune conditions, including Crohn diseases, making TLR4 a viable candidate gene for investigation. The aim of this study was to investigate polymorphisms across the TLR4 region with a high-density single nucleotide polymorphism (SNP) panel in a large cohort of patients with chronic plaque type psoriasis. Twenty SNPs were successfully genotyped using Sequenom iPLEX Gold platform in 2826 UK chronic plaque type psoriasis patients including subgroup data on presence of confirmed psoriatic arthritis (n = 1839) and early-onset psoriasis (n = 1466) was available. Allele frequencies for psoriasis patients were compared against imputed Wellcome Trust Case Control Consortium controls (n = 4861). Significant association was observed between a missense variant rs4986790 of TLR4 (Asp229Gly) and plaque type psoriasis (p = 2 × 10(-4)) which was also notable in those with psoriatic arthritis (p = 2 × 10(-4)) and early-onset psoriasis (p = 8 × 10(-4)). We present data suggestive of an association between a functional variant and an intronic variant of TLR4 and chronic plaque type psoriasis and psoriatic arthritis. However, validation of this association in independent cohorts will be necessary.

Identification of loci associated with late-onset psoriasis using dense genotyping of immune-related regions.

BACKGROUND: Chronic plaque psoriasis can be subdivided into two groups according to the age of onset: type 1 (early onset, before 40 years) and type 2 (late onset, at or beyond 40 years). So far, 36 genetic loci have been associated with early-onset psoriasis in genome-wide association studies of white populations, while few studies have investigated genetic susceptibility to late-onset psoriasis. OBJECTIVES: To characterize the genetics underpinning late-onset psoriasis. METHODS: We genotyped 543 cases of late-onset psoriasis and 4373 healthy controls using the Immunochip array, a dense genotyping chip containing single-nucleotide polymorphisms previously associated with autoimmune diseases. Imputation using SNP2HLA and stepwise logistic regression analysis was performed for markers spanning the human leucocyte antigen gene region. RESULTS: Two loci (HLA-C and IL12B) previously associated with early-onset psoriasis showed significant association at a genome-wide threshold in the current study (P < 5 × 10(-8)). Six more loci (TRAF3IP2, IL23R, RNF114, IFIH1, IL23A and HLA-A) showed study-wide significant association (P < 2·3 × 10(-5); calculated using Genetic type 1 error calculator). Additionally, we identified an association at IL1R1 on chromosome 2q13, which is not associated with early-onset disease. CONCLUSIONS: This is the largest study to date of genetic loci in late-onset psoriasis, and demonstrates the overlap that exists with early-onset psoriasis. It also suggests that some loci are associated exclusively with late-onset psoriasis.

Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays.

Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies - radioligand-binding or automated electrophysiology - was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost.

Investigation of connexin 43 uncoupling and prolongation of the cardiac QRS complex in preclinical and marketed drugs.

BACKGROUND AND PURPOSE: Prolongation of the cardiac QRS complex is linked to increased mortality and may result from drug-induced inhibition of cardiac sodium channels (hNaV 1.5). There has been no systematic evaluation of preclinical and marketed drugs for their additional potential to cause QRS prolongation via gap junction uncoupling. EXPERIMENTAL APPROACH: Using the human cardiac gap junction connexin 43 (hCx43), a dye transfer 'parachute' assay to determine IC50 values for compound ranking was validated with compounds known to uncouple gap junctions. Uncoupling activity (and hNaV 1.5 inhibition by automated patch clamp) was determined in a set of marketed drugs and preclinical candidate drugs, each with information regarding propensity to prolong QRS. KEY RESULTS: The potency of known gap junction uncouplers to uncouple hCx43 was ranked (according to IC50 ) as phorbol ester>digoxin>meclofenamic acid>carbenoxolone>heptanol. Among the drugs associated with QRS prolongation, 29% were found to uncouple hCx43 (IC50 < 50 µM), whereas no uncoupling activity was observed in drugs not associated with QRS prolongation. In preclinical candidate drugs, hCx43 and hNaV 1.5 IC50 values were similar (within threefold). No consistent margin over preclinical Cmax (free) was apparent for QRS prolongation associated with Cx43 inhibition. However, instances were found of QRS prolonging compounds that uncoupled hCx43 with significantly less activity at hNaV 1.5. CONCLUSION AND IMPLICATIONS: These results demonstrate that off-target uncoupling activity is apparent in drug and drug-like molecules. Although the full ramifications of Cx inhibition remain to be established, screening for hCx43 off-target activity could reduce the likelihood of developing candidate drugs with a risk of causing QRS prolongation.

Enrichment of vitamin D response elements in RA-associated loci supports a role for vitamin D in the pathogenesis of RA.

The aim of this study was to explore the role of vitamin D in rheumatoid arthritis (RA) pathogenesis by investigating the enrichment of vitamin D response elements (VDREs) in confirmed RA susceptibility loci and testing variants associated with vitamin D levels for association with RA. Bioinformatically, VDRE genomic positions were overlaid with non-HLA (human leukocyte antigen)-confirmed RA susceptibility regions. The number of VDREs at RA loci was compared to a randomly selected set of genomic loci to calculate an average relative risk (RR). Single-nucleotide polymorphisms (SNPs) in the DHCR7/NADSYN1 (nicotinamide adenine dinucleotide synthase 1) and CYP2R1 loci, previously associated with circulating vitamin D levels, were tested in UK RA cases (n=3870) and controls (n=8430). Significant enrichment of VDREs was seen at RA loci (P=9.23 × 10(-8)) when regions were defined either by gene (RR 5.50) or position (RR 5.86). SNPs in the DHCR7/NADSYN1 locus showed evidence of positive association with RA, rs4944076 (P=0.008, odds ratio (OR) 1.14, 95% confidence interval (CI) 1.03-1.24). The significant enrichment of VDREs at RA-associated loci and the modest association of variants in loci-controlling levels of circulating vitamin D supports the hypothesis that vitamin D has a role in the development of RA.

An investigation of rheumatoid arthritis loci in patients with early-onset psoriasis validates association of the REL gene.

BACKGROUND: Phenotypically diverse autoimmune conditions share common genetic susceptibility loci and underlying molecular pathways. OBJECTIVES: By systematically searching for single nucleotide polymorphisms (SNPs) associated with another autoimmune disease, rheumatoid arthritis (RA), we aimed to elucidate novel genetic markers of psoriasis. METHODS: We investigated 18 SNPs, previously confirmed as being associated with RA, in a U.K. cohort of 623 patients with early-onset psoriasis (presenting before age 40 years), comparing them with 2662 control subjects. RESULTS: Our findings confirm the association of early-onset psoriasis with REL (rs13031237, P=0·0027). The minor allele of REL had opposing effects upon susceptibility to disease in patients with psoriasis and RA. CONCLUSION: Similar exploration of additional autoimmune loci and fine mapping of such regions may provide further insight into the genetics and molecular pathophysiology of psoriasis.

MTHFR gene polymorphisms and outcome of methotrexate treatment in patients with rheumatoid arthritis: analysis of key polymorphisms and meta-analysis of C677T and A1298C polymorphisms.

Association of two key variants mapping to the MTHFR gene (C677T (rs1801133) and A1298C (rs1801131)) with response to methotrexate (MTX) remains controversial. We investigated these and other markers spanning the gene as predictors of MTX efficacy and adverse events in a UK rheumatoid arthritis (RA) patient cohort and performed a meta-analysis of the two key variants using all published data. The tagging single nucleotide polymorphisms (SNPs) were genotyped in 309 patients with well-defined outcomes to MTX treatment and 17 studies were included in the meta-analysis. No association of the SNPs tested was detected with MTX efficacy or toxicity in our UK cohort. After combining our data with previous studies by meta-analysis, the random effects pooled odds ratios (OR) for both C677T and A1298C showed no association with efficacy or toxicity for either of the SNPs (efficacy: OR=1.05 (95% confidence interval (CI) 0.83-1.32) and OR=0.81 (95% CI 0.53-1.24), respectively; toxicity: OR=1.38 (95% CI 0.90-2.12) and OR=1.19 (95% CI 0.80-1.78), respectively). The available evidence suggests that the MTHFR C677T and A1298C gene polymorphisms are not reliable predictors of response to MTX treatment in RA patients.

Information and professional support: key factors in the provision of family-centred early childhood intervention services.

BACKGROUND: Much has been written on the principles of family-centred practice and on the service delivery methods and skills required of its practitioners. Far less has been written from the perspective of families whose children have a disability. The aims of this study were twofold: firstly to understand families' experiences of family-centred early childhood intervention services and secondly to explore other factors that might impact on these experiences. METHODS: One hundred and thirty families attending two established early childhood intervention services in New South Wales, Australia completed a survey incorporating the Measure of Processes of Care-56, the Family Empowerment Scale, the Family Support Scale and the Parenting Daily Hassles Scale. RESULTS: Consistent with previous research using the Measure of Processes of Care-56, 'respectful and supportive care' was the domain of care families rated to occur most and 'provision of general information' was the domain they rated to occur least. Significant positive relationships existed between families' ratings of family-centred care and feelings of empowerment. Being provided with general information was strongly correlated with family empowerment. Families' social support networks played an important role but support from professionals was most strongly correlated with families' experiences of family-centred care. Finally, families whose children's early intervention services were co-ordinated by a professional experienced significantly better care. CONCLUSIONS: The provision of general information and professional support are key components of family-centred early childhood intervention services.

Bowel cancer screening is safe, detects earlier stage cancer and adenomas in 50% of cases: experience of the prevalent round of screening from two first wave centres in the North East of England.

OBJECTIVE: The NHS Bowel Cancer Screening Programme (BCSP) began roll-out in 2006 aiming to reduce cancer mortality through detection at an earlier stage. We report results from the prevalent round of screening at two first wave centres and compare with the UK pilot study. DESIGN: This is a service evaluation study. Data were collected prospectively for all individuals undergoing faecal occult blood testing (FOBt) and colonoscopy including: uptake and outcomes of FOBt, colonoscopic performance, findings, histological data and complications. Continuous data were compared using a two-tailed test of two proportions. SETTING: The South of Tyne and Tees Bowel Cancer Screening centres. PATIENTS: Participants of the BCSP. MAIN OUTCOME MEASURES: 1) Colonoscopy Quality Assurance and 2) Cancer stage shift. RESULTS: 195,772 individuals were invited to participate. Uptake was 54% and FOBt positivity 1.7%. 1524 underwent colonoscopy with caecal intubation in 1485 (97%). 180 (12%) cancers were detected. Dukes stages were: 76 (42%) A; 47 (26%) B; 47 (26%) C; 8 (4%) D and 2 (1%) unknown. This demonstrates a significantly earlier stage at diagnosis compared with data from 2867 non-screening detected cancers (p<0.001). Adenomas were detected in 758 (50%). One perforation occurred (0.07%) and two intermediate bleeds requiring transfusion only (0.12%). Both caecal intubation and adenoma detection were significantly higher than in the UK pilot study (p<0.001). CONCLUSIONS: The prevalent round of screening demonstrates a high adenoma and cancer detection rate and significantly earlier stage at diagnosis. Complications were few providing reassurance regarding safety. Efforts are required to improve uptake.

Genetic susceptibility to psoriasis and psoriatic arthritis: implications for therapy.

The era of genome-wide association studies has revolutionized the search for genetic susceptibility loci in complex genetic conditions such as psoriasis. There are currently 16 loci with confirmed evidence for association with psoriasis susceptibility but there is the potential for more to be discovered as the genetic heritability of the disease has not yet been fully explained. Many of the associated loci overlap with those for psoriatic arthritis. In contrast to psoriasis susceptibility, few studies have been performed to identify predictors of drug response in psoriasis. As large-scale collaborations and registries for psoriasis and psoriatic arthritis are established, it is likely that a genome-wide approach may be used as a more effective method of searching for genetic predictors of treatment response. However, candidate gene studies will still have a role; for example, it is likely that some disease susceptibility genes will also be markers of treatment response, based on evidence from other diseases. This review summarizes recent advances in investigating the role genetics plays in psoriasis susceptibility and contrasts these to advances made in psoriatic arthritis. Further, it describes the genetics of treatment response in the two diseases and indicates how susceptibility loci could be used to identify drug response in the future.

A systematic investigation of confirmed autoimmune loci in early-onset psoriasis reveals an association with IL2/IL21.

BACKGROUND: Many autoimmune diseases share common susceptibility loci suggesting similar underlying cellular mechanisms involved in disease expression. OBJECTIVES: The purpose of this investigation was to study 21 genetic variants in 14 genes that are confirmed autoimmune loci in a cohort of patients with early-onset psoriasis. METHODS: Patients with early-onset psoriasis (n = 750) and controls (n = 3531) were genotyped using the Sequenom(®) MassArray™ iPLEX Gold platform. RESULTS: We found strong evidence of association with two variants in the IL2/IL21 (rs6822844, genotypic P = 3·3 × 10(-4) ; rs2069778, genotypic P = 7·86 × 10(-4)) region. CONCLUSIONS: The findings, although requiring replication, suggest that IL2/IL21 may play a key role in the pathogenesis of psoriasis as well as in other diverse autoimmune diseases.

Examining the overlap between genome-wide rare variant association signals and linkage peaks in rheumatoid arthritis.

OBJECTIVE: With the exception of the major histocompatibility complex (MHC) and STAT4, no other rheumatoid arthritis (RA) linkage peak has been successfully fine-mapped to date. This apparent failure to identify association under peaks of linkage could be ascribed to the examination of common variation, when linkage is likely to be driven by rare variants. The purpose of this study was to investigate the overlap between genome-wide rare variant RA association signals observed in the Wellcome Trust Case Control Consortium (WTCCC) study and 11 replicating RA linkage peaks, defined as regions with evidence for linkage in >1 study. METHODS: The WTCCC data set contained 40,482 variants with minor allele frequency of ≤0.05 in 1,860 RA patients and 2,938 controls. Genotypes of all rare variants within a given gene region were collapsed into a single locus and a global P value was calculated per gene. RESULTS: The distribution of rare variant signals (association P≤10(-5)) was found to differ significantly between regions with and without linkage evidence (P=2×10(-17) by Fisher's exact test). No significant difference was observed after data from the MHC region were removed or when the effect of the HLA-DRB1 locus was accounted for. CONCLUSION: The results suggest that rare variant association signals are significantly overrepresented under linkage peaks in RA, but the effect is driven by the MHC. This is the first study to examine the overlap between linkage peaks and rare variant association signals genome-wide in a complex disease.

Recent advances in the genetics of RA susceptibility.

RA is a common autoimmune disease with a complex aetiology in which genetic and environmental factors contribute to disease. The genetic component of RA is largely undefined and, up until very recently, there were only two reproducible associations. The strongest of these associations is of genes within the HLA region, particularly the HLA-DRB1 gene. A second, more modest, association identified has been of the protein tyrosine phosphatase non-receptor 22 (PTPN22) gene. Advances in genotyping technology have facilitated the application of whole genome association approaches to identify disease causal variants. This, coupled with the availability of large case and control collections has enabled the identification of low-to-moderate risk loci. These newer study designs combined with traditional linkage and association studies have accelerated the identification of novel risk loci. The past few months alone have witnessed the identification of three new RA risk loci. In this review, we aim to give an update on recent progress in RA genetics, focusing mainly on the identification of novel loci.

Investigation of polymorphisms in the PADI4 gene in determining severity of inflammatory polyarthritis.

BACKGROUND: A functional haplotype of the peptidylarginine deiminase 4 (PADI4) gene has recently been identified as a rheumatoid arthritis susceptibility gene in a Japanese but not in a UK population. One possible explanation for this disparity is that the gene determines severity of rather than susceptibility to inflammatory polyarthritis (IP) and that the UK and Japanese cohorts differed in terms of outcome. AIM: To examine the association between individual PADI4 single nucleotide polymorphisms (SNPs) and haplotypes, with the development and severity of erosions by five years in patients with IP. METHODS: 438 patients from the NOAR inception cohort of patients with IP were x rayed five years after presentation with early IP. Association with four exonic SNPs (padi4_89*G/A, padi4_90*T/C, padi4_92*G/C, and padi4_104*T/C), mapping to the PADI4 gene and defining a haplotype previously reported to be associated with rheumatoid arthritis, was investigated. Patients were compared for the presence, extent, and progression of erosions by five years and the presence of antibodies to citrullinated peptide (anti-CCP antibodies). RESULTS: There was no association between individual PADI4 SNPs or haplotypes and the development or extent of erosions by five years. Restricting analysis to patients who satisfied ACR criteria for rheumatoid arthritis by five years did not alter the conclusions. No association with presence of anti-CCP antibodies was detected. CONCLUSIONS: No evidence was found for association of the PADI4 gene with severity as assessed by erosive outcome at five years or with presence of anti-CCP antibodies in patients with IP.

Differential effects of repeated restraint stress on pulsatile lutenizing hormone secretion in female Fischer, Lewis and Wistar rats.

Stress activates the hypothalamic-pituitary-adrenocortical (HPA) axis and can suppress pulsatile luteinizing hormone (LH) secretion, resulting in reproductive dysfunction. The histocompatible inbred Fischer and Lewis rat strains exhibit marked phenotypic differences in the activity of the HPA axis, the former being more reactive. Using Fischer, Lewis and Wistar rats, we assessed effects of repeated restraint stress on pulsatile LH secretion. Adult rats were ovariectomized and fitted with cardiac catheters. Blood samples were collected at 5-min intervals for 3-5 h for detection of LH. Less frequent samples were collected for corticosterone measurement. After 2 h, rats were restrained for 60 min. The same regimen was repeated four times at 6-day intervals. The mean peak corticosterone levels achieved during the first restraint in Fischer rats were significantly higher than those in Lewis and Wistar rats. By the time of the fourth episode of restraint, there had been some adaptation of the corticosterone response in the Fischer, but not in the Lewis or Wistar rats. LH pulses were interrupted during the 1st restraint in all experimental groups, although only Fischer rats showed suppression of LH pulses during the subsequent 2-h postrestraint period. During the fourth restraint, LH pulse frequency was still reduced in Wistar, but not in Fischer and Lewis rats, both of which showed a complete habituation. These results suggest that differential control mechanisms underlie the response of the HPA and HPG axes to repeated restraint stress.

Using variance tracking to improve outcomes and reduce costs.

Clinical pathways outline patient-care delivery over time for specific patient populations, but their true utility is derived from information obtained through variance tracking-documenting when and why a patient's care varies from the clinical pathway. This article describes one health care facility's variance tracking for pulmonary, medical oncology, and nephrology patients and their associated measured outcomes. Avoidable hospital days were tracked and a performance improvement initiative, focused on the pathways most often used on the medical unit, was undertaken, resulting in decreased length of patient stay and cost savings of more than $160,000.