Replication and fine-mapping of a QTL for recurrent airway obstruction in European Warmblood horses.

Recurrent airway obstruction (RAO), or 'heaves', is a common performance-limiting allergic respiratory disease of mature horses. It is related to sensitization and exposure to mouldy hay and has a familial basis with a complex mode of inheritance. In a previous study, we detected a QTL for RAO on ECA 13 in a half-sib family of European Warmblood horses. In this study, we genotyped additional markers in the family and narrowed the QTL down to about 1.5 Mb (23.7-25.2 Mb). We detected the strongest association with SNP BIEC2-224511 (24,309,405 bp). We also obtained SNP genotypes in an independent cohort of 646 unrelated Warmblood horses. There was no genome-wide significant association with RAO in these unrelated horses. However, we performed a genotypic association study of the SNPs on ECA 13 in these unrelated horses, and the SNP BIEC2-224511 also showed the strongest association with RAO in the unrelated horses (p(raw) = 0.00037). The T allele at this SNP was associated with RAO both in the family and the unrelated horses. Thus, the association study in the unrelated animals provides independent support for the previously detected QTL. The association study allows further narrowing of the QTL interval to about 0.5 Mb (24.0-24.5 Mb). We sequenced the coding regions of the genes in the critical region but did not find any associated coding variants. Therefore, the causative variant underlying this QTL is likely to be a regulatory mutation.

Noninvasive pressure difference mapping derived from 4D flow MRI in patients with unrepaired and repaired aortic coarctation.

PURPOSE To develop a method for computing and visualizing pressure differences derived from time-resolved velocity-encoded three-dimensional phase-contrast magnetic resonance imaging (4D flow MRI) and to compare pressure difference maps of patients with unrepaired and repaired aortic coarctation to young healthy volunteers. METHODS 4D flow MRI data of four patients with aortic coarctation either before or after repair (mean age 17 years, age range 3-28, one female, three males) and four young healthy volunteers without history of cardiovascular disease (mean age 24 years, age range 20-27, one female, three males) was acquired using a 1.5-T clinical MR scanner. Image analysis was performed with in-house developed image processing software. Relative pressures were computed based on the Navier-Stokes equation. RESULTS A standardized method for intuitive visualization of pressure difference maps was developed and successfully applied to all included patients and volunteers. Young healthy volunteers exhibited smooth and regular distribution of relative pressures in the thoracic aorta at mid systole with very similar distribution in all analyzed volunteers. Patients demonstrated disturbed pressures compared to volunteers. Changes included a pressure drop at the aortic isthmus in all patients, increased relative pressures in the aortic arch in patients with residual narrowing after repair, and increased relative pressures in the descending aorta in a patient after patch aortoplasty. CONCLUSIONS Pressure difference maps derived from 4D flow MRI can depict alterations of spatial pressure distribution in patients with repaired and unrepaired aortic coarctation. The technique might allow identifying pathophysiological conditions underlying complications after aortic coarctation repair.

Laparoscopic Indocyanine Green Sentinel Lymph Node Mapping in Pregnant Cervical Cancer Patients.

We present cases of 2 pregnant patients with early-stage cervical cancer who have undergone indocyanine green (ICG) sentinel lymph node (SLN) mapping followed by laparoscopic SLN biopsy, pelvic lymphadenectomy, and cervical conization. Eight milliliters of ICG were injected in the 4 quadrants of the cervix after having obtained an adequate pneumoperitoneum and having inspected the abdominal cavity. SLNs were identified in both hemipelvises in both patients. In the final pathologic analysis, both SLNs and non-SLNs were negative for metastatic disease. No adverse events from ICG injection were recorded. ICG SLN mapping seems to be feasible in pregnant cervical cancer patients.

A Comparison of Radiocolloid and Indocyanine Green Fluorescence Imaging, Sentinel Lymph Node Mapping in Patients with Cervical Cancer Undergoing Laparoscopic Surgery.

BACKGROUND AND PURPOSE (99)TC combined with blue-dye mapping is considered the best sentinel lymph node (SLN) mapping technique in cervical cancer. Indocyanine green (ICG) with near infrared fluorescence imaging has been introduced as a new methodology for SLN mapping. The aim of this study was to compare these two techniques in the laparoscopic treatment of cervical cancer. METHODS Medical records of patients undergoing laparoscopic SLN mapping for cervical cancer with either (99)Tc and patent blue dye (Group 1) or ICG (Group 2) from April 2008 until August 2012 were reviewed. Sensitivity, specificity, and overall and bilateral detection rates were calculated and compared. RESULTS Fifty-eight patients were included in the study-36 patients in Group 1 and 22 patients in Group 2. Median tumor diameter was 25 and 29 mm, and mean SLN count was 2.1 and 3.7, for Groups 1 and 2, respectively. Mean non-SLN (NSLN) count was 39 for both groups. SLNs were ninefold more likely to be affected by metastatic disease compared with NSLNs (p < 0.005). Sensitivity and specificity were both 100 %. Overall detection rates were 83 and 95.5 % (p = nonsignificant), and bilateral detection rates were 61 and 95.5 % (p < 0.005), for Groups 1 and 2, respectively. In 75 % of cases, SLNs were located along the external or internal iliac nodal basins. CONCLUSIONS ICG SLN mapping in cervical cancer provides high overall and bilateral detection rates that compare favorably with the current standard of care.

Physical and functional mapping of a novel tumor suppressor locus for prostate cancer within chromosome 10p12.31-q11

Prostate cancer remains the second leading cause of male cancer deaths in the United States, yet the molecular mechanisms underlying this disease remain largely unknown. Cytogenetic and molecular analyses of prostate tumors suggest a consistent association with the loss of chromosome 10. Previously, we have defined a novel tumor suppressor locus PAC-1 within chromosome 10pter-q11. Introduction of the short arm of chromosome 10 into a prostatic adenocarcinoma cell line PC-3H resulted in dramatic tumor suppression and restoration of a programmed cell death pathway. Using a combined approach of comparative genomic hybridization and microsatellite analysis of PC-3H, I have identified a region of hemizygosity within 10p12-p15. This region has been shown to be involved in frequent loss of heterozygosity in gliomas and melanoma. To functionally dissect the region within chromosome 10p containing PAC-1, we developed a strategy of serial microcell fusion, a technique that allows the transfer of defined fragments of chromosome 10p into PC-3H. Serial microcell fusion was used to transfer defined 10p fragments into a mouse A9 fibrosarcoma cell line. Once characterized by FISH and microsatellite analyses, the 10p fragments were subsequently transferred into PC-3H to generate a panel of microcell hybrid clones containing overlapping deletions of chromosome 10p. In vivo and microsatellite analyses of these PC hybrids identified a small chromosome 10p fragment (an estimated 31 Mb in size inclusive of the centromere) that when transferred into the PC-3H background, resulted in significant tumor suppression and limited a region of functional tumor suppressor activity to chromosome 10p12.31-q11. This region coincides with a region of LOH demonstrated in prostate cancer. These studies demonstrate the utility of this approach as a powerful tool to limit regions of functional tumor suppressor activity. Furthermore, these data used in conjunction with data generated by the Human Genome Project lent a focused approach to identify candidate tumor suppressor genes involved in prostate cancer. ^

The genetic basis of thoracic aortic aneurysms and dissections: Genetic heterogeneity and mapping of TAAD1 and TAAD2 loci

Thoracic aortic aneurysms leading to aortic dissections (TAAD) are a major cause of morbidity and mortality in the United States. TAAD is a complication of some known genetic disorders, such as Marfan syndrome and Turner syndrome, but the majority of familial cases are not due to a known genetic syndrome. Previous studies by our group have established that nonsyndromic, familial TAAD is inherited in an autosomal dominant manner with decreased penetrance and variable expression. Using one large family with multiple members with TAAD for the genome wide scan, a major locus for familial TAAD was mapped to 5q13–14 (TAAD1). Nine out of 15 families studied were linked to this locus, establishing that TAAD1 was a major locus, and that there was genetic heterogeneity for the condition. Mapping of TAAD2 locus was accomplished using a single large family with multiple members with TAAD not linked to known loci of aneurysm formation. This established a second novel locus for familial TAAD on 3p24–25 (LOD score of 4.3), termed the TAAD2 locus. Two putative loci with suggestive LOD scores were mapped on 4q and 12q through a genome scan carried out using three families. TAAD phenotype in 12 families did not segregate with known loci, indicating further genetic heterogeneity. An STS-tagged BAC based contig was constructed for 7.8Mb and 25Mb critical interval of TAAD1 and TAAD2 respectively and characterized to identify the defective gene. The hypothesis that the defective genes responsible for the TAAD1 and TAAD2 encoded extracellular matrix (ECM) proteins, the major components of the elastic fiber system in the aortic media was tested. Four genes encoding ECM proteins, versican, thrombospondin-3, CRTL1, on TAAD1 and FBLN2 at TAAD2 were sequenced, but no disease-causing mutations were identified. Studies to identify the defective gene are initiated through the positional candidate gene approach using combination of bioinformatics and expression studies. The identification of the TAAD susceptibility genes will allow for presymptomatic diagnosis of individuals at risk for this life threatening disease. The identification of the molecular defects that contribute to TAAD will also further our understanding of the proteins that provide structural integrity to the aortic wall. ^

Nonlinear tests for genetic studies of complex disease

Linkage and association studies are major analytical tools to search for susceptibility genes for complex diseases. With the availability of large collection of single nucleotide polymorphisms (SNPs) and the rapid progresses for high throughput genotyping technologies, together with the ambitious goals of the International HapMap Project, genetic markers covering the whole genome will be available for genome-wide linkage and association studies. In order not to inflate the type I error rate in performing genome-wide linkage and association studies, multiple adjustment for the significant level for each independent linkage and/or association test is required, and this has led to the suggestion of genome-wide significant cut-off as low as 5 × 10 −7. Almost no linkage and/or association study can meet such a stringent threshold by the standard statistical methods. Developing new statistics with high power is urgently needed to tackle this problem. This dissertation proposes and explores a class of novel test statistics that can be used in both population-based and family-based genetic data by employing a completely new strategy, which uses nonlinear transformation of the sample means to construct test statistics for linkage and association studies. Extensive simulation studies are used to illustrate the properties of the nonlinear test statistics. Power calculations are performed using both analytical and empirical methods. Finally, real data sets are analyzed with the nonlinear test statistics. Results show that the nonlinear test statistics have correct type I error rates, and most of the studied nonlinear test statistics have higher power than the standard chi-square test. This dissertation introduces a new idea to design novel test statistics with high power and might open new ways to mapping susceptibility genes for complex diseases. ^

A Bayesian analysis for spatial processes with application to mapping

In geographical epidemiology, maps of disease rates and disease risk provide a spatial perspective for researching disease etiology. For rare diseases or when the population base is small, the rate and risk estimates may be unstable. Empirical Bayesian (EB) methods have been used to spatially smooth the estimates by permitting an area estimate to "borrow strength" from its neighbors. Such EB methods include the use of a Gamma model, of a James-Stein estimator, and of a conditional autoregressive (CAR) process. A fully Bayesian analysis of the CAR process is proposed. One advantage of this fully Bayesian analysis is that it can be implemented simply by using repeated sampling from the posterior densities. Use of a Markov chain Monte Carlo technique such as Gibbs sampler was not necessary. Direct resampling from the posterior densities provides exact small sample inferences instead of the approximate asymptotic analyses of maximum likelihood methods (Clayton & Kaldor, 1987). Further, the proposed CAR model provides for covariates to be included in the model. A simulation demonstrates the effect of sample size on the fully Bayesian analysis of the CAR process. The methods are applied to lip cancer data from Scotland, and the results are compared. ^

Predicting EQ-5D from the Parkinson's disease questionnaire PDQ-8 using multi-dimensional Bayesian network classifiers

The impact of the Parkinson's disease and its treatment on the patients' health-related quality of life can be estimated either by means of generic measures such as the european quality of Life-5 Dimensions (EQ-5D) or specific measures such as the 8-item Parkinson's disease questionnaire (PDQ-8). In clinical studies, PDQ-8 could be used in detriment of EQ-5D due to the lack of resources, time or clinical interest in generic measures. Nevertheless, PDQ-8 cannot be applied in cost-effectiveness analyses which require generic measures and quantitative utility scores, such as EQ-5D. To deal with this problem, a commonly used solution is the prediction of EQ-5D from PDQ-8. In this paper, we propose a new probabilistic method to predict EQ-5D from PDQ-8 using multi-dimensional Bayesian network classifiers. Our approach is evaluated using five-fold cross-validation experiments carried out on a Parkinson's data set containing 488 patients, and is compared with two additional Bayesian network-based approaches, two commonly used mapping methods namely, ordinary least squares and censored least absolute deviations, and a deterministic model. Experimental results are promising in terms of predictive performance as well as the identification of dependence relationships among EQ-5D and PDQ-8 items that the mapping approaches are unable to detect

Mapping the active site in vasoactive intestinal peptide to a core of four amino acids: Neuroprotective drug design

The understanding of the molecular mechanisms leading to peptide action entails the identification of a core active site. The major 28-aa neuropeptide, vasoactive intestinal peptide (VIP), provides neuroprotection. A lipophilic derivative with a stearyl moiety at the N-terminal and norleucine residue replacing the Met-17 was 100-fold more potent than VIP in promoting neuronal survival, acting at femtomolar–picomolar concentration. To identify the active site in VIP, over 50 related fragments containing an N-terminal stearic acid attachment and an amidated C terminus were designed, synthesized, and tested for neuroprotective properties. Stearyl-Lys-Lys-Tyr-Leu-NH2 (derived from the C terminus of VIP and the related peptide, pituitary adenylate cyclase activating peptide) captured the neurotrophic effects offered by the entire 28-aa parent lipophilic derivative and protected against β-amyloid toxicity in vitro. Furthermore, the 4-aa lipophilic peptide recognized VIP-binding sites and enhanced choline acetyltransferase activity as well as cognitive functions in Alzheimer’s disease-related in vivo models. Biodistribution studies following intranasal administration of radiolabeled peptide demonstrated intact peptide in the brain 30 min after administration. Thus, lipophilic peptide fragments offer bioavailability and stability, providing lead compounds for drug design against neurodegenerative diseases.

Linkage disequilibrium mapping in isolated populations: The example of Finland revisited

Linkage disequilibrium analysis can provide high resolution in the mapping of disease genes because it incorporates information on recombinations that have occurred during the entire period from the mutational event to the present. A circumstance particularly favorable for high-resolution mapping is when a single founding mutation segregates in an isolated population. We review here the population structure of Finland in which a small founder population some 100 generations ago has expanded into 5.1 million people today. Among the 30-odd autosomal recessive disorders that are more prevalent in Finland than elsewhere, several appear to have segregated for this entire period in the “panmictic” southern Finnish population. Linkage disequilibrium analysis has allowed precise mapping and determination of genetic distances at the 0.1-cM level in several of these disorders. Estimates of genetic distance have proven accurate, but previous calculations of the confidence intervals were too small because sampling variation was ignored. In the north and east of Finland the population can be viewed as having been “founded” only after 1500. Disease mutations that have undergone such a founding bottleneck only 20 or so generations ago exhibit linkage disequilibrium and haplotype sharing over long genetic distances (5–15 cM). These features have been successfully exploited in the mapping and cloning of many genes. We review the statistical issues of fine mapping by linkage disequilibrium and suggest that improved methodologies may be necessary to map diseases of complex etiology that may have arisen from multiple founding mutations.

The Endogenous and Cell Cycle-dependent Phosphorylation of tau Protein in Living Cells: Implications for Alzheimer’s Disease

In Alzheimer’s disease the neuronal microtubule-associated protein tau becomes highly phosphorylated, loses its binding properties, and aggregates into paired helical filaments. There is increasing evidence that the events leading to this hyperphosphorylation are related to mitotic mechanisms. Hence, we have analyzed the physiological phosphorylation of endogenous tau protein in metabolically labeled human neuroblastoma cells and in Chinese hamster ovary cells stably transfected with tau. In nonsynchronized cultures the phosphorylation pattern was remarkably similar in both cell lines, suggesting a similar balance of kinases and phosphatases with respect to tau. Using phosphopeptide mapping and sequencing we identified 17 phosphorylation sites comprising 80–90% of the total phosphate incorporated. Most of these are in SP or TP motifs, except S214 and S262. Since phosphorylation of microtubule-associated proteins increases during mitosis, concomitant with increased microtubule dynamics, we analyzed cells mitotically arrested with nocodazole. This revealed that S214 is a prominent phosphorylation site in metaphase, but not in interphase. Phosphorylation of this residue strongly decreases the tau–microtubule interaction in vitro, suppresses microtubule assembly, and may be a key factor in the observed detachment of tau from microtubules during mitosis. Since S214 is also phosphorylated in Alzheimer’s disease tau, our results support the view that reactivation of the cell cycle machinery is involved in tau hyperphosphorylation.

Cloning, expression, and genetic mapping of Sema W, a member of the semaphorin family

The semaphorins comprise a large family of membrane-bound and secreted proteins, some of which have been shown to function in axon guidance. We have cloned a transmembrane semaphorin, Sema W, that belongs to the class IV subgroup of the semaphorin family. The mouse and rat forms of Sema W show 97% amino acid sequence identity with each other, and each shows about 91% identity with the human form. The gene for Sema W is divided into 15 exons, up to 4 of which are absent in the human cDNAs that we sequenced. Unlike many other semaphorins, Sema W is expressed at low levels in the developing embryo but was found to be expressed at high levels in the adult central nervous system and lung. Functional studies with purified membrane fractions from COS7 cells transfected with a Sema W expression plasmid showed that Sema W has growth-cone collapse activity against retinal ganglion-cell axons, indicating that vertebrate transmembrane semaphorins, like secreted semaphorins, can collapse growth cones. Genetic mapping of human SEMAW with human/hamster radiation hybrids localized the gene to chromosome 2p13. Genetic mapping of mouse Semaw with mouse/hamster radiation hybrids localized the gene to chromosome 6, and physical mapping placed the gene on bacteria artificial chromosomes carrying microsatellite markers D6Mit70 and D6Mit189. This localization places Semaw within the locus for motor neuron degeneration 2, making it an attractive candidate gene for this disease.

Mapping the parameters of prion-induced neuropathology

We present a theoretical framework that enables us to dissect out the parametric dependencies of the pathogenesis of prion diseases. We are able to determine the influence of both host-dependent factors (connectivity, cell density, protein synthesis rate, and cell death) and strain-dependent factors (cell tropism, virulence, and replication rate). We use a model based on a linked system of differential equations on a lattice to explore how the regional distribution of central nervous system pathology in Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome, and fatal familial insomnia relates to each of these factors. The model then is used to make qualitative predictions about the pathology for two possible hypothetical triggers of neuronal loss in prion diseases. Pathological progression in overexpressing mouse models has been shown to depend on the site of initial infection. The model allows us to compare the pathologies resulting from different inoculation routes.

Mapping the early steps in the pH-induced conformational conversion of the prion protein

Under certain conditions, the prion protein (PrP) undergoes a conformational change from the normal cellular isoform, PrPC, to PrPSc, an infectious isoform capable of causing neurodegenerative diseases in many mammals. Conversion can be triggered by low pH, and in vivo this appears to take place in an endocytic pathway and/or caveolae-like domains. It has thus far been impossible to characterize the conformational change at high resolution by experimental methods. Therefore, to investigate the effect of acidic pH on PrP conformation, we have performed 10-ns molecular dynamics simulations of PrPC in water at neutral and low pH. The core of the protein is well maintained at neutral pH. At low pH, however, the protein is more dynamic, and the sheet-like structure increases both by lengthening of the native β-sheet and by addition of a portion of the N terminus to widen the sheet by another two strands. The side chain of Met-129, a polymorphic codon in humans associated with variant Creutzfeldt–Jakob disease, pulls the N terminus into the sheet. Neutralization of Asp-178 at low pH removes interactions that inhibit conversion, which is consistent with the Asp-178–Asn mutation causing human prion diseases.