Demethylation analysis of the FOXP3 locus shows quantitative defects of regulatory T cells in IPEX-like syndrome.

Immune dysregulation, Polyendocrinopathy, Enteropathy X-linked (IPEX) syndrome is a unique example of primary immunodeficiency characterized by autoimmune manifestations due to defective regulatory T (Treg) cells, in the presence of FOXP3 mutations. However, autoimmune symptoms phenotypically resembling IPEX often occur in the absence of detectable FOXP3 mutations. The cause of this "IPEX-like" syndrome presently remains unclear. To investigate whether a defect in Treg cells sustains the immunological dysregulation in IPEX-like patients, we measured the amount of peripheral Treg cells within the CD3(+) T cells by analysing demethylation of the Treg cell-Specific-Demethylated-Region (TSDR) in the FOXP3 locus and demethylation of the T cell-Specific-Demethylated-Region (TLSDR) in the CD3 locus, highly specific markers for stable Treg cells and overall T cells, respectively. TSDR demethylation analysis, alone or normalized for the total T cells, showed that the amount of peripheral Treg cells in a cohort of IPEX-like patients was significantly reduced, as compared to both healthy subjects and unrelated disease controls. This reduction could not be displayed by flow cytometric analysis, showing highly variable percentages of FOXP3(+) and CD25(+)FOXP3(+) T cells. These data provide evidence that a quantitative defect of Treg cells could be considered a common biological hallmark of IPEX-like syndrome. Since Treg cell suppressive function was not impaired, we propose that this reduction per se could sustain autoimmunity.

Discovery and fine mapping of serum protein loci through transethnic meta-analysis.

Many disorders are associated with altered serum protein concentrations, including malnutrition, cancer, and cardiovascular, kidney, and inflammatory diseases. Although these protein concentrations are highly heritable, relatively little is known about their underlying genetic determinants. Through transethnic meta-analysis of European-ancestry and Japanese genome-wide association studies, we identified six loci at genome-wide significance (p < 5 × 10(-8)) for serum albumin (HPN-SCN1B, GCKR-FNDC4, SERPINF2-WDR81, TNFRSF11A-ZCCHC2, FRMD5-WDR76, and RPS11-FCGRT, in up to 53,190 European-ancestry and 9,380 Japanese individuals) and three loci for total protein (TNFRS13B, 6q21.3, and ELL2, in up to 25,539 European-ancestry and 10,168 Japanese individuals). We observed little evidence of heterogeneity in allelic effects at these loci between groups of European and Japanese ancestry but obtained substantial improvements in the resolution of fine mapping of potential causal variants by leveraging transethnic differences in the distribution of linkage disequilibrium. We demonstrated a functional role for the most strongly associated serum albumin locus, HPN, for which Hpn knockout mice manifest low plasma albumin concentrations. Other loci associated with serum albumin harbor genes related to ribosome function, protein translation, and proteasomal degradation, whereas those associated with serum total protein include genes related to immune function. Our results highlight the advantages of transethnic meta-analysis for the discovery and fine mapping of complex trait loci and have provided initial insights into the underlying genetic architecture of serum protein concentrations and their association with human disease.

In vitro whole-genome analysis identifies a susceptibility locus for HIV-1.

Advances in large-scale analysis of human genomic variability provide unprecedented opportunities to study the genetic basis of susceptibility to infectious agents. We report here the use of an in vitro system for the identification of a locus on HSA8q24.3 associated with cellular susceptibility to HIV-1. This locus was mapped through quantitative linkage analysis using cell lines from multigeneration families, validated in vitro, and followed up by two independent association studies in HIV-positive individuals. Single nucleotide polymorphism rs2572886, which is associated with cellular susceptibility to HIV-1 in lymphoblastoid B cells and in primary T cells, was also associated with accelerated disease progression in one of two cohorts of HIV-1-infected patients. Biological analysis suggests a role of the rs2572886 region in the regulation of the LY6 family of glycosyl-phosphatidyl-inositol (GPI)-anchored proteins. Genetic analysis of in vitro cellular phenotypes provides an attractive approach for the discovery of susceptibility loci to infectious agents.

Using high-resolution quantitative mapping of R1 as an index of cortical myelination

A fundamental tenet of neuroscience is that cortical functional differentiation is related to the cross-areal differences in cyto-, receptor-, and myeloarchitectonics that are observed in ex-vivo preparations. An ongoing challenge is to create noninvasive magnetic resonance (MR) imaging techniques that offer sufficient resolution, tissue contrast, accuracy and precision to allow for characterization of cortical architecture over an entire living human brain. One exciting development is the advent of fast, high-resolution quantitative mapping of basic MR parameters that reflect cortical myeloarchitecture. Here, we outline some of the theoretical and technical advances underlying this technique, particularly in terms of measuring and correcting for transmit and receive radio frequency field inhomogeneities. We also discuss new directions in analytic techniques, including higher resolution reconstructions of the cortical surface. We then discuss two recent applications of this technique. The first compares individual and group myelin maps to functional retinotopic maps in the same individuals, demonstrating a close relationship between functionally and myeloarchitectonically defined areal boundaries (as well as revealing an interesting disparity in a highly studied visual area). The second combines tonotopic and myeloarchitectonic mapping to localize primary auditory areas in individual healthy adults, using a similar strategy as combined electrophysiological and post-mortem myeloarchitectonic studies in non-human primates.

Quantitative Mapping of Waterways Characteristics at Bridge Sites, TR-569, 2008

Iowa state, county, and city engineering offices expend considerable effort monitoring the state’s approximately 25,000 bridges, most of which span small waterways. In fact, the need for monitoring is actually greater for bridges over small waterways because scour processes are exacerbated by the close proximity of abutments, piers, channel banks, approach embankments, and other local obstructions. The bridges are customarily inspected biennially by the county’s road department bridge inspectors. It is extremely time consuming and difficult to obtain consistent, reliable, and timely information on bridge-waterway conditions for so many bridges. Moreover, the current approaches to gather survey information is not uniform, complete, and quantitative. The methodology and associated software (DIGIMAP) developed through the present project enable a non-intrusive means to conduct fast, efficient, and accurate inspection of the waterways in the vicinity of the bridges and culverts using one technique. The technique combines algorithms image of registration and velocimetry using images acquired with conventional devices at the inspection site. The comparison of the current bridge inspection and monitoring methods with the DIGIMAP methodology enables to conclude that the new procedure assembles quantitative information on the waterway hydrodynamic and morphologic features with considerable reduced effort, time, and cost. It also improves the safety of the bridge and culvert inspections conducted during normal and extreme hydrologic events. The data and information are recorded in a digital format, enabling immediate and convenient tracking of the waterway changes over short or long time intervals.

Heritability and quantitative genetic divergence of serotiny, a fire-persistence plant trait.

BACKGROUND AND AIMS: Although it is well known that fire acts as a selective pressure shaping plant phenotypes, there are no quantitative estimates of the heritability of any trait related to plant persistence under recurrent fires, such as serotiny. In this study, the heritability of serotiny in Pinus halepensis is calculated, and an evaluation is made as to whether fire has left a selection signature on the level of serotiny among populations by comparing the genetic divergence of serotiny with the expected divergence of neutral molecular markers (QST-FST comparison). METHODS: A common garden of P. halepensis was used, located in inland Spain and composed of 145 open-pollinated families from 29 provenances covering the entire natural range of P. halepensis in the Iberian Peninsula and Balearic Islands. Narrow-sense heritability (h(2)) and quantitative genetic differentiation among populations for serotiny (QST) were estimated by means of an 'animal model' fitted by Bayesian inference. In order to determine whether genetic differentiation for serotiny is the result of differential natural selection, QST estimates for serotiny were compared with FST estimates obtained from allozyme data. Finally, a test was made of whether levels of serotiny in the different provenances were related to different fire regimes, using summer rainfall as a proxy for fire regime in each provenance. KEY RESULTS: Serotiny showed a significant narrow-sense heritability (h(2)) of 0·20 (credible interval 0·09-0·40). Quantitative genetic differentiation among provenances for serotiny (QST = 0·44) was significantly higher than expected under a neutral process (FST = 0·12), suggesting adaptive differentiation. A significant negative relationship was found between the serotiny level of trees in the common garden and summer rainfall of their provenance sites. CONCLUSIONS: Serotiny is a heritable trait in P. halepensis, and selection acts on it, giving rise to contrasting serotiny levels among populations depending on the fire regime, and supporting the role of fire in generating genetic divergence for adaptive traits.

Detection and mapping of a lethal locus in a eucalyptus hybrid population

The objective of this work was to verify the existence of a lethal locus in a eucalyptus hybrid population, and to quantify the segregation distortion in the linkage group 3 of the Eucalyptus genome. A E. grandis x E. urophylla hybrid population, which segregates for rust resistance, was genotyped with 19 microsatellite markers belonging to linkage group 3 of the Eucalyptus genome. To quantify the segregation distortion, maximum likelihood (ML) models, specific to outbreeding populations, were used. These models consider the observed marker genotypes and the lethal locus viability as parameters. The ML solutions were obtained using the expectation‑maximization algorithm. A lethal locus in the linkage group 3 was verified and mapped, with high confidence, between the microssatellites EMBRA 189 e EMBRA 122. This lethal locus causes an intense gametic selection from the male side. Its map position is 25 cM from the locus which controls the rust resistance in this population.

Trans-Ancestral Studies Fine Map the SLE-Susceptibility Locus TNFSF4

We previously established an 80 kb haplotype upstream of TNFSF4 as a susceptibility locus in the autoimmune disease SLE. SLE-associated alleles at this locus are associated with inflammatory disorders, including atherosclerosis and ischaemic stroke. In Europeans, the TNFSF4 causal variants have remained elusive due to strong linkage disequilibrium exhibited by alleles spanning the region. Using a trans-ancestral approach to fine-map the locus, utilising 17,900 SLE and control subjects including Amerindian/Hispanics (1348 cases, 717 controls), African-Americans (AA) (1529, 2048) and better powered cohorts of Europeans and East Asians, we find strong association of risk alleles in all ethnicities; the AA association replicates in African-American Gullah (152,122). The best evidence of association comes from two adjacent markers: rs2205960-T (P = 1.71×10-34, OR = 1.43[1.26-1.60]) and rs1234317-T (P = 1.16×10-28, OR = 1.38[1.24-1.54]). Inference of fine-scale recombination rates for all populations tested finds the 80 kb risk and non-risk haplotypes in all except African-Americans. In this population the decay of recombination equates to an 11 kb risk haplotype, anchored in the 5′ region proximal to TNFSF4 and tagged by rs2205960-T after 1000 Genomes phase 1 (v3) imputation. Conditional regression analyses delineate the 5′ risk signal to rs2205960-T and the independent non-risk signal to rs1234314-C. Our case-only and SLE-control cohorts demonstrate robust association of rs2205960-T with autoantibody production. The rs2205960-T is predicted to form part of a decameric motif which binds NF-κBp65 with increased affinity compared to rs2205960-G. ChIP-seq data also indicate NF-κB interaction with the DNA sequence at this position in LCL cells. Our research suggests association of rs2205960-T with SLE across multiple groups and an independent non-risk signal at rs1234314-C. rs2205960-T is associated with autoantibody production and lymphopenia. Our data confirm a global signal at TNFSF4 and a role for the expressed product at multiple stages of lymphocyte dysregulation during SLE pathogenesis. We confirm the validity of trans-ancestral mapping in a complex trait. © 2013 Manku et al.

Mapping the platelet profile for functional genomic studies and demonstration of the effect size of the GP6 locus

BACKGROUND: Evidence suggests the wide variation in platelet response within the population is genetically controlled. Unraveling the complex relationship between sequence variation and platelet phenotype requires accurate and reproducible measurement of platelet response. OBJECTIVE: To develop a methodology suitable for measuring signaling pathway-specific platelet phenotype, to use this to measure platelet response in a large cohort, and to demonstrate the effect size of sequence variation in a relevant model gene. METHODS: Three established platelet assays were evaluated: mobilization of [Ca(2+)](i), aggregometry and flow cytometry, each in response to adenosine 5'-diphosphate (ADP) or the glycoprotein (GP) VI-specific crosslinked collagen-related peptide (CRP). Flow cytometric measurement of fibrinogen binding and P-selectin expression in response to a single, intermediate dose of each agonist gave the best combination of reproducibility and inter-individual variability and was used to measure the platelet response in 506 healthy volunteers. Pathway specificity was ensured by blocking the main subsidiary signaling pathways. RESULTS: Individuals were identified who were hypo- or hyper-responders for both pathways, or who had differential responses to the two agonists, or between outcomes. 89 individuals, retested three months later using the same methodology, showed high concordance between the two visits in all four assays (r(2) = 0.872, 0.868, 0.766 and 0.549); all subjects retaining their phenotype at recall. The effect of sequence variation at the GP6 locus accounted for approximately 35% of the variation in the CRP-XL response. CONCLUSION: Genotyping-phenotype association studies in a well-characterized, large cohort provides a powerful strategy to measure the effect of sequence variation in genes regulating the platelet response.

A new three-locus model for rootstock-induced dwarfing in apple revealed by genetic mapping of root bark percentage

Rootstock-induced dwarfing of apple scions revolutionized global apple production during the twentieth century, leading to the development of modern intensive orchards. A high root bark percentage (the percentage of the whole root area constituted by root cortex) has previously been associated with rootstock induced dwarfing in apple. In this study, the root bark percentage was measured in a full-sib family of ungrafted apple rootstocks and found to be under the control of three loci. Two QTL for root bark percentage were found to co-localise to the same genomic regions on chromosome 5 and chromosome 11 previously identified as controlling dwarfing, Dw1 and Dw2, respectively. A third QTL was identified on chromosome 13 in a region that has not been previously associated with dwarfing. The development of closely linked 3 Sequence-tagged site STS markers improved the resolution of allelic classes thereby allowing the detection of dominance and epistatic interactions between loci, with high root bark percentage only occurring in specific allelic combinations. In addition, we report a significant negative correlation between root bark percentage and stem diameter (an indicator of tree vigour), measured on a clonally propagated grafted subset of the mapping population. The demonstrated link between root bark percentage and rootstock-induced dwarfing of the scion leads us to propose a three-locus model that is able to explain levels of dwarfing from the dwarf ‘M.27’ to the semi-invigorating rootstock ‘M.116’. Moreover, we suggest that the QTL on chromosome 13 (Rb3) might be analogous to a third dwarfing QTL, Dw3 that has not previously been identified.

Auriculo-condylar syndrome: mapping of a first locus and evidence for genetic heterogeneity

Auriculo-condylar syndrome (ACS), an autosomal dominant disorder of first and second pharyngeal arches, is characterized by malformed ears (`question mark ears`), prominent cheeks, microstomia, abnormal temporomandibular joint, and mandibular condyle hypoplasia. Penetrance seems to be complete, but there is high inter-and intra-familial phenotypic variation, with no evidence of genetic heterogeneity. We herein describe a new multigeneration family with 11 affected individuals (F1), in whom we confirm intra-familial clinical variability. Facial asymmetry, a clinical feature not highlighted in other ACS reports, was highly prevalent among the patients reported here. The gene responsible for ACS is still unknown and its identification will certainly contribute to the understanding of human craniofacial development. No chromosomal rearrangements have been associated with ACS, thus mapping and positional cloning is the best approach to identify this disease gene. To map the ACS gene, we conducted linkage analysis in two large ACS families, F1 and F2 (F2; reported elsewhere). Through segregation analysis, we first excluded three known loci associated with disorders of first and second pharyngeal arches (Treacher Collins syndrome, oculo-auriculo-vertebral spectrum, and Townes-Brocks syndrome). Next, we performed a wide genome search and we observed evidence of linkage to 1p21.1-q23.3 in F2 (LOD max 3.01 at theta = 0). Interestingly, this locus was not linked to the phenotype segregating in F1. Therefore, our results led to the mapping of a first locus of ACS (ACS1) and also showed evidence for genetic heterogeneity, suggesting that there are at least two loci responsible for this phenotype.

Mapping of Diepoxybutane Damage in the Cytochrome b Domain of Mitochondrial DNA and the β-globin Domain of Nuclear Chicken DNA Using Quantitative PCR

Diepoxybutane (DEB), a known industrial carcinogen, reacts with DNA primarily at the N7 position of deoxyguanosine residues and creates interstrand cross-links at the sequence 5'-GNC. Since N7-N7 cross-links cause DNA to fragment upon heating, quantative polymerase chain reaction (QPCR) is being used in this experiment to measure the amount of DEB damage (lesion frequency) with three different targets-mitochondrial (unpackaged), open chromatin region, and closed chromatin region. Initial measurements of DEB damage within these three targets were not consistent because the template DNA was not the limiting reagent in the PCR. Follow-up PCR trials using a limiting amount of DNA are still in progress although initial experimentation looks promising. Sequencing of these three targets to confirm the primer targets has only been successfully performed for the closed chromatin target and does not match the sequence from NIH used to design that primer pair. Further sequencing trials need to be conducted on all three targets to assure that a mitochondrial, open chromatin, and closed chromatin region are actually being amplified in this experimental series.