Detection of ApoE E2, E3 and E4 alleles using MALDI-TOF mass spectrometry and the homogeneous mass-extend technology

Apolipoprotein (Apo) E is one of the five main types of blood lipoproteins (A–E). It is synthesized primarily in the liver and brain and helps in transporting lipids from one place to another as well as facilitates the clearing of dietary fats, such as triglycerides, from the blood. The ApoE gene exists in three different forms: E2, E3 and E4. E3 is considered to be the normal form. Variants of the ApoE gene have been associated with various diseases. Developing an assay for the genotyping of ApoE variants for use both in clinical and large cohort based association settings would be extremely valuable and would require the use of a platform that has high-throughput capabilities and is highly accurate. Here we describe an assay for the simultaneous genotyping of the ApoE variants in a single bi-plex reaction and a single well using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and the homogeneous mass-extend (hME) technology. The assay is robust, highly accurate and suitable for both clinical applications and for the genotyping of large disease cohorts. Moreover, the prevalence of ApoE variants in a cohort of Caucasians from the central Wisconsin area is outlined

Killing the umpire: cooperative defects in mitotic checkpoint and BRCA2 genes on the road to transformation

Recent findings from mouse models of BRCA2 genetic lesions have provided intriguing insights and important questions concerning modes of tumor development in familial breast and ovarian cancers. Fibroblasts from mice homozygous for the BRCA2(Tr) allele grow poorly and display an array of chromosomal abnormalities that are consistent with a role for BRCA2 in DNA repair. This growth defect can be overcome and cellular transformation promoted by the expression of defective, dominant negative alleles of p53 and of the mitotic checkpoint gene Bub1, both of which are known to induce chromosome instability. These findings are mirrored in the genetic lesions sustained in tumors found in the rare BRCA2(Tr/Tr)mice that survive to adulthood, which include defects in p53 as well as the mitotic checkpoint proteins Bub1 and Mad3L. Together, these data hint that tumors in these mice evolve from an unusually intense selective pressure to remove DNA damage checkpoints, which in turn might be facilitated by chromosomal abolition of mitotic checkpoints and the consequent increase in shuffling of genetic information. How these genetic lesions co-operate to yield transformed cells and how these data relate to BRCA1 and BRCA2 defects in the human population are important questions raised by this work

Autophagy in Premature Senescent Cells Is Activated via AMPK Pathway

Autophagy is a highly regulated intracellular process involved in the turnover of most cellular constituents and in the maintenance of cellular homeostasis. In this study, we show that the activity of autophagy increases in H2O2 or RasV12-induced senescent fibroblasts. Inhibiting autophagy promotes cell apoptosis in senescent cells, suggesting that autophagy activation plays a cytoprotective role. Furthermore, our data indicate that the increase of autophagy in senescent cells is linked to the activation of transcription factor FoxO3A, which blocks ATP generation by transcriptionally up-regulating the expression of PDK4, an inhibitor of pyruvate dehydrogenase complex, thus leading to AMPK activation and mTOR inhibition. These findings suggest a novel mechanism by which FoxO3A factors can activate autophagy via metabolic alteration

Speech delays and behavioral problems are the predominant features in individuals with developmental delays and 16p11.2 microdeletions and microduplications

Microdeletions and microduplications encompassing a ~593-kb region of 16p11.2 have been implicated as one of the most common genetic causes of susceptibility to autism/autism spectrum disorder (ASD). We report 45 microdeletions and 32 microduplications of 16p11.2, representing 0.78% of 9,773 individuals referred to our laboratory for microarray-based comparative genomic hybridization (aCGH) testing for neurodevelopmental and congenital anomalies. The microdeletion was de novo in 17 individuals and maternally inherited in five individuals for whom parental testing was available. Detailed histories of 18 individuals with 16p11.2 microdeletions were reviewed; all had developmental delays with below-average intelligence, and a majority had speech or language problems or delays and various behavioral problems. Of the 16 individuals old enough to be evaluated for autism, the speech/behavior profiles of seven did not suggest the need for ASD evaluation. Of the remaining nine individuals who had speech/behavior profiles that aroused clinical suspicion of ASD, five had formal evaluations, and three had PDD-NOS. Of the 19 microduplications with parental testing, five were de novo, nine were maternally inherited, and five were paternally inherited. A majority with the microduplication had delayed development and/or specific deficits in speech or language, though these features were not as consistent as seen with the microdeletions. This study, which is the largest cohort of individuals with 16p11.2 alterations reported to date, suggests that 16p11.2 microdeletions and microduplications are associated with a high frequency of cognitive, developmental, and speech delay and behavior abnormalities. Furthermore, although features associated with these alterations can be found in individuals with ASD, additional factors are likely required to lead to the development of ASD

Low Levels of Genetic Divergence across Geographically and Linguistically Diverse Populations from India

Ongoing modernization in India has elevated the prevalence of many complex genetic diseases associated with a western lifestyle and diet to near-epidemic proportions. However, although India comprises more than one sixth of the world's human population, it has largely been omitted from genomic surveys that provide the backdrop for association studies of genetic disease. Here, by genotyping India-born individuals sampled in the United States, we carry out an extensive study of Indian genetic variation. We analyze 1,200 genome-wide polymorphisms in 432 individuals from 15 Indian populations. We find that populations from India, and populations from South Asia more generally, constitute one of the major human subgroups with increased similarity of genetic ancestry. However, only a relatively small amount of genetic differentiation exists among the Indian populations. Although caution is warranted due to the fact that United States–sampled Indian populations do not represent a random sample from India, these results suggest that the frequencies of many genetic variants are distinctive in India compared to other parts of the world and that the effects of population heterogeneity on the production of false positives in association studies may be smaller in Indians (and particularly in Indian-Americans) than might be expected for such a geographically and linguistically diverse subset of the human population

Sequence variants of interleukin 6 (IL-6) are significantly associated with a decreased risk of late-onset Alzheimer's disease

<p>Abstract</p> <p>Background</p> <p>Interleukin 6 (IL-6) has been related to beta-amyloid aggregation and the appearance of hyperphosphorylated tau in Alzheimer's disease (AD) brain. However, previous studies relating <it>IL-6 </it>genetic polymorphisms to AD included few and unrepresentative single nucleotide polymorphisms (SNPs) and the results were inconsistent.</p> <p>Methods</p> <p>This is a case-control study. A total of 266 patients with AD, aged≧65, were recruited from three hospitals in Taiwan (2007-2010). Controls (n = 444) were recruited from routine health checkups and volunteers of the hospital during the same period of time. Three common <it>IL-6 </it>haplotype-tagging SNPs were selected to assess the association between <it>IL-6 </it>polymorphisms and the risk of late-onset AD (LOAD).</p> <p>Results</p> <p>Variant carriers of <it>IL-6 </it>rs1800796 and rs1524107 were significantly associated with a reduced risk of LOAD [(GG + GC vs. CC): adjusted odds ratio (AOR) = 0.64 and (CC + CT vs. TT): AOR = 0.60, respectively]. Haplotype CAT was associated with a decreased risk of LOAD (0 and 1 copy vs. 2 copies: AOR = 0.65, 95% CI = 0.44-0.95). These associations remained significant in <it>ApoE e4 </it>non-carriers only. Hypertension significantly modified the association between rs2069837 polymorphisms and the risk of LOAD (<it>p</it>_interaction= 0.03).</p> <p>Conclusions</p> <p><it>IL-6 </it>polymorphisms are associated with reduced risk of LOAD, especially in <it>ApoE e4 </it>non-carriers. This study identified genetic markers for predicting LOAD in <it>ApoE e4 </it>non-carriers.</p

Zebrafish homologs of 16p11.2, a genomic region associated with brain disorders, are active during brain development, and include two deletion dosage sensor genes

Deletion or duplication of one copy of the human 16p11.2 interval is tightly associated with impaired brain function, including autism spectrum disorders (ASDs), intellectual disability disorder (IDD) and other phenotypes, indicating the importance of gene dosage in this copy number variant region (CNV). The core of this CNV includes 25 genes; however, the number of genes that contribute to these phenotypes is not known. Furthermore, genes whose functional levels change with deletion or duplication (termed 'dosage sensors'), which can associate the CNV with pathologies, have not been identified in this region. Using the zebrafish as a tool, a set of 16p11.2 homologs was identified, primarily on chromosomes 3 and 12. Use of 11 phenotypic assays, spanning the first 5 days of development, demonstrated that this set of genes is highly active, such that 21 out of the 22 homologs tested showed loss-of-function phenotypes. Most genes in this region were required for nervous system development - impacting brain morphology, eye development, axonal density or organization, and motor response. In general, human genes were able to substitute for the fish homolog, demonstrating orthology and suggesting conserved molecular pathways. In a screen for 16p11.2 genes whose function is sensitive to hemizygosity, the aldolase a (aldoaa) and kinesin family member 22 (kif22) genes were identified as giving clear phenotypes when RNA levels were reduced by ~50%, suggesting that these genes are deletion dosage sensors. This study leads to two major findings. The first is that the 16p11.2 region comprises a highly active set of genes, which could present a large genetic target and might explain why multiple brain function, and other, phenotypes are associated with this interval. The second major finding is that there are (at least) two genes with deletion dosage sensor properties among the 16p11.2 set, and these could link this CNV to brain disorders such as ASD and IDD.Simons Foundation (Grant Number 95091

Exposure to N-Ethyl-N-Nitrosourea in Adult Mice Alters Structural and Functional Integrity of Neurogenic Sites

BACKGROUND: Previous studies have shown that prenatal exposure to the mutagen N-ethyl-N-nitrosourea (ENU), a N-nitroso compound (NOC) found in the environment, disrupts developmental neurogenesis and alters memory formation. Previously, we showed that postnatal ENU treatment induced lasting deficits in proliferation of neural progenitors in the subventricular zone (SVZ), the main neurogenic region in the adult mouse brain. The present study is aimed to examine, in mice exposed to ENU, both the structural features of adult neurogenic sites, incorporating the dentate gyrus (DG), and the behavioral performance in tasks sensitive to manipulations of adult neurogenesis. METHODOLOGY/PRINCIPAL FINDINGS: 2-month old mice received 5 doses of ENU and were sacrificed 45 days after treatment. Then, an ultrastructural analysis of the SVZ and DG was performed to determine cellular composition in these regions, confirming a significant alteration. After bromodeoxyuridine injections, an S-phase exogenous marker, the immunohistochemical analysis revealed a deficit in proliferation and a decreased recruitment of newly generated cells in neurogenic areas of ENU-treated animals. Behavioral effects were also detected after ENU-exposure, observing impairment in odor discrimination task (habituation-dishabituation test) and a deficit in spatial memory (Barnes maze performance), two functions primarily related to the SVZ and the DG regions, respectively. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that postnatal exposure to ENU produces severe disruption of adult neurogenesis in the SVZ and DG, as well as strong behavioral impairments. These findings highlight the potential risk of environmental NOC-exposure for the development of neural and behavioral deficits

Necdin, a p53-Target Gene, Is an Inhibitor of p53-Mediated Growth Arrest

In vitro, cellular immortalization and transformation define a model for multistep carcinogenesis and current ongoing challenges include the identification of specific molecular events associated with steps along this oncogenic pathway. Here, using NIH3T3 cells, we identified transcriptionally related events associated with the expression of Polyomavirus Large-T antigen (PyLT), a potent viral oncogene. We propose that a subset of these alterations in gene expression may be related to the early events that contribute to carcinogenesis. The proposed tumor suppressor Necdin, known to be regulated by p53, was within a group of genes that was consistently upregulated in the presence of PyLT. While Necdin is induced following p53 activation with different genotoxic stresses, Necdin induction by PyLT did not involve p53 activation or the Rb-binding site of PyLT. Necdin depletion by shRNA conferred a proliferative advantage to NIH3T3 and PyLT-expressing NIH3T3 (NIHLT) cells. In contrast, our results demonstrate that although overexpression of Necdin induced a growth arrest in NIH3T3 and NIHLT cells, a growing population rapidly emerged from these arrested cells. This population no longer showed significant proliferation defects despite high Necdin expression. Moreover, we established that Necdin is a negative regulator of p53-mediated growth arrest induced by nutlin-3, suggesting that Necdin upregulation could contribute to the bypass of a p53-response in p53 wild type tumors. To support this, we characterized Necdin expression in low malignant potential ovarian cancer (LMP) where p53 mutations rarely occur. Elevated levels of Necdin expression were observed in LMP when compared to aggressive serous ovarian cancers. We propose that in some contexts, the constitutive expression of Necdin could contribute to cancer promotion by delaying appropriate p53 responses and potentially promote genomic instability