Genome-wide association with select biomarker traits in the Framingham Heart Study

Background

Systemic biomarkers provide insights into disease pathogenesis, diagnosis, and risk stratification. Many systemic biomarker concentrations are heritable phenotypes. Genome-wide association studies (GWAS) provide mechanisms to investigate the genetic contributions to biomarker variability unconstrained by current knowledge of physiological relations.

Methods

We examined the association of Affymetrix 100K GeneChip single nucleotide polymorphisms (SNPs) to 22 systemic biomarker concentrations in 4 biological domains: inflammation/oxidative stress; natriuretic peptides; liver function; and vitamins. Related members of the Framingham Offspring cohort (n = 1012; mean age 59 ± 10 years, 51% women) had both phenotype and genotype data (minimum-maximum per phenotype n = 507–1008). We used Generalized Estimating Equations (GEE), Family Based Association Tests (FBAT) and variance components linkage to relate SNPs to multivariable-adjusted biomarker residuals. Autosomal SNPs (n = 70,987) meeting the following criteria were studied: minor allele frequency ≥ 10%, call rate ≥ 80% and Hardy-Weinberg equilibrium p ≥ 0.001.

Results

With GEE, 58 SNPs had p < 10^-6: the top SNPs were rs2494250 (p = 1.00*10^-14) and rs4128725 (p = 3.68*10^-12) for monocyte chemoattractant protein-1 (MCP1), and rs2794520 (p = 2.83*10^-8) and rs2808629 (p = 3.19*10^-8) for C-reactive protein (CRP) averaged from 3 examinations (over about 20 years). With FBAT, 11 SNPs had p < 10^-6: the top SNPs were the same for MCP1 (rs4128725, p = 3.28*10^-8, and rs2494250, p = 3.55*10^-8), and also included B-type natriuretic peptide (rs437021, p = 1.01*10^-6) and Vitamin K percent undercarboxylated osteocalcin (rs2052028, p = 1.07*10^-6). The peak LOD (logarithm of the odds) scores were for MCP1 (4.38, chromosome 1) and CRP (3.28, chromosome 1; previously described) concentrations; of note the 1.5 support interval included the MCP1 and CRP SNPs reported above (GEE model). Previous candidate SNP associations with circulating CRP concentrations were replicated at p < 0.05; the SNPs rs2794520 and rs2808629 are in linkage disequilibrium with previously reported SNPs. GEE, FBAT and linkage results are posted at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007.

Conclusion

The Framingham GWAS represents a resource to describe potentially novel genetic influences on systemic biomarker variability. The newly described associations will need to be replicated in other studies.

     

Storage of heat-fixed unstained sputum AFB smears for panel testing in a tuberculosis unit in South India.

To evaluate the suitability for panel testing of heat-fixed unstained sputum AFB smears stored for up to 10 months, panels of slides were prepared at the national laboratory and stored under ambient conditions. Every month, three slides were utilised for panel testing in each of 12 microscopy centres; 70 smears were checked in a blinded fashion after 10 months. Reading errors occurred in 15/360 slides used in panel testing and in 4/70 slides used in blinded checking. The quality and grading of heat-fixed unstained smears were unaffected for up to 10 months and were found suitable for panel testing.     

Accelerating the return to work (RTW) chances of coronary heart disease (CHD) patients: part 1--development and validation of a training programme

PURPOSE: Conventional phase II cardiac rehabilitation (CR) programmes have not resulted in an improvement in returning coronary heart disease (CHD) patients to work in over 35 years. This 4 year field-initiated research, sponsored by the National Institute on Disability and Rehabilitation Research, compares conventional CR programmes with a low-intensity CR programme that simulates elements of work (job-simulated CR programme) in terms of return to work (RTW) and physiological conditioning. The effect of training on physical capabilities of patients participating in the job-simulated CR programme was also of equal interest. METHOD: Thirty patients (15 bypass and 15 angioplasty; 15 males and 15 females) participated in a conventional CR programme (control group). The job-simulated CR programme included 15 male and 2 female bypass and angioplasty patients (experimental group). Patients in the control group underwent regular aerobic exercise training (treadmill and bicycle). Experimental group patients participated in a series of low-intensity exercises such as progressive time exercises, flexibility exercises, and dexterity exercises. RESULTS: All patients participating in the low-intensity job-simulated CR programme returned to the same job they held at the onset of myocardial infarction (MI). In contrast, only 60% of the control group patients returned to work; at least one-third of these did not go back to the same job they held at the onset of M1. Patients in both groups achieved the same level of physiological conditioning. The physical functional capabilities of the experimental group patients improved significantly throughout training. CONCLUSION: The results of this field-study lead to the conclusion that a low-intensity phase II cardiac rehabilitation programme that simulates elements of work may be far superior to conventional endurance exercise-based cardiac rehabilitation programmes in terms of returning patients to work. Such a programme also strengthens patients, improving their physical capabilities, without compromising their physiological conditioning.     

Cytosolic reducing power preserves glutamate in retina

Glutamate in neurons is an important excitatory neurotransmitter, but it also is a key metabolite. We investigated how glutamate in a neural tissue is protected from catabolism. Flux analysis using ¹³C-labeled fuels revealed that retinas use activities of the malate aspartate shuttle to protect >98% of their glutamate from oxidation in mitochondria. Isolation of glutamate from the oxidative pathway relies on cytosolic NADH/NAD⁺, which is influenced by extracellular glucose, lactate, and pyruvate.Glutamate is especially important as a metabolite because it is required for the synthesis of glutathione, other amino acids, and proteins. Glutamate also is a key intermediate in glutamine-dependent anaplerosis, now known to be a principal source of citric acid cycle intermediates in cancer cells (1).When it is released as a neurotransmitter at brain synapses, glutamate that escapes from the synapse is taken up by astrocytes. There it is converted to glutamine and is delivered back to neurons in a process called the “glutamate/glutamine cycle” (2). Uptake of glutamate and conversion to glutamine within astrocytes stimulates glycolysis and synthesis of lactate. Astrocytes export the lactate to neurons as fuel in a process called the “astrocyte neuron lactate shuttle” (ANLS) (3).Synaptic terminals of rod and cone photoreceptors have characteristics that appear incompatible with the ANLS. The photoreceptor terminal is enriched with transporters for reuptake of glutamate (4), and it encapsulates the synapse. It is unlikely that much glutamate can escape the synapse before being sequestered back into the photoreceptor. We initiated a study to evaluate the role of ANLS in retina. However, the unusual metabolic features of retina revealed a surprising feature of neuronal metabolism, that >98% of glutamate is protected from catabolism. We investigated this protection and show here that the protection is provided by activities associated with the metabolic pathway known as the “malate aspartate shuttle” (MAS) (shown schematically in Fig. 1).Open in a separate windowFig. 1.How the malate aspartate shuttle isolates glutamate. The glutamate/α-ketoglutarate cycle in retina isolates the carbon atoms of glutamate from the oxidative pathway inside mitochondria. In this report we demonstrate the influence that cytosolic reducing power and Aralar/AGC1 activity have on this pathway. ASP, aspartate; OAA, oxaloacetate; MAL, malate; GLU, glutamate; AKG, α-ketoglutarate; OGC, oxoglutarate carrier, IPM, interphotoreceptor matrix.MAS activity regenerates cytosolic NAD⁺ that is needed to support glycolysis. To do so, it uses two important transporters to trap the reducing power from cytosolic NADH and shuttle it into the mitochondrial matrix. One transporter is the neuronal aspartate/glutamate carrier (AGC1 or Aralar) (Fig. 1, orange circle); the other transporter is the oxoglutarate carrier (OGC) (Fig. 1, light blue circle). AGC1 transports glutamate from the cytoplasm into the mitochondrial matrix in exchange for aspartate from the matrix (Fig. 1). OGC transports α-ketoglutarate from the matrix into the cytoplasm in exchange for malate from the cytoplasm (Fig. 1) (5). An important consequence of MAS activity is that it diverts metabolic flux in mitochondria away from succinyl CoA, succinate, and fumarate (Fig. 1). Most importantly, glutamate that completes a MAS cycle functions as a catalyst for the importation of reducing power into the mitochondria. The carbon atoms of glutamate are isolated from the oxidative pathway in the mitochondrial matrix. To determine the extent of that isolation in a neuronal tissue, we used ¹³C-labeled fuels to identify metabolic networks in mouse retinas and quantify their metabolic flux.     

Mycoses in India—study in Madras

The incidence of mycoses is high in Madras accounting for over 13% of dermatoses diagnosed in a three-year period. Dermatophytoses (tinea corporis and tinea cruris) and pityriasis versicolor were most common in May and October. A correlation was observed between these infections and environmental temperature, humidity and rainfall. Most cases of all dermatophytoses except tinea capitis were recorded between 10 and 30 years of age but the latter was most common between one and 10 years. Males were predominantly affected with all except tinea axillaris, candidiasis and piedra. The incidence of piedra and deep mycoses was low.     

Multiple High-Energy Open Injuries in the Same Limb: Comparison of Outcomes Between Salvage and Amputation Groups

AimTo analyze the functional outcomes between limb salvage and amputation patients who had multiple open injuries in the same lower limb.Materials and MethodsThis observational study analyzed 21 patients who were admitted with multiple open injuries in the same lower limb between January 2012 and December 2015 in our unit. Twelve patients underwent limb salvage and nine patients underwent amputation. The total number of surgeries, duration of hospital stays, ICU admission, complications, time to return to work and costs of inpatient treatment were analyzed. The functional outcome was assessed by using the lower extremity functional scale (LEFS) in both groups, SF-12 score was done for both groups and amputation specific scoring was done by using locomotors capabilities index (LCI).ResultsThe LEFS was lower in salvage group than amputation group. The SF-12 score was close to normal population in the amputation group and was higher than salvage group. The duration of hospital stays, total number of surgeries and the costs of inpatient admission were higher in salvage group. The time to return to work was earlier in amputation group. Sixty-seven percentage of patients in the salvage group developed complications.ConclusionThe functional outcome and SF-12 score was better in amputation group. Patients who had amputation returned to work earlier, had smaller number of secondary hospitalization and has less complications and incurred less expenditure for treatment. The treatment decision should be periodically reviewed when an initial choice of salvage is made. Amputation must be looked at as a treatment for early rehabilitation.     

Changes in Magnetic Resonance Imaging and Sex Behavior After 6-OHDA Injection in the Medial Preoptic Area

Magnetic resonance imaging (MRI) of the brains of male rats was done before and after destroying the catecholamine (CA) fibers by local application of 6-hydroxydopamine (6-OHDA) in the medial preoptic area (mPOA). The male sexual behavior was also assessed before and after injection of this toxic drug. The administration of 6-OHDA (8 μg) resulted in highly variable lesions, as shown by MRI and confirmed by histological examination. A hyperintense area was visible either on one or on both sides, about 1–3 h after the administration of the drug. Postmortem histofluorescence showed destruction of CA fibers in the mPOA on those sides that showed hyperintense areas in the MRI. No CA fiber destruction was seen in those rats that had shown no change in MRI after 6-OHDA injection. There was a transient reduction in sex drive score in all the 6-OHDA-treated rats. The present findings point out a correlation between the MRI changes and CA fiber destruction, whereas the transient reduction in the sexual behavior was not related to these changes. It is suggested that some biochemical events related to 6-OHDA destruction of CA fibers may have been responsible for the hyperintensity seen in the MRI.     

Evidence for frequency-dependent arterial damage in vibrated rat tails

The effects of single 4-hr bouts of continuous 30, 60, 120, and 800 Hz tail vibration (49 m/sec2, root mean squared) were compared to assess frequency-amplitude-related structural damage of the ventral caudal artery. Amplitudes were 3.9, 0.98, 0.24, and 0.0055 mm, respectively. Vibrated, sham-vibrated, and normal arteries were processed for light and electron microscopy. The Curry rat tail model of hand-arm vibration (Curry et al. Muscle Nerve 2002;25:527-534) proved well-suited for testing multiple frequencies. NFATc3 immunostaining, an early marker of cell damage, increased in smooth muscle and endothelial cells after 30, 60, and 120 Hz but not 800 Hz. Increased vacuolization, which is indicative of smooth muscle contraction, occurred for all frequencies except 800 Hz. Vacuoles increased in both endothelial and smooth muscle cells after 60 and 120 Hz. Only 30 Hz showed pronounced smooth muscle cell vacuolization along the internal and external elastic membranes, suggesting stretch-mediated contraction from the large amplitude shear stress. Discontinuities in toluidine blue staining of the internal elastic membrane (IEM) increased for all frequencies, indicating vibration-induced structural weakening of this structure. Patches of missing IEM and overlying endothelium occurred in approximately 5% of arteries after 60, 120, and 800 Hz. The pattern of damage after 800 Hz suggests that the IEM is disrupted because it resonates at this frequency. Vibration acceleration stress and smooth muscle contraction appear to be the major contributors to arterial damage. The pattern of vibration-induced arterial damage of smooth muscle and endothelial cells is frequency-amplitude-dependent.