David A. Mrazek

Implementing genomic medicine in the clinic: the future is here

Although the potential for genomics to contribute to clinical care has long been anticipated, the pace of defining the risks and benefits of incorporating genomic findings into medical practice has been relatively slow. Several institutions have recently begun genomic medicine programs, encountering many of the same obstacles and developing the same solutions, often independently. Recognizing that successful early experiences can inform subsequent efforts, the National Human Genome Research Institute brought together a number of these groups to describe their ongoing projects and challenges, identify common infrastructure and research needs, and outline an implementation framework for investigating and introducing similar programs elsewhere. Chief among the challenges were limited evidence and consensus on which genomic variants were medically relevant; lack of reimbursement for genomically driven interventions; and burden to patients and clinicians of assaying, reporting, intervening, and following up genomic findings. Key infrastructure needs included an openly accessible knowledge base capturing sequence variants and their phenotypic associations and a framework for defining and cataloging clinically actionable variants. Multiple institutions are actively engaged in using genomic information in clinical care. Much of this work is being done in isolation and would benefit from more structured collaboration and sharing of best practices.Genet Med 2013:15(4):258–267

Novel mechanism for age-related macular degeneration : An equilibrium shift between the angiogenesis factors VEGF and PEDF

We investigated gene expression profiles of vascular endothelial growth factor (VEGF) and pigment epithelium‐derived factor (PEDF) in differentiated and non‐differentiated retinal pigment epithelial (RPE) cells during oxidative stress. Human RPE cells were grown in culture on laminin‐coated flasks to obtain differentiated features. Cells cultured on plastic were used as non‐differentiated controls. After confluence, hydrogen peroxide (H2O2) was added for 48 h, then, total RNA was extracted and used for RT‐PCR and Northern blot analysis. Medium conditioned by RPE was used for ELISA, Western blotting, and in vitro angiogenesis assay. As a result, differentiated RPE cells expressed significantly higher levels of VEGF protein, as compared to their non‐differentiated counterparts. The expression pattern remained consistent even after cellular exposure to H2O2. Conversely, while elevated levels of PEDF transcript and protein were seen in differentiated RPE cells, compared to non‐differentiated cells, a marked decrease at both PEDF mRNA and protein levels was seen after treatment with H2O2. Moreover, this decrease in PEDF expression was dosage dependent. In in vitro angiogenesis assay, conditioned medium from differentiated human RPE cells after exposure to H2O2 showed a dramatic increase in tubular formation and migratory activity of microvascular endothelial cells. These data suggest that, in physiological conditions, a critical balance between PEDF and VEGF exists, and PEDF may counteract the angiogenic potential of VEGF. Under oxidative stress, PEDF decreases disrupting this balance. This equilibrium shift may be significant in promoting a pathological condition of RPE cells and contributing to choroidal neovascularization in age‐related macular degeneration.

Pigment epithelium-derived factor protects cultured retinal neurons against hydrogen peroxide-induced cell death

Pigment epithelium‐derived factor (PEDF) is a neurotrophic protein synthesized and secreted by retinal pigment epithelial (RPE) cells in early embryogenesis and has been shown to be present in the extracellular matrix between the RPE cells and the neural retina. It induces neuronal differentiation and promotes survival of neurons of the central nervous system from degeneration caused by serum withdrawal or glutamate cytotoxicity. Because the role of PEDF in the retina is still unknown, we examined its ability to protect cultured retinal neurons against hydrogen peroxide (H2O2)‐induced cell death. Retinas of 0–2‐day‐old Sprague‐Dawley rats were isolated and dissociated, and the neurons were maintained for 2 weeks in a synthetic serum‐free medium. Immunocytochemical labeling showed that 50–60% of the cultured cells were rod photoreceptors. Treatment with H2O2 induced significant death of retinal neurons in a dose‐ and time‐dependent manner. Pretreatment with PEDF prior to insult greatly attenuated H2O2‐induced cytotoxicity, and its effect was shown to be dose dependent. Cytotoxicity was determined by 3,(4,5‐dimethylthiazol‐2‐yl)2,5‐diphenyl‐tetrazolium bromide and lactate dehydrogenase assays, and apoptotic cell death was evaluated by the TdT‐mediated digoxigenin‐dUTP nick‐end labeling assay. The present study also showed that H2O2‐induced retinal neuron death was by apoptosis that could be inhibited by PEDF. Combination of PEDF with basic fibroblast growth factor, brain‐derived neurotrophic factor, or ciliary neurotrophic factor improves the protection. These data strongly suggest that PEDF is a potential neuroprotective agent in the treatment of retinal degeneration. J. Neurosci. Res. 57:789–800, 1999.

SLC6A4 variation and citalopram response.

The influence of genetic variations in SLC6A4 (serotonin transporter gene) on citalopram treatment of depression using the Sequenced Treatment to Relieve Depression (STAR*D) sample was assessed. Of primary interest were three previously studied polymorphisms: 1) the VNTR variation of the second intron, 2) the indel promoter polymorphism (5HTTLPR or SERT), and 3) a single nucleotide polymorphism (SNP) rs25531. Additionally, SLC6A4 was resequenced to identify new SNPs for exploratory analyses. DNA from 1914 subjects in the STAR*D study were genotyped for the intron 2 VNTR region, the indel promoter polymorphism, and rs25531. Associations of these variants with remission of depressive symptoms were evaluated following citalopram treatment. In white non‐Hispanic subjects, variations in the intron 2 VNTR (point‐wise P = 0.041) and the indel promoter polymorphism (point‐wise P = 0.039) were associated with remission following treatment with citalopram. The haplotype composed of the three candidate loci was also associated with remission, with a global p‐value of 0.040 and a maximum statistic simulation p‐value of 0.0031 for the S‐a‐12 haplotype, under a dominant model. One SNP identified through re‐sequencing the SLC6A4 gene, Intron7‐83‐TC, showed point‐wise evidence of association, which did not remain significant after correction for the number of SNPs evaluated in this exploratory analysis. No associations between these SLC6A4 variations and remission were found in the white Hispanic or black subjects. These findings suggest that multiple variations in the SLC6A4 gene are associated with remission in white non‐Hispanic depressed adults treated with citalopram. The mechanism of action of these variants remains to be determined.

Glycine and a glycine dehydrogenase (GLDC) SNP as citalopram/escitalopram response biomarkers in depression: pharmacometabolomics-informed pharmacogenomics.

Major depressive disorder (MDD) is a common psychiatric disease. Selective serotonin reuptake inhibitors (SSRIs) are an important class of drugs used in the treatment of MDD. However, many patients do not respond adequately to SSRI therapy. We used a pharmacometabolomics‐informed pharmacogenomic research strategy to identify citalopram/escitalopram treatment outcome biomarkers. Metabolomic assay of plasma samples from 20 escitalopram remitters and 20 nonremitters showed that glycine was negatively associated with treatment outcome (P = 0.0054). This observation was pursued by genotyping tag single‐nucleotide polymorphisms (SNPs) for genes encoding glycine synthesis and degradation enzymes, using 529 DNA samples from SSRI‐treated MDD patients. The rs10975641 SNP in the glycine dehydrogenase (GLDC) gene was associated with treatment outcome phenotypes. Genotyping for rs10975641 was carried out in 1,245 MDD patients in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study, and its presence was significant (P = 0.02) in DNA taken from these patients. These results highlight a possible role for glycine in SSRI response and illustrate the use of pharmacometabolomics to “inform” pharmacogenomics.

De novo genesis of neuropsychiatric symptoms in Mild Cognitive Impairment (MCI)

BACKGROUND There is inadequate information regarding the neuropsychiatric aspect of Mild Cognitive Impairment (MCI). OBJECTIVE To determine the neuropsychiatric profile of MCI, and compare this with normal controls and patients with mild Alzheimers Disease (AD). DESIGN Cross-sectional assessment of psychiatric symptoms in subjects that are enrolled in Mayo Clinics longitudinal study of normal aging, MCI and dementia. METHODS AND PARTICIPANTS The Neuropsychiatric Inventory (NPI) was administered to normal control subjects, MCI subjects and patients with early AD. Individual NPI domain scores and total NPI scores were compared among the three groups after controlling for age, educational status, Dementia Rating Scale (DRS) and Mini-Mental State Examination (MMSE) scores. Statistical analysis was performed by utilizing ANOVA, chi2 and Fishers exact test. RESULTS Data were analyzed on 514 normal controls, 54 MCI subjects, and 87 subjects with mild AD (CDR of 0.5 or 1); females consisted of 60.3%, 53.7% and 57.5%; and, the average ages (SD) were 77.8 (1.95), 79 (4.6), 80.5 (14.6) respectively. ANOVA pair-wise comparison revealed that both MMSE and DRS differences among the three groups were significantly different at (p = 0.05). The total NPI scores were significantly different (p =0.0001, F = 107.93) among the three groups using ANOVA. Pair-wise comparison of individual behavioral domain of NPI showed statistically significant differences between MCI and normals; and MCI and AD (p = 0.001). Group differences on NPI remained after controlling for age and education at p = 0.0375 and p = 0.0050 respectively. CONCLUSION The neuropsychiatric pattern is reminiscent of the clinical, neuroimaging and neuropsychological profile of MCI. It gives further credence to the view that MCI is indeed the gray zone, with overlap on both ends of the pole.

Pigment epithelium derived factor as a neuroprotective agent against ischemic retinal injury.

Purpose. Pigment epithelium-derived factor (PEDF) is a protein shown to have neurotrophic activity. The purpose of this study was to determine whether PEDF is neuroprotective of retinal neurons that are exposed to transient ischemia-reperfusion. Methods. Transient retinal ischemia was produced by increasing the intraocular pressure for 45 min in albino rats eyes. Immediately after reperfusion, PEDF was injected intravitreally into the experimental eyes. Injury was evaluated morphologically and by measuring the thickness of the inner retinal layers (IRL) and by counting the number of retinal ganglion cells (RGC) in epon embedded sections. Results. Morphologic and morphometric analysis of the thickness of the IRL and the counting of RGC demonstrated that PEDF injected immediately after reperfusion protected the eyes partially but significantly from the ischemic injury. Cocnlusions. Intravitreal injection of PEDF even after the ischemia can ameliorate retinal injury. PEDF may be useful in preventing neuronal degeneration in the inner retina resulting from ischemia.

Prediction of early-onset asthma in genetically at-risk children.

The W.T. Grant Foundation Asthma Risk Study was designed to prospectively examine children who were considered at a genetically increased risk for the development of asthma. The respective contributions of 11 potential risk factors, both environmental and biological, were assessed in order to determine their relative roles in affecting the early onset of asthma. This is a report of an inception cohort of children born to asthmatic mothers and followed for a 3‐year period. All 150 families were recruited from the general community and living within 2 h of the National Jewish Center for Immunology and Respiratory Medicine (Denver, CO). Mothers in the index risk sample had been previously diagnosed with asthma and were recruited during their pregnancy through physician referrals and media solicitation. The index sample of 150 families was 92% Caucasian and predominantly middle class.

Upregulation of pigment epithelium-derived factor after laser photocoagulation

PURPOSE To determine the changes in the expression of pigment epithelium-derived factor in cultured human retinal pigment epithelial cells and rat retinas after laser photocoagulation. METHODS Experimental study of laser photocoagulation on human retinal pigment epithelial cells in culture and on adult rats. Reverse transcription-polymerase chain reaction and semiquantitative polymerase chain reaction analysis were used. RESULTS After photocoagulation, the mRNA expression of pigment epithelium-derived factor was upregulated in human retinal pigment epithelial cells at 6 hours and then gradually decreased. Compared with controls, significantly higher levels of pigment epithelium-derived factor were observed in rat retinas from 6 to 24 hours after laser photocoagulation (P <.005), and they were still higher than before photocoagulation at 2 weeks. CONCLUSION An upregulation of pigment epithelium-derived factor in retinal pigment epithelial cells and in the retina after photocoagulation suggests that pigment epithelium-derived factor plays a role in inhibiting neovascularization by its antiangiogenic activity.

Utility of integrated pharmacogenomic testing to support the treatment of major depressive disorder in a psychiatric outpatient setting

Objective The objective was to evaluate the potential benefit of an integrated, five-gene pharmacogenomic test and interpretive report (GeneSight) for the management of psychotropic medications used to treat major depression in an outpatient psychiatric practice. Methods The open-label study was divided into two groups. In the first (unguided) group (n=113), pharmacogenomic information was not shared until all participants completed the study. In the second (guided) group (n=114), the pharmacogenomic report was provided to physicians for clinical use. Three depression ratings, the 17-item Hamilton Rating Scale for Depression (HAMD-17), the Quick Inventory of Depressive Symptomatology – Clinician Rated (QIDS-C16), and the Patient Health Questionnaire (PHQ-9), were collected at baseline, and at 2, 4, and 8 weeks. Results The guided group experienced greater percent improvement in depression scores from baseline on all three depression instruments (HAMD-17, P<0.0001; QIDS-C16, P<0.0001; PHQ-9, P<0.0001) compared with the unguided group. Eight-week response rates were higher in the guided group than in the unguided group on all three measurements (HAMD-17, P=0.03; QIDS-C16, P=0.005; PHQ-9, P=0.01). Eight-week QIDS-C16 remission rates were higher in the guided group (P=0.03). Participants in the unguided group who at baseline were prescribed a medication that was most discordant with their genotype experienced the least improvement compared with other unguided participants (HAMD-17, P=0.007). Participants in the guided group and on a baseline medication most discordant with their genotype showed the greatest improvement compared with the unguided cohort participants (HAMD-17, P=0.01). Conclusion These findings replicate previous studies and demonstrate significantly improved depression outcomes with use of GeneSight, an integrated, multigenetic pharmacogenomic testing platform.