Targeted array CGH as a valuable molecular diagnostic approach: experience in the diagnosis of mitochondrial and metabolic disorders

Oligonucleotide array-based comparative genomic hybridization (aCGH) targeted to coding exons of genes of interest has been proven to be a valuable diagnostic tool to complement with Sanger sequencing for the detection of large deletions/duplications. We have developed a custom designed oligonucleotide aCGH platform for this purpose. This array platform provides tiled coverage of the entire mitochondrial genome and high-density coverage of a set of nuclear genes involving mitochondrial and metabolic disorders and can be used to evaluate large deletions in targeted genes. A total of 1280 DNA samples from patients suspected of having mitochondrial or metabolic disorders were evaluated using this targeted aCGH. We detected 40 (3%) pathogenic large deletions in unrelated individuals, including 6 in genes responsible for mitochondrial DNA (mtDNA) depletion syndromes, 23 in urea cycle genes, 11 in metabolic and related genes. Deletion breakpoints have been confirmed in 31 cases by PCR and sequencing. The possible deletion mechanism has been discussed. These results illustrate the successful utilization of targeted aCGH to detect large deletions in nuclear and mitochondrial genomes. This technology is particularly useful as a complementary diagnostic test in the context of a recessive disease when only one mutant allele is found by sequencing. For female carriers of X-linked disorders, if sequencing analysis does not detect point mutations, targeted aCGH should be considered for the detection of large heterozygous deletions.     

Quantitative principal component model for skin chromophore mapping using multi-spectral images and spatial priors

We describe a novel reconstruction algorithm based on Principal Component Analysis (PCA) applied to multi-spectral imaging data. Using numerical phantoms, based on a two layered skin model developed previously, we found analytical expressions, which convert qualitative PCA results into quantitative blood volume and oxygenation values, assuming the epidermal thickness to be known. We also evaluate the limits of accuracy of this method when the value of the epidermal thickness is not known. We show that blood volume can reliably be extracted (less than 6% error) even if the assumed thickness deviates 0.04mm from the actual value, whereas the error in blood oxygenation can be as large as 25% for the same deviation in thickness. This PCA based reconstruction was found to extract blood volume and blood oxygenation with less than 8% error, if the underlying structure is known. We then apply the method to in vivo multi-spectral images from a healthy volunteer's lower forearm, complemented by images of the same area using Optical Coherence Tomography (OCT) for measuring the epidermal thickness. Reconstruction of the imaging results using a two layered analytical skin model was compared to PCA based reconstruction results. A point wise correlation was found, showing the proof of principle of using PCA based reconstruction for blood volume and oxygenation extraction.     

NIH Consensus Development Conference statement: inhaled nitric-oxide therapy for premature infants

Premature birth is a major public health problem in the United States and internationally. Infants born at or before 32 weeks' gestation (2% of all births in the United States in 2007) are at extremely high risk for death in the neonatal period or for pulmonary, visual, and neurodevelopmental morbidities with lifelong consequences including bronchopulmonary dysplasia, retinopathy of prematurity, and brain injury. Risks for adverse outcomes increase with decreasing gestational age. The economic costs to care for these infants are also substantial (estimated at $26 billion in 2005 in the United States). It is clear that the need for strategies to improve outcomes for this high-risk population is great, and this need has prompted testing of new therapies with the potential to decrease pulmonary and other complications of prematurity. Inhaled nitric oxide (iNO) emerged as one such therapy. To provide health care professionals, families, and the general public with a responsible assessment of currently available data regarding the benefits and risks of iNO in premature infants, the Eunice Kennedy Shriver National Institute of Child Health and Human Development, the National Heart, Lung, and Blood Institute, and the Office of Medical Applications of Research of the National Institutes of Health convened a consensus-development conference. Findings from a substantial body of experimental work in developing animals and other model systems suggest that nitric oxide may enhance lung growth and reduce lung inflammation independently of its effects on blood vessel resistance. Although this work demonstrates biological plausibility and the results of randomized controlled trials in term and near-term infants were positive, combined evidence from the 14 randomized controlled trials of iNO treatment in premature infants of ≤ 34 weeks' gestation shows equivocal effects on pulmonary outcomes, survival, and neurodevelopmental outcomes.     

Economics at the frontline: Tools and tips for busy clinicians

Frontline providers of neonatal care have a moral imperative to enhance value and inform senior administrators of how to most efficiently spend healthcare dollars. This article argues that the frontline is the ideal setting to pursue these efforts, offers recommendations for how to measure value, and describes five simple yet effective concrete tools that can improve value. It concludes with tips on advancing a value-added agenda through the Model for Improvement and advice for teams on ways of approaching senior leaders to help align unit-level aims with system-level goals and mission. Armed with these instruments, multidisciplinary teams can help ensure that neonatal care remains at the forefront of high-value healthcare.     

Multiple Redundant Medulla Projection Neurons Mediate Color Vision in Drosophila

The receptor mechanism for color vision has been extensively studied. In contrast, the circuit(s) that transform(s) photoreceptor signals into color percepts to guide behavior remain(s) poorly characterized. Using intersectional genetics to inactivate identified subsets of neurons, we have uncovered the first-order interneurons that are functionally required for hue discrimination in Drosophila. We developed a novel aversive operant conditioning assay for intensity-independent color discrimination (true color vision) in Drosophila. Single flying flies are magnetically tethered in an arena surrounded by blue and green LEDs (light-emitting diodes). The flies’ optomotor response is used to determine the blue-green isoluminant intensity. Flies are then conditioned to discriminate between equiluminant blue or green stimuli. Wild-type flies are successfully trained in this paradigm when conditioned to avoid either blue or green. Functional color entrainment requires the function of the narrow-spectrum photoreceptors R8 and/or R7, and is within a limited range, intensity independent, suggesting that it is mediated by a color vision system. The medulla projection neurons, Tm5a/b/c and Tm20, receive direct inputs from R7 or R8 photoreceptors and indirect input from the broad-spectrum photoreceptors R1–R6 via the lamina neuron L3. Genetically inactivating these four classes of medulla projection neurons abolished color learning. However, inactivation of subsets of these neurons is insufficient to block color learning, suggesting that true color vision is mediated by multiple redundant pathways. We hypothesize that flies represent color along multiple axes at the first synapse in the fly visual system. The apparent redundancy in learned color discrimination sharply contrasts with innate ultraviolet (UV) spectral preference, which is dominated by a single pathway from the amacrine neuron Dm8 to the Tm5c projection neurons.     

Evaluation of the effect of naproxen on the pharmacokinetics and pharmacodynamics of apixaban

Aim

To assess pharmacokinetic and pharmacodynamic interactions between naproxen (a non-steroidal anti-inflammatory drug) and apixaban (an oral, selective, direct factor-Xa inhibitor).

Method

In this randomized, three period, two sequence study, 21 healthy subjects received a single oral dose of apixaban 10 mg, naproxen 500 mg or co-administration of both. Blood samples were collected for determination of apixaban and naproxen pharmacokinetics and pharmacodynamics (anti-Xa activity, international normalized ratio [INR] and arachidonic acid–induced platelet aggregation [AAI-PA]). Adverse events, bleeding time and routine safety assessments were also evaluated.

Results

Apixaban had no effect on naproxen pharmacokinetics. However, following co-administration, apixaban AUC(0,∞), AUC(0,t) and C_max were 54% (geometric mean ratio 1.537; 90% confidence interval (CI) 1.394, 1.694), 55% (1.549; 90% CI 1.400, 1.713) and 61% (1.611; 90% CI 1.417, 1.831) higher, respectively. Mean (standard deviation [SD]) anti-Xa activity at 3 h post-dose was approximately 60% higher following co-administration compared with apixaban alone, 4.4 [1.0] vs. 2.7 [0.7] IU ml⁻¹, consistent with the apixaban concentration increase following co-administration. INR was within the normal reference range after all treatments. AAI-PA was reduced by approximately 80% with naproxen. Co-administration had no impact beyond that of naproxen. Mean [SD] bleeding time was higher following co-administration (9.1 [4.1] min) compared with either agent alone (5.8 [2.3] and 6.9 [2.6] min for apixaban and naproxen, respectively).

Conclusion

Co-administration of naproxen with apixaban results in higher apixaban exposure and appears to occur through increased apixaban bioavailability. The effects on anti-Xa activity, INR and inhibition of AAI-PA observed in this study were consistent with the individual pharmacologic effects of apixaban and naproxen.     

Detection of clinically relevant exonic copy‐number changes by array CGH

Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy‐number variation (CNV). However, intragenic deletions or duplications—those including genomic intervals of a size smaller than a gene—have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy‐number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom‐designed, exon‐targeted oligonucleotide array to detect intragenic copy‐number changes in patients with various clinical phenotypes. Hum Mutat 31:1–17, 2010. © 2010 Wiley‐Liss, Inc.     

Comorbidity as a predictor of stage of illness for patients with breast cancer

OBJECTIVE: The purpose of this research was to determine whether comorbidity affects the stage at which breast cancer is diagnosed. METHODS: Data from the Surveillance, Epidemiology and End Results (SEER) program of the National Cancer Institute (NCI) was merged with Medicare claims for 17,468 women diagnosed with breast cancer from 1993 to 1995. RESULTS: Women with cardiovascular disease, musculoskeletal disorders, mild-to-moderate gastrointestinal disease, and nonmalignant benign breast disease had a 13%, 7%, 14%, and 24% lower odds, respectively, of being diagnosed with advanced breast cancer. Women with diabetes, other endocrine disorders, psychiatric disorders, or hematologic disorders increased the odds of a late-stage diagnosis by 19%, 11%, 20%, and 19% respectively. Mammography screening and contact with the medical care system decreased the odds of late-stage diagnosis. DISCUSSION: Four hypotheses are suggested to explain this link between comorbid illness and stage at diagnosis: (1) the "surveillance" hypothesis, (2) the "physiological" hypothesis, (3) the "competing demand" hypothesis, and (4) the "death from other causes" hypothesis. CONCLUSIONS: Comorbidity may complicate the diagnostic decision-making process for breast cancer. The results suggest that contact with the medical care system improves the odds of early-stage diagnosis. Thus, barriers to access for people with chronic conditions may exacerbate those chronic conditions and increase the odds of late-stage breast cancer.     

REM latency in Alzheimer's disease

Latency to the first episode of rapid eye movement sleep (REML) has been proposed as a potential biomarker for Alzheimer's disease (AD). In this study, we compared REML values from 28 AD patients and 28 age- and sex-matched controls. We employed multiple definitions of REML and multiple cutoffs to classify patients and controls. Results indicated that the best REML definition and optimal cutoff criterion resulted in only 65% correct classifications. We discuss the longer REML in AD patients relative to controls in terms of both overall sleep disturbance and selective deterioration of the REM-cholinergic system. As REML may be relatively short in other forms of psychopathology (e.g., affective disorders), REML may still hold promise in the differential diagnosis of dementia and pseudodementia.