Alzheimer's disease markers in the aged sheep (Ovis aries)

This study reports the identification and characterization of markers of Alzheimer's disease (AD) in aged sheep (Ovis aries) as a preliminary step toward making a genetically modified large animal model of AD. Importantly, the sequences of key proteins involved in AD pathogenesis are highly conserved between sheep and human. The processing of the amyloid-β (Aβ) protein is conserved between sheep and human, and sheep Aβ1–42/Aβ1–40 ratios in cerebrospinal fluid (CSF) are also very similar to human. In addition, total tau and neurofilament light levels in CSF are comparable with those found in human. The presence of neurofibrillary tangles in aged sheep brain has previously been established; here, we report for the first time that plaques, the other pathologic hallmark of AD, are also present in the aged sheep brain. In summary, the biological machinery to generate the key neuropathologic features of AD is conserved between the human and sheep, making the sheep a good candidate for future genetic manipulation to accelerate the condition for use in pathophysiological discovery and therapeutic testing

Molecular diagnosis of Huntington disease in Portugal : implications for genetic counselling and clinical practice

Huntington disease (HD) is a eurodegenerative, autosomal dominant disorder of late-onset, caused by the expansion of a CAG repeat in the coding region of the gene. Ours is the reference laboratory for genetic testing in HD, in Portugal, since 1998; 90.1% of all 158 families known were identified for the first time, including patients with unusual presentation or without family history. A total of 338 genetic tests were performed: 234 for diagnosis, 96 for presymptomatic and four for prenatal testing (four were done for family studies). Most referring physicians were neurologists (90.6%); 82.8% of all clinical diagnosis were confirmed, while 83.1% of those sent for exclusion were in fact excluded. In presymptomatic testing, an excess of female subjects (59.4%) was again verified; 37.5% of the consultands were found to be carriers. None of the foetuses, in four prenatal tests, were mutation carriers. One juvenile case was inherited from her mother. Our patient population is very similar to others described so far, namely in terms of mean age at onset and (CAG)n distribution, except perhaps for a higher frequency of large normal (class 2) alleles (3.7%). We also identify cases posing particular problems for genetic counselling, such as, ‘homozygosity’ that can pose a serious ethical dilemma, carriers of large normal alleles, and ‘homoallelism’ for a normal gene, which will demand further procedures and may delay results in presymptomatic and prenatal testing

Minimum sample size for external validation of a clinical prediction model with a binary outcome

In prediction model research, external validation is needed to examine an existing model's performance using data independent to that for model development. Current external validation studies often suffer from small sample sizes and consequently imprecise predictive performance estimates. To address this, we propose how to determine the minimum sample size needed for a new external validation study of a prediction model for a binary outcome. Our calculations aim to precisely estimate calibration (Observed/Expected and calibration slope), discrimination (C-statistic), and clinical utility (net benefit). For each measure, we propose closed-form and iterative solutions for calculating the minimum sample size required. These require specifying: (i) target SEs (confidence interval widths) for each estimate of interest, (ii) the anticipated outcome event proportion in the validation population, (iii) the prediction model's anticipated (mis)calibration and variance of linear predictor values in the validation population, and (iv) potential risk thresholds for clinical decision-making. The calculations can also be used to inform whether the sample size of an existing (already collected) dataset is adequate for external validation. We illustrate our proposal for external validation of a prediction model for mechanical heart valve failure with an expected outcome event proportion of 0.018. Calculations suggest at least 9835 participants (177 events) are required to precisely estimate the calibration and discrimination measures, with this number driven by the calibration slope criterion, which we anticipate will often be the case. Also, 6443 participants (116 events) are required to precisely estimate net benefit at a risk threshold of 8%. Software code is provided.</p

Variation within the Huntington's Disease Gene Influences Normal Brain Structure

Genetics of the variability of normal and diseased brain structure largely remains to be elucidated. Expansions of certain trinucleotide repeats cause neurodegenerative disorders of which Huntington's disease constitutes the most common example. Here, we test the hypothesis that variation within the IT15 gene on chromosome 4, whose expansion causes Huntington's disease, influences normal human brain structure. In 278 normal subjects, we determined CAG repeat length within the IT15 gene on chromosome 4 and analyzed high-resolution T1-weighted magnetic resonance images by the use of voxel-based morphometry. We found an increase of GM with increasing long CAG repeat and its interaction with age within the pallidum, which is involved in Huntington's disease. Our study demonstrates that a certain trinucleotide repeat influences normal brain structure in humans. This result may have important implications for the understanding of both the healthy and diseased brain

Quantification of Age-Dependent Somatic CAG Repeat Instability in Hdh CAG Knock-In Mice Reveals Different Expansion Dynamics in Striatum and Liver

Age at onset of Huntington's disease (HD) is largely determined by the CAG trinucleotide repeat length in the HTT gene. Importantly, the CAG repeat undergoes tissue-specific somatic instability, prevalent in brain regions that are disease targets, suggesting a potential role for somatic CAG repeat instability in modifying HD pathogenesis. Thus, understanding underlying mechanisms of somatic CAG repeat instability may lead to discoveries of novel therapeutics for HD. Investigation of the dynamics of the CAG repeat size changes over time may provide insights into the mechanisms underlying CAG repeat instability.To understand how the HTT CAG repeat length changes over time, we quantified somatic instability of the CAG repeat in Huntington's disease CAG knock-in mice from 2-16 months of age in liver, striatum, spleen and tail. The HTT CAG repeat in spleen and tail was very stable, but that in liver and striatum expanded over time at an average rate of one CAG per month. Interestingly, the patterns of repeat instability were different between liver and striatum. Unstable CAG repeats in liver repeatedly gained similar sizes of additional CAG repeats (approximately two CAGs per month), maintaining a distinct population of unstable repeats. In contrast, unstable CAG repeats in striatum gained additional repeats with different sizes resulting in broadly distributed unstable CAG repeats. Expanded CAG repeats in the liver were highly enriched in polyploid hepatocytes, suggesting that the pattern of liver instability may reflect the restriction of the unstable repeats to a unique cell type.Our results are consistent with repeat expansion occurring as a consequence of recurrent small repeat insertions that differ in different tissues. Investigation of the specific mechanisms that underlie liver and striatal instability will contribute to our understanding of the relationship between instability and disease and the means to intervene in this process

Functional genomics reveals serine synthesis is essential in PHGDH-amplified breast cancer

Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation[superscript 1, 2]. RNA interference (RNAi)-based loss-of-function screening has proven powerful for the identification of new and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumour suppressor genes[superscript 3]. Here we developed a method for identifying novel cancer targets via negative-selection RNAi screening using a human breast cancer xenograft model at an orthotopic site in the mouse. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumorigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of oestrogen receptor (ER)-negative breast cancers. PHGDH catalyses the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have increased serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not in those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of α-ketoglutarate, another output of the pathway and a tricarboxylic acid (TCA) cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH overexpression and demonstrate the utility of in vivo negative-selection RNAi screens for finding potential anticancer targets.Susan G. Komen Breast Cancer Foundation (Fellowship)Life Sciences Research Foundation (Fellowship)W. M. Keck FoundationDavid H. Koch Cancer Research FundAlexander and Margaret Stewart TrustNational Institutes of Health (U.S.) (Grant CA103866

Onset and Progression of Behavioral and Molecular Phenotypes in a Novel Congenic R6/2 Line Exhibiting Intergenerational CAG Repeat Stability

In the present study we report on the use of speed congenics to generate a C57BL/6J congenic line of HD-model R6/2 mice carrying 110 CAG repeats, which uniquely exhibits minimal intergenerational instability. We also report the first identification of the R6/2 transgene insertion site. The relatively stable line of 110 CAG R6/2 mice was characterized for the onset of behavioral impairments in motor, cognitive and psychiatric-related phenotypes as well as the progression of disease-related impairments from 4 to 10 weeks of age. 110Q mice exhibited many of the phenotypes commonly associated with the R6/2 model including reduced activity and impairments in rotarod performance. The onset of many of the phenotypes occurred around 6 weeks and was progressive across age. In addition, some phenotypes were observed in mice as early as 4 weeks of age. The present study also reports the onset and progression of changes in several molecular phenotypes in the novel R6/2 mice and the association of these changes with behavioral symptom onset and progression. Data from TR-FRET suggest an association of mutant protein state changes (soluble versus aggregated) in disease onset and progression

Ischemia of the lung causes extensive long-term pulmonary injury: an experimental study

Background: Lung ischemia-reperfusion injury (LIRI) is suggested to be a major risk factor for development of primary acute graft failure (PAGF) following lung transplantation, although other factors have been found to interplay with LIRI. The question whether LIRI exclusively results in PAGF seems difficult to answer, which is partly due to the lack of a long-term experimental LIRI model, in which PAGF changes can be studied. In addition, the long-term effects of LIRI are unclear and a detailed description of the immunological changes over time after LIRI is missing. Therefore our purpose was to establish a long-term experimental model of LIRI, and to study the impact of LIRI on the development of PAGF, using a broad spectrum of LIRI parameters including leukocyte kinetics.Methods: Male Sprague-Dawley rats (n = 135) were subjected to 120 minutes of left lung warm ischemia or were sham-operated. A third group served as healthy controls. Animals were sacrificed 1, 3, 7, 30 or 90 days after surgery. Blood gas values, lung compliance, surfactant conversion, capillary permeability, and the presence of MMP-2 and MMP-9 in broncho-alveolar-lavage flui