Utilizing Multiplex Ligation-dependent Probe Amplification to Detect Novel X-linked Microduplications Which Cause Intellectual Disability

ABSTRACT A number of conditions related to X-linked intellectual disabilities (XLID) are in part due to microduplications that are not visible cytogenetically. With the focus on Rho, Ras and Rab genes, a family of genes known to be associated with intellectual disabilities, were screened for dosage aberrations (Leeuwen, F. N. 1997), (Ng, E. L. 2008), (Gissen, P. 2007), (Gurkan, C. 2005). Cohorts of intellectually disabled ID individuals were explored with new technologies. These new technologies include comparative genomic hybridization (CGH), multiplex ligation dependent probe amplification (MLPA) and quantitative PCR (qPCR) (Madrigal, I. 2007), (Hermsen, M. A. 2005), (Morey, J. S. 2006). The first screening was of two groups of individuals, one group with hypotonia and varying degrees of ID and the other of individuals with nonsyndromic ID and a suspected X-linked etiology. These cohorts were screened using the Mental Retardation on the X chromosome (MRX) kit, which focuses on genes that cause intellectual disability and are located on the X chromosome. The second screening consisted of the two former groups and 5 additional cohorts totaling 1152 patients, using a synthetic probe kit that was designed to target primarily Ras, Rab and Rho X-linked genes that were not covered by the MRX kit. The 5 additional cohorts were individuals that had normal sequencing results for one of the following X-linked genes XNP, L1CAM, UBE3A, FGD1, and STK9. The MRX screening produced a GDI1 duplication, deletion in FACL4 and an FMR2 missense mutation (c.474C\u3eT). The synthetic MLPA screening found a partial XNP duplication (248kb), a 1p36 duplication/deletion complex rearrangement and a greater than 3Mb 1p36 deletion. It has been concluded from this study that duplications in these genes are rare, appearing in less than 1% in these chosen populations. Another section of this project is the characterization of a 275kb Xq25 duplication found during routine MLPA testing for MECP2. An Xq25 control peak on the MRC Holland MECP2 MLPA revealed a duplication in a female that presented with a MECP2 phenotype (Chahrour, M. 2007). This duplication spanned four genes (AIF, ELF4, BCORL1 and RAB33A) and of these four, two were over-expressed (AIF and RAB33A). Using qPCR to look for the link that may cause the similar phenotype to Rett syndrome in this patient, 26 Ras, Rab and Rho genes were tested in patients with Rett syndrome, Fragile X syndrome, ID with unknown etiology and the Xq25 patient. A similar pattern of expression was seen in this small cohort with ID. The CREB1 gene, the co-activator of MECP2, part of the transcription factor complex for 21 of the 26 genes screened, plays a role in all of these conditions and may be the linking factor in producing these patterns. The over expression of the AIF gene seemed to play a role in the mis-regulation of many genes, but with uncertainty on how it led to any affect on the phenotype. In this study duplications that play a role in the causation of ID were found using MLPA technology. As array CGH becomes more refined, with higher coverage and better software, the finding of microduplications that cause ID will increase

Developmental Origins of Cardiovascular Disease: Impact of Early Life Stress in Humans and Rodents

The Developmental Origins of Health and Disease (DOHaD) hypothesizes that environmental insults during childhood programs the individual to develop chronic disease in adulthood. Emerging epidemiological data strongly supports that early life stress (ELS) given by the exposure to adverse childhood experiences is regarded as an independent risk factor capable of predicting future risk of cardiovascular disease. Experimental animal models utilizing chronic behavioral stress during postnatal life, specifically maternal separation (MatSep) provides a suitable tool to elucidate molecular mechanisms by which ELS increases the risk to develop cardiovascular disease, including hypertension. The purpose of this review is to highlight current epidemiological studies linking ELS to the development of cardiovascular disease and to discuss the potential molecular mechanisms identified from animal studies. Overall, this review reveals the need for future investigations to further clarify the molecular mechanisms of ELS in order to develop more personalized therapeutics to mitigate the long-term consequences of chronic behavioral stress including cardiovascular and heart disease in adulthood

Oxidative Stress Accumulates in Adipose Tissue during Aging and Inhibits Adipogenesis

Aging constitutes a major independent risk factor for the development of type 2 diabetes and is accompanied by insulin resistance and adipose tissue dysfunction. One of the most important factors implicitly linked to aging and age-related chronic diseases is the accumulation of oxidative stress. However, the effect of increased oxidative stress on adipose tissue biology remains elusive. In this study, we demonstrate that aging in mice results in a loss of fat mass and the accumulation of oxidative stress in adipose tissue. In vitro, increased oxidative stress through glutathione depletion inhibits preadipocyte differentiation. This inhibition of adipogenesis is at least in part the result of reduced cell proliferation and an inhibition of G1→S-phase transition during the initial mitotic clonal expansion of the adipocyte differentiation process. While phosphorylation of the retinoblastoma protein (Rb) by cyclin/cdk complexes remains unaffected, oxidative stress decreases the expression of S-phase genes downstream of Rb. This silencing of S phase gene expression by increased oxidative stress is mediated through a transcriptional mechanism involving the inhibition of E2F recruitment and transactivation of its target promoters. Collectively, these data demonstrate a previously unrecognized role of oxidative stress in the regulation of adipogenesis which may contribute to age-associated adipose tissue dysfunction

‘Is this a time of beautiful chaos?’: reflecting on international feminist legal methods

This article considers how Margaret Jane Radin’s theory of the feminist double bind can bring conceptual clarity to the difficulties feminisms face in engaging with political and legal institutions of global governance. I draw on her theory to reinitiate a conversation on ideal and nonideal theory, in order to answer the call of key proponents in international legal feminism to reevaluate methodologies in critiquing mainstream institutions. By providing an account of how to navigate the double bind, this article brings conceptual clarity to the tension between resistance and compliance that has been argued to lie at the heart of the feminist project in international law. I demonstrate how this theoretical framework can foster greater pluralist perspectives in feminist engagement of ideal theories to temper the deradicalising and conservative risk of navigating feasibility constrained nonideal strategies

Defining the effect and mediators of two knowledge translation strategies designed to alter knowledge, intent and clinical utilization of rehabilitation outcome measures: a study protocol [NCT00298727]

BACKGROUND: A substantial number of valid outcome measures have been developed to measure health in adult musculoskeletal and childhood disability. Regrettably, national initiatives have merely resulted in changes in attitude, while utilization remains unacceptably low. This study will compare the effectiveness and mediators of two different knowledge transfer (KT) interventions in terms of their impact on changing knowledge and behavior (utilization and clinical reasoning) related to health outcome measures. METHOD/DESIGN: Physical and occupational therapists (n = 144) will be recruited in partnership with the national professional associations to evaluate two different KT interventions with the same curriculum: 1) Stakeholder-Hosted Interactive Problem-Based Seminar (SHIPS), and 2) Online Problem-Based course (e-PBL). SHIPS will consist of face-to-face problem-based learning (PBL) for 2 1/2 days with outcome measure developers as facilitators, using six problems generated in consultation with participants. The e-PBL will consist of a 6-week web-based course with six generic problems developed by content experts. SHIPS will be conducted in three urban centers in Canada. Participants will be block-allocated by a minimization procedure to either of the two interventions to minimize any prognostic differences. Trained evaluators at each site will conduct chart audits and chart-stimulated recall. Trained interviewers will conduct semi-structured interviews focused on identifying critical elements in KT and implementing practice changes. Interviews will be transcribed verbatim. Baseline predictors including demographics, knowledge, attitudes/barriers regarding outcome measures, and Readiness to Change will be assessed by self-report. Immediately post-intervention and 6 months later, these will be re-administered. Primary qualitative and quantitative evaluations will be conducted 6-months post-intervention to assess the relative effectiveness of KT interventions and to identify elements that contribute to changing clinical behavior. Chart audits will determine the utilization of outcome measures (counts). Incorporation of outcome measures into clinical reasoning will be assessed using an innovative technique: chart-stimulated recall. DISCUSSION: A strategy for optimal transfer of health outcome measures into practice will be developed and shared with multiple disciplines involved in primary and specialty management of musculoskeletal and childhood disability

Large Prospective Study of Ovarian Cancer Screening in High-Risk Women: CA125 Cut-Point Defined by Menopausal Status

Previous screening trials for early detection of ovarian cancer in postmenopausal women have used the standard CA125 cut-point of 35 U/mL, the 98th percentile in this population yielding a 2% false positive rate, while the same cut-point in trials of premenopausal women results in substantially higher false positive rates. We investigated demographic and clinical factors predicting CA125 distributions, including 98th percentiles, in a large population of high-risk women participating in two ovarian cancer screening studies with common eligibility criteria and screening protocols

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead