Using ancestry-informative markers to identify fine structure across 15 populations of European origin

The Wellcome Trust Case Control Consortium 3 anorexia nervosa genome-wide association scan includes 2907 cases from 15 different populations of European origin genotyped on the Illumina 670K chip. We compared methods for identifying population stratification, and suggest list of markers that may help to counter this problem. It is usual to identify population structure in such studies using only common variants with minor allele frequency (MAF) >5%; we find that this may result in highly informative SNPs being discarded, and suggest that instead all SNPs with MAF >1% may be used. We established informative axes of variation identified via principal component analysis and highlight important features of the genetic structure of diverse European-descent populations, some studied for the first time at this scale. Finally, we investigated the substructure within each of these 15 populations and identified SNPs that help capture hidden stratification. This work can provide information regarding the designing and interpretation of association results in the International Consortia. 

Variants in the 1q21 risk region are associated with a visual endophenotype of autism and schizophrenia

Deficits in sensitivity to visual stimuli of low spatial frequency and high temporal frequency (so-called frequency-doubled gratings) have been demonstrated both in schizophrenia and in autism spectrum disorder (ASD). Such basic perceptual functions are ideal candidates for molecular genetic study, because the underlying neural mechanisms are well characterized; but they have sometimes been overlooked in favor of cognitive and neurophysiological endophenotypes, for which neural substrates are often unknown. Here, we report a genome-wide association study of a basic visual endophenotype associated with psychological disorder. Sensitivity to frequency-doubled gratings was measured in 1060 healthy young adults, and analyzed for association with genotype using linear regression at 642758 single nucleotide polymorphism (SNP) markers. A significant association (P=7.9×10) was found with the SNP marker rs1797052, situated in the 5′-untranslated region of PDZK1; each additional copy of the minor allele was associated with an increase in sensitivity equivalent to more than half a standard deviation. A permutation procedure, which accounts for multiple testing, showed that the association was significant at the α=0.005 level. The region on chromosome 1q21.1 surrounding PDZK1 is an established susceptibility locus both for schizophrenia and for ASD, mirroring the common association of the visual endophenotype with the two disorders. PDZK1 interacts with N-methyl-d-aspartate receptors and neuroligins, which have been implicated in the etiologies of schizophrenia and ASD. These findings suggest that perceptual abnormalities observed in two different disorders may be linked by common genetic elements. © 2013 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Population pharmacokinetic and pharmacogenetic analysis of tacrolimus in paediatric liver transplant patients

Aims: To build a population pharmacokinetic model that describes the apparent clearance of tacrolimus and the potential demographic, clinical and genetically controlled factors that could lead to inter-patient pharmacokinetic variability within children following liver transplantation.

Methods: The present study retrospectively examined tacrolimus whole blood pre-dose concentrations (n = 628) of 43 children during their first year post-liver transplantation. Population pharmacokinetic analysis was performed using the non-linear mixed effects modelling program (nonmem) to determine the population mean parameter estimate of clearance and influential covariates.

Results: The final model identified time post-transplantation and CYP3A5*1 allele as influential covariates on tacrolimus apparent clearance according to the following equation:

TVCL=12.9×(Weight /13.2)^0.75×EXP(-0.00158×TPT)×EXP(0.428×CYP3A5)

where TVCL is the typical value for apparent clearance, TPT is time post-transplantation in days and the CYP3A5 is 1 where*1 allele is present and 0 otherwise. The population estimate and inter-individual variability (%CV) of tacrolimus apparent clearance were found to be 0.977 l h kg (95% CI 0.958, 0.996) and 40.0%, respectively, while the residual variability between the observed and predicted concentrations was 35.4%.

Conclusion: Tacrolimus apparent clearance was influenced by time post-transplantation and CYP3A5 genotypes. The results of this study, once confirmed by a large scale prospective study, can be used in conjunction with therapeutic drug monitoring to recommend tacrolimus dose adjustments that take into account not only body weight but also genetic and time-related changes in tacrolimus clearance. © 2013 The British Pharmacological Society.

The Prevalence of Age-Related Macular Degeneration in Alzheimer's Disease

BACKGROUND:
Age-related macular degeneration (AMD) and Alzheimer's disease (AD) share several features, including the presence of extracellular abnormal deposits associated with neuronal degeneration, drusen, and plaques, respectively. Investigation of any association of AMD and specifically AD is worthwhile but has rarely been done.
OBJECTIVES:
The aim of this study was to determine the prevalence of AMD in subjects with AD in comparison with an age-matched cognitively normal cohort.
METHODS:
Cases were defined as those diagnosed with AD using standardized criteria as part of their clinical care, while controls were cognitively intact individuals aged 65 years or more. Dilated retinal photographs were taken, and a range of potentially confounding factors measured including APOE genotype. AMD features were recorded and AMD grades given.
RESULTS:
Data was collected on 322 controls and 258 cases. While AMD was associated with AD, and the proportion of cases of advanced AMD in AD cases was twice that of controls, when corrected the association was lost. AD was associated with age, the presence of an APOE allele, and smoking, while being 'generally unwell recently' was associated with a reduced risk of AD.
CONCLUSION:
AD and AMD are both associated with age, but our study does not find evidence they are associated with each other. However the retina offers an opportunity to non-invasively image neuronal tissue, and more sophisticated imaging techniques may shed light on ocular biomarkers of AD.

Genome-wide significant associations in schizophrenia to ITIH3/4, CACNA1C and SDCCAG8, and extensive replication of associations reported by the Schizophrenia PGC

The Schizophrenia Psychiatric Genome-Wide Association Study Consortium (PGC) highlighted 81 single-nucleotide polymorphisms (SNPs) with moderate evidence for association to schizophrenia. After follow-up in independent samples, seven loci attained genome-wide significance (GWS), but multi-locus tests suggested some SNPs that did not do so represented true associations. We tested 78 of the 81 SNPs in 2640 individuals with a clinical diagnosis of schizophrenia attending a clozapine clinic (CLOZUK), 2504 cases with a research diagnosis of bipolar disorder, and 2878 controls. In CLOZUK, we obtained significant replication to the PGC-associated allele for no fewer than 37 (47%) of the SNPs, including many prior GWS major histocompatibility complex (MHC) SNPs as well as 3/6 non-MHC SNPs for which we had data that were reported as GWS by the PGC. After combining the new schizophrenia data with those of the PGC, variants at three loci (ITIH3/4, CACNA1C and SDCCAG8) that had not previously been GWS in schizophrenia attained that level of support. In bipolar disorder, we also obtained significant evidence for association for 21% of the alleles that had been associated with schizophrenia in the PGC. Our study independently confirms association to three loci previously reported to be GWS in schizophrenia, and identifies the first GWS evidence in schizophrenia for a further three loci. Given the number of independent replications and the power of our sample, we estimate 98% (confidence interval (CI) 78-100%) of the original set of 78 SNPs represent true associations. We also provide strong evidence for overlap in genetic risk between schizophrenia and bipolar disorder.

SORBS1 gene, a new candidate for diabetic nephropathy: results from a multi-stage genome-wide association study in patients with type 1 diabetes

Aims/hypothesis

Conserved histidine of metal transporter AtNRAMP1 is crucial for optimal plant growth under manganese deficiency at chilling temperatures

Manganese (Mn) is an essential nutrient required for plant growth, in particular in the process of photosynthesis. Plant performance is influenced by various environmental stresses including contrasting temperatures, light or nutrient deficiencies. The molecular responses of plants exposed to such stress factors in combination are largely unknown. 

Screening of 108 Arabidopsis thaliana (Arabidopsis) accessions for reduced photosynthetic performance at chilling temperatures was performed and one accession (Hog) was isolated. Using genetic and molecular approaches, the molecular basis of this particular response to temperature (GxE interaction) was identified. 

Hog showed an induction of a severe leaf chlorosis and impaired growth after transfer to lower temperatures. We demonstrated that this response was dependent on the nutrient content of the soil. Genetic mapping and complementation identified NRAMP1 as the causal gene. Chlorotic phenotype was associated with a histidine to tyrosine (H239Y) substitution in the allele of Hog NRAMP1. This led to lethality when Hog seedlings were directly grown at 4 degrees C. 

Chemical complementation and hydroponic culture experiments showed that Mn deficiency was the major cause of this GxE interaction. For the first time, the NRAMP-specific highly conserved histidine was shown to be crucial for plant performance.

Replication of the association of GLT6D1 with aggressive periodontitis in a Sudanese population

BACKGROUND: Susceptibility to aggressive periodontitis (AgP) is influenced by genetic as well as environmental factors. Studies linking gene variants to AgP have been mainly centred in developed countries with limited data from Africa.
AIM: To investigate whether previously reported candidate gene associations with AgP could be replicated in a population from Sudan.


METHODS: The investigation was a case-control design. Cases with AgP (n = 132) and controls (n = 136) were identified from patients attending the Periodontal Department in Khartoum Dental Hospital. Genotyping was performed using the Sequenom MassARRAY iPLEX platform. Analysis focused on gene variants with a minor allele frequency (MAF) > 25% in the Sudanese subjects that had previously been reported to be associated with AgP.


RESULTS: One candidate gene rs1537415 (GLT6D1) was significantly associated with AgP, OR = 1.50 (95% CI 1.04-2.17), p = 0.0295 (increasing to p = 0.09 after correction for multiple testing). The association strengthened to OR = 1.56 (95% CI 1.15-2.16), p = 0.0042 when the controls were supplemented with data from the Hap map for the Yoruba in Ibadan (n = 147) and remained significant (p = 0.013) after correction for multiple testing.


CONCLUSION: The study independently replicated the finding that rs1537415, a variant in glycosyl transferase gene GLT6D1, is associated with AgP and provided the first report of genetic associations with AgP in a Sudanese population.

Plasma Clusterin Levels and the rs11136000 Genotype in Individuals with Mild Cognitive Impairment and Alzheimer's Disease

Aim: Substantial evidence links atherosclerosis and Alzheimer's disease (AD). Apolipoproteins, such as apolipoprotein E, have a causal relationship with both diseases. The rs11136000 SNP within the CLU gene, which encodes clusterin (apolipoprotein J), is also associated with increased AD risk. The aim of this study was to investigate the relationship between plasma clusterin and the rs11136000 genotype in mild cognitive impairment (MCI) and AD.

Methods: Plasma and DNA samples were collected from control, MCI and AD subjects (n=142, 111, 154, respectively). Plasma clusterin was determined by ELISA and DNA samples were genotyped for rs11136000 by TaqMan assay.

Results: Plasma clusterin levels were higher in MCI and AD subjects vs. controls (222.3 +/- 61.3 and 193.6 +/- 58.2 vs. 178.6 +/- 52.3 mu g/ml, respectively; p

Conclusion: This study examined control, MCI and AD subjects, identifying for the first time that plasma clusterin levels were influenced, not only by the presence of AD, but also the transitional stage of MCI, while rs11136000 genotype only influenced plasma clusterin levels in the control group. The increase in plasma clusterin in MCI and AD subjects may occur in response to the disease process and would be predicted to increase binding capacity for amyloid-beta peptides in plasma, enhancing their removal from the brain.

Assessment of clinical response to ivacaftor with lung clearance index in cystic fibrosis patients with a G551D-CFTR mutation and preserved spirometry:A randomised controlled trial

Background: Ivacaftor has shown a clinical benefit in patients with cystic fibrosis who have the G551D-CFTR mutation and reduced lung function. Lung clearance index (LCI) using multiple-breath washout might be an alternative to and more sensitive method than forced expiratory volume in 1 s (FEV₁) to assess treatment response in the growing number of children and young adults with cystic fibrosis who have normal spirometry. The aim of the study was to assess the treatment effects of ivacaftor on LCI in patients with cystic fibrosis, a G551D-CFTR mutation, and an FEV₁ >90% predicted. Methods: This phase 2, multicentre, placebo-controlled, double-blind 2×2 crossover study of ivacaftor treatment was conducted in patients with cystic fibrosis, at least one G551D-CFTR allele, and an FEV₁ >90% predicted. Patients also had to have an LCI higher than 7·4 at screening, age of 6 years or older, and a weight higher than or equal to 15 kg. Eligible patients were randomly allocated to receive one of two treatment sequences (placebo first followed by ivacaftor 150 mg twice daily [sequence 1] or ivacaftor 150 mg twice daily first followed by placebo [sequence 2]) of 28 days' treatment in each period, with a 28-day washout between the two treatment periods. Randomisation (ratio 1:1) was done with block sizes of 4, and all site personnel including the investigator, the study monitor, and the Vertex study team were masked to treatment assignment. The primary outcome measure was change from baseline in LCI. The study is registered at ClinicalTrials.gov, NCT01262352. Findings: Between February and November, 2011, 21 patients were enrolled, of which 11 were assigned to the sequence 1 group, and 10 to the sequence 2 group. 20 of these patients received treatment and 17 completed the trial (eight in sequence 1 group and 9 in sequence 2 group). Treatment with ivacaftor led to significant improvements compared with placebo in LCI (difference between groups in the average of mean changes from baseline at days 15 and 29 was -2·16 [95% CI -2·88 to -1·44]; p<0·0001). Adverse events experienced by study participants were similar between treatment groups; at least one adverse event was reported by 15 (79%) of 19 patients who received placebo and 13 (72%) of 18 patients who received ivacaftor. No deaths occurred during study period. Interpretation: In patients with cystic fibrosis aged 6 years or older who have at least one G551D-CFTR allele, ivacaftor led to improvements in LCI. LCI might be a more sensitive alternative to FEV₁ in detecting response to intervention in these patients with mild lung disease. Funding: Vertex Pharmaceuticals Incorporated. © 2013 Elsevier Ltd.

Variation in DNA repair genes XRCC3, XRCC4, XRCC5 and susceptibility to myeloma

Cytogenetic analysis in myeloma reveals marked chromosomal instability. Both widespread genomic alterations and evidence of aberrant class switch recombination, the physiological process that regulates maturation of the antibody response, implicate the DNA repair pathway in disease pathogenesis. We therefore assessed 27 SNPs in three genes (XRCC3, XRCC4 and XRCC5) central to DNA repair in patients with myeloma and controls from the EpiLymph study and from an Irish hospital registry (n = 306 cases, 263 controls). For the haplotype-tagging SNP (htSNP) rs963248 in XRCC4, Allele A was significantly more frequent in cases than in controls (86.4 versus 80.8%; odds ratio 1.51; 95% confidence interval 1.10-2.08; P = 0.0133), as was the AA genotype (74 versus 65%) (P = 0.026). Haplotype analysis was performed using Unphased for rs963248 in combination with additional SNPs in XRCC4. The strongest evidence of association came from the A-T haplotype from rs963248-rs2891980 (P = 0.008). For XRCC5, the genotype GG from rs1051685 was detected in 10 cases from different national populations but in only one control (P = 0.015). This SNP is located in the 3'-UTR of XRCC5. Overall, these data provide support for the hypothesis that common variation in the genes encoding DNA repair proteins contributes to susceptibility to myeloma.

Thermolabile methylenetetrahydrofolate reductase (C677T):frequency in the Irish population

BACKGROUND: The genetic variation which underlies the thermolability and low enzyme activity of 5,10-methylenetetrahydrofolate reductase (MTHFR; C677T) has been extensively studied in many populations, including the Irish population.

AIM: To describe the examination of the C677T substitution in two new control samples drawn from the Irish population.

METHODS: A collection of 487 serum samples was obtained through the blood transfusion services of both the Republic of Ireland and Northern Ireland and a further 115 samples from volunteers.

RESULTS: In both samples, the frequency of the thermolabile/low enzyme activity allele (T) was higher than that previously reported for the Irish population.

CONCLUSION: This finding thus supports the need for a greater use of internal control/family-based association studies, as opposed to the classic case control study design, when assessing the contribution of the MTHFR T allele to disease processes.

Prevalence of the factor VR506Q mutation in two Irish control populations:use of a novel nested polymerase chain reaction approach

The prevalence of factor V (FV) Leiden among normal populations has primarily been determined using blood donors. This control group is carefully selected and therefore may not accurately reflect the true prevalence within the population. We assessed the prevalence of FV Leiden within the Irish population using Guthrie card samples randomly selected from all newborns. We compared this result with the prevalence of FV Leiden within blood donors. A novel nested polymerase chain reaction (PCR) method for FV Leiden was developed for analysis of the Guthrie card samples. There was no significant difference between the allele frequency within the Guthrie card samples and blood donors (2.07% vs. 2.35%, P = 0.66)

Prevalence of the prothrombin G20210A mutation in the Irish populations:use of a novel polymerase chain reaction approach

The prothrombin G20210A polymorphism is associated with a threefold-increased risk of venous thrombosis. There is considerable variation in the reported prevalence of this polymorphism within normal populations, ranging from 0 to 6.5%. The prevalence within the Irish population has not been determined. A restriction fragment length polymorphism (RFLP)-based assay is commonly used for the detection of the prothrombin 20210A allele. This assay does not include a control restriction digest fragment and, consequently, failure of the enzyme activity or lack of addition of enzyme to the sample cannot be distinguished from wild-type prothrombin. We developed a RFLP-based assay, which incorporates an invariant digest site, resulting in the generation of a control digest fragment. Furthermore, we developed a nested polymerase chain reaction (PCR) method for the amplification and digestion of poor-quality or low-concentration DNA. In the Irish population studied, five of 385 (1.29%) were heterozygous and one patient was homozygous for the prothrombin 20210A polymorphism. This is the first reported data on an Irish or Celtic population and suggests that the allele frequency is similar to Anglo-Saxon populations. The nested PCR method successfully amplified and digested 100/100 (100%) of the archived samples; none of these samples could be analyzed by the standard single-round PCR method. In conclusion, nested PCR should be considered in the analysis of archived samples. Single-round PCR is appropriate for recently collected samples; however, an invariant control digest site should be incorporated in RFLP-based assays to validate the integrity of the digestion enzyme and limit the risk of false-negative results.

Sorting out mix-ups. The provenance of tissue sections may be confirmed by PCR using microsatellite markers

Standard identification systems usually ensure that biopsy material is correctly associated with a given patient. Sometimes, as when a tumor is unexpectedly found, the provenance (proof of origin) of a tissue sample may be questioned; the tissue may have been mislabelled or contaminated with tissue from another patient. Techniques used to confirm tissue provenance include comparing either tissue markers of gender or ABO blood groups; however, these methods have weak confirmatory power. Recently, the use of DNA-based polymerase chain reaction (PCR) techniques has been reported. Paired, formalin-fixed, paraffin-embedded, 10 microns tissue sections were selected from 17 patients, 8 of whom had carcinoma, either by dividing a biopsy section, using sequential biopsies, or sequential biopsy and autopsy tissue. The resulting 36 samples were coded before analysis. In two additional cases, 1-mm fragments of tumor from one patient were included in the tissue block of benign tissue from another patient, the tumor fragments were identified on hematoxylin-and-eosin-stained sections, separately scraped off the glass slide, and analyzed. Tissue from two clinical cases, one of suspected mislabelling and one with a suspected carry-over of malignant tissue were also investigated. Short tandem repeat sequences (STR) or microsatellites, are 2-5 base pair repeats that vary in their repeat number between individuals. This variation (polymorphism) can be assessed using a PCR. A panel of markers of 3 STRs; ACPP, INT 2, and CYP 19 (on chromosomes 3, 11, and 15, respectively) were used. DNA was isolated from the samples after xylene deparaffinization and proteinase digestion, and was then amplified in a radioactive PCR using primers selected to give a product size ranging from 136-178 bases. Amplified products were electrophoresed on denaturing polyacrylamide gels, dried, and autoradiographed. DNA segments were successfully extracted from all samples but one, which was fixed in Bouin's fluid. By comparing allele sizes from the panel, all tissue pairs (other than the Bouin's pair) were successfully matched, the 1-mm tumor fragments were correctly assigned, and the two clinical problems were solved. STRs are highly informative and robust markers, well suited to PCR of small portions of tissue sections, and are an effective method to confirm the provenance of benign and malignant biopsy and autopsy material.