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One of the most abundant flavonoids present in cacao is (-)-epicatechin (Epi) and this flavanol has been linked to the cardiovascular health promoting actions of cocoa products. We previously demonstrated that Epi reduces infarct size in rodent models of ischemia/reperfusion and permanent coronary occlusion. Reduced infarct size was associated with decreased left ventricular (LV) oxidative stress (OS) and indicators of inflammation factors, which foster myocardial fibrosis. In this study, we examine the antifibrotic actions of Epi in an aging female rat model of pre-heart failure with preserved ejection fraction (pre-HFpEF) as well as its potential to mitigate plasma levels of OS, proinflammatory/profibrotic cytokines, and improve passive and active LV function. Epi treatment [1 mg/(kg·d)] was provided daily by gavage from 21 to 22 months of age, whereas controls received water. A Millar catheter was used to assess hemodynamic function. Subsequently, hearts were arrested in diastole, a balloon inserted into the LV and passive pressure-volume curves generated. Fixed LV sections were processed for collagen area fraction quantification using Sirius Red staining. Treatment with Epi did not lead to detectable changes in LV contractile function. However, passive LV pressure volume curves were significantly right shifted with Epi. Collagen area fraction values indicated that Epi treatment significantly reduces LV fibrosis. Epi also significantly reduced plasma OS markers and levels of profibrotic and proinflammatory cytokines. In conclusion, Epi reduces cardiac fibrosis in an aged, female rat model of pre-HFpEF, which correlates with significant reductions in OS and cytokine levels in the absence of changes in LV contractile function.

The identification of clinically significant genes recurrently mutated in myeloid malignancies necessitates expanding diagnostic testing with higher throughput, such as targeted next-generation sequencing. We present validation of the Thermo Fisher Oncomine Myeloid Next-Generation Sequencing Panel (OMP), targeting 40 genes and 29 fusion drivers recurrently mutated in myeloid malignancies. The study includes data from a sample exchange between two Canadian hospitals demonstrating high concordance for detection of DNA and RNA aberrations. Clinical validation demonstrates high accuracy, sensitivity, and specificity of the OMP, with a lower limit of detection of 5% for single-nucleotide variants and 10% for insertions/deletions. Prospective sequencing was performed for 187 samples from 168 unique patients presenting with suspected or confirmed myeloid malignancy and other hematological conditions to assess clinical impact of identifying variants. Of detected variants, 48% facilitated or clarified diagnoses, 29% affected prognoses, and 25% had the potential to influence clinical management. Of note, OMP was essential to identifying patients with premalignant clonal states likely contributing to cytopenias. We also found that the detection of even a single variant by the OMP assay, versus 0 variants, was predictive of overall survival, independent of age, sex, or diagnosis (P = 0.03). This study demonstrates that molecular profiling of myeloid malignancies with the OMP represents a promising strategy to advance molecular diagnostics.

Formyl peptide receptor 2 (FPR2) plays an integral role in the transition of macrophages from a pro-inflammatory program to one that is pro-resolving. FPR2-mediated stimulation of resolution post myocardial infarction has demonstrated efficacy in rodent models and is hypothesized to reduce progression into heart failure. FPR2 agonists that promote long-lasting receptor internalization can lead to persistent desensitization and diminished therapeutic benefits. In vitro signaling profiles and propensities for receptor desensitization of two clinically studied FPR2 agonists, namely, BMS-986235 and ACT-389949, were evaluated. In contrast to BMS-986235, pre-stimulation with ACT-389949 led to a decrease in its potency to inhibit cAMP production. Moreover, ACT-389949 displayed greater efficacy for β-arrestin recruitment, while efficacy of Gi activation was similar for both agonists. Following agonist-promoted FPR2 internalization, effective recycling to the plasma membrane was observed only with BMS-986235. Use of G protein-coupled receptor kinase (GRK) knock-out cells revealed a differential impact of GRK2 versus GRK5/6 on β-arrestin recruitment and Gi activation promoted by the two FPR2 agonists. In vivo, decreases of granulocytes in circulation were greatly diminished in mice treated with ACT-389949 but not for BMS-986235. With short-term dosing, both compounds induced a pro-resolution polarization state in cardiac monocyte/macrophages post myocardial infarction. By contrast, with long-term dosing, only BMS-986235 preserved the infarct wall thickness and increased left ventricular ejection fraction in a rat model of myocardial infarction. Altogether, the study shows that differences in the desensitization profiles induced by ACT-389949 and BMS-986235 at the molecular level may explain their distinct inflammatory/pro-resolving activities in vivo.

Background: The incidence of cardiovascular disease is higher in HIV-positive (HIV + ) patients than it is in the average population, and combination antiretroviral therapy (cART) is a recognized risk factor for cardiovascular disease. However, the molecular mechanisms that link cART and cardiovascular disease are currently unknown. Our study explores the role of the activation of p90RSK, a reactive oxygen species-sensitive kinase, in engendering senescent phenotype in macrophages and accelerating atherogenesis in patients undergoing cART. Methods: Peripheral whole blood from cART-treated HIV + individuals and nontreated HIV-negative individuals was treated with H 2 O 2 (200 µmol/L) for 4 minutes, and p90RSK activity in CD14 + monocytes was measured. Plaque formation in the carotids was also analyzed in these individuals. Macrophage senescence was determined by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. The involvement of p90RSK-NRF2 signaling in cART-induced senescence was assessed by p90RSK-specific inhibitor (FMK-MEA) or dominant-negative p90RSK (DN-p90RSK) and NRF2 activator (NRF2A). Further, the severity of atherosclerosis was determined in myeloid cell-specific wild-type and DN-p90RSK transgenic mice. Results: Monocytes from HIV + patients exhibited higher levels of p90RSK activity and were also more sensitive to reactive oxygen species than monocytes from HIV-negative individuals. A multiple linear regression analysis involving cART, Reynolds cardiovascular risk score, and basal p90RSK activity revealed that cART and basal p90RSK activity were the 2 significant determinants of plaque formation. Many of the antiretroviral drugs individually activated p90RSK, which simultaneously triggered all components of the macrophage senescent phenotype. cART inhibited antioxidant response element reporter activity via ERK5 S496 phosphorylation. NRF2A reversed the H 2 O 2 -induced overactivation of p90RSK in cART-treated macrophages by countering the induction of senescent phenotype. Last, the data obtained from our gain- or loss-of-function mice conclusively showed the crucial role of p90RSK in inducing senescent phenotype in macrophages and atherogenesis. Conclusions: cART increased monocyte/macrophage sensitivity to reactive oxygen species- in HIV + individuals by suppressing NRF2-ARE activity via p90RSK-mediated ERK5 S496 phosphorylation, which coordinately elicited senescent phenotypes and proinflammatory responses. As such, our report underscores the importance of p90RSK regulation in monocytes/macrophages as a viable biomarker and therapeutic target for preventing cardiovascular disease, especially in HIV + patients treated with cART.

Background Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy (LVH) in the absence of predisposing cardiovascular conditions. Pathogenic variants in at least 16 cardiac sarcomeric genes have been implicated in HCM, most of which act in a dominant‐negative fashion. However loss‐of‐function (haploinsufficiency) is the most common disease mechanism for pathogenic variants in MYBPC3, suggesting that MYBPC3 complete deletion may play a role in HCM pathogenesis. Here, we investigate MYBPC3 complete deletion as a disease mechanism in HCM by analyzing two unrelated patients with confirmed diagnosis of HCM that tested negative by Sanger sequencing analysis. Methods MYBPC3 complete deletion was investigated by Multiplex ligation‐dependent probe amplification (MLPA) and microarray analyses. The mechanism of deletion was investigated by interrogating the SINEBase database. Results Patient‐1 was diagnosed with nonobstructive HCM in his mid‐40s while undergoing assessment for palpitations, and patient‐2 with obstructive HCM in his late‐20s while undergoing systolic heart murmur assessment for an unrelated illness. MLPA testing revealed a heterozygous deletion of all MYBPC3 exons in both patients. Subsequent microarray testing confirmed these deletions which extended beyond the 5′ and 3′ ends of MYBPC3. Genomic assessment suggested that these deletions resulted from Alu/Alu‐homologous recombination. Conclusion Our results demonstrate that haploinsufficiency resulting from MYBPC3 complete deletion, potentially mediated by Alu recombination, is an important disease mechanism in cardiomyopathy and emphasizes the importance of copy number variation analysis in patients clinically suspected of HCM., We report on the complete deletion of MYBPC3, potentially mediated through Alu recombination, in two patients with confirmed diagnosis of hypertrophic cardiomyopathy (HCM). Our result demonstrates for the first time that MYBPC3 complete deletion is an important disease mechanism in cardiomyopathy and emphasizes the importance of copy number variation analysis in patients clinically suspected of HCM.

Background A homozygous loss-of-function mutation p.(Arg316Gln) in the fat mass and obesity-associated ( FTO ) gene, which encodes for an iron and 2-oxoglutarate-dependent oxygenase, was previously identified in a large family in which nine affected individuals present with a lethal syndrome characterised by growth retardation and multiple malformations. To date, no other pathogenic mutation in FTO has been identified as a cause of multiple congenital malformations. Methods We investigated a 21-month-old girl who presented distinctive facial features, failure to thrive, global developmental delay, left ventricular cardiac hypertrophy, reduced vision and bilateral hearing loss. We performed targeted next-generation sequencing of 4813 clinically relevant genes in the patient and her parents. Results We identified a novel FTO homozygous missense mutation (c.956C>T; p.(Ser319Phe)) in the affected individual. This mutation affects a highly conserved residue located in the same functional domain as the previously characterised mutation p.(Arg316Gln). Biochemical studies reveal that p.(Ser319Phe) FTO has reduced 2-oxoglutarate turnover and N -methyl-nucleoside demethylase activity. Conclusion Our findings are consistent with previous reports that homozygous mutations in FTO can lead to rare growth retardation and developmental delay syndrome, and further support the proposal that FTO plays an important role in early development of human central nervous and cardiovascular systems.

We present the investigation and management of a premature, hypotensive neonate born after a pregnancy complicated by anhydramnios to highlight the impact of early and informed management for rare kidney disease. Vasopressin was used to successfully treat refractory hypotension and anuria in the neonate born at 27 weeks of gestation. Next generation sequencing of a targeted panel of genes was then performed in the neonate and parents. Subsequently, two compound heterozygous deletions leading to frameshift mutations were identified in the angiotensin 1-converting enzyme gene ACE; exon 5:c.820_821delAG (p.Arg274Glyfs*117) and exon24: c.3521delG (p.Gly1174Alafs*12), consistent with a diagnosis of renal tubular dysgenesis. In light of the molecular diagnosis, identification, and treatment of associated low aldosterone level resulted in further improvement in renal function and only mild residual chronic renal failure is present at 14 months of age. Truncating alterations in ACE most often result in fetal demise during gestation or in the first days of life and typically as a result of the Potter sequence. The premature delivery, and serendipitous early treatment with vasopressin, and then later fludrocortisone, resulted in an optimal outcome in an otherwise lethal condition.

Individuals infected with the human immunodeficiency virus (HIV) are prone to develop cardiovascular disease (CVD) because the HIV infection and combination antiretroviral therapy (cART) produces atherosclerosis. Clinicians need sensitive tests to monitor interventions designed to prevent CVD in patients with HIV on long term cART. We hypothesize that vascular elastography can fulfill this role. To corroborate this hypothesis, we performed vascular elastography on 60 volunteers (mean age of 55.5 y), which we divided into four groups: (a) HIV positive with CVD (HIV++), (b) HIV positive without CVD (HIV+−), (c) HIV negative with CVD (HIV-+), and (c) HIV negative without CVD (HIV−−).

PurposeThe purpose of this study was to develop a national program for Canadian diagnostic laboratories to compare DNA-variant interpretations and resolve discordant-variant classifications using the BRCA1 and BRCA2 genes as a case study.MethodsBRCA1 and BRCA2 variant data were uploaded and shared through the Canadian Open Genetics Repository (COGR; http://www.opengenetics.ca). A total of 5,554 variant observations were submitted; classification differences were identified and comparison reports were sent to participating laboratories. Each site had the opportunity to reclassify variants. The data were analyzed before and after the comparison report process to track concordant- or discordant-variant classifications by three different models.ResultsVariant-discordance rates varied by classification model: 38.9% of variants were discordant when using a five-tier model, 26.7% with a three-tier model, and 5.0% with a two-tier model. After the comparison report process, the proportion of discordant variants dropped to 30.7% with the five-tier model, to 14.2% with the three-tier model, and to 0.9% using the two-tier model.ConclusionWe present a Canadian interinstitutional quality improvement program for DNA-variant interpretations. Sharing of variant knowledge by clinical diagnostic laboratories will allow clinicians and patients to make more informed decisions and lead to better patient outcomes.

It is difficult to produce reliable polar strain elastograms (radial and circumferential) because the center of carotid artery is typically unknown. Principal strain imaging can overcome this limitation, but suboptimal lateral displacement estimates measured with conventional ultrasonic imaging methods make this an impractical approach for visualizing the mechanical properties within the carotid artery. We hypothesize that compounded plane wave imaging can minimize this problem. To corroborate this hypothesis, we performed (a) simulations with vessels of varying morphology and mechanical behavior (i.e., isotropic and transversely isotropic), and (b) a pilot study with 10 healthy volunteers. The accuracy of principal and polar strain (computed using knowledge of the precise vessel center) elastograms varied between 7–17%. In both types of elastograms, strain concentrated at the junction between the fibrous cap and the vessel wall, and the strain magnitude decreased with increasing fibrous cap thickness. Elastograms obtained from healthy volunteers were consistent with those obtained from transversely isotropic homogeneous vessels; they were spatially asymmetric–a trend that was common to both principal and polar strains. No significant differences were observed in the mean strain recovered from principal and polar strains (p > 0.05). This investigation demonstrates that principal strain elastograms measured with compounding plane wave imaging overcame problems incurred when polar strain elastograms are computed with imprecise estimates of the vessel center.

OBJECTIVE. The purpose of this article is to review scrotal and penile anatomy, the role of ultrasound in evaluating scrotal and penile trauma, and the vast spectrum of sonographic manifestations of scrotal and penile trauma. CONCLUSION. Scrotal and penile trauma is an uncommon type of trauma injury. However, knowledge of scrotal and penile anatomy and the appropriate imaging findings associated with acute traumatic injuries is important in establishing the correct diagnosis. Sonography is considered the first choice of imaging modalities in establishing a diagnosis and triaging patients into surgical and nonsurgical treatment.

Objectives/Hypothesis: Congenital deafness occurs in approximately 1 in 1,000 live births, and 50% of these cases are hereditary. Connexin mutations have been identified as the most common cause of hereditary hearing loss in many populations. The prevalence of this mutation in African patients has not been adequately studied. The objective of this study was to determine the prevalence of connexin 26 and 30 mutations in a population of hearing-impaired patients from Uganda. Study Design: This is an observational study. Methods: Coding regions of both GJB2 and GJB6, noncoding exon 1 of GJB2, and 30 nucleotides of intronic sequence bordering the exons were analyzed in 126 subjects from Uganda with confirmed bilateral, severe-to-profound sensorineural hearing loss. All variants were analyzed for possible clinical significance using a combination of database searches and in silico tools. Results: Complete sequence data were obtained on 115/126 individuals; 11 had only partial or no results. Only one reported pathogenic variant was found in GJB2 (c.208C>G; p.Pro70Ala) and none in GJB6. Three reported variants and two novel variants within intron 1 of GJB2 and two variants within exon 3 of GJB6 were also found. Conclusions: None of the most common types of deletions in the GJB2 gene (c.35delG, c.167delT or c.235delC) were found in this large cohort of deaf children from Uganda. This prompts a search for genetic causes of deafness among this and other previously studied African populations.

Stroke may occur when an atherosclerotic plaque ruptures in the carotid artery. Noninvasive vascular elastography (NIVE) visualizes the strain distribution within the carotid artery, which is related to its mechanical properties that govern plaque rupture. Strain elastograms obtained from the transverse plane of the carotid artery are difficult to interpret, because strain is estimated in Cartesian coordinates. Sparsearray (SA) elastography overcomes this problem by transforming shear and normal strain to polar coordinates. However, the SA's transmit power may be too weak to produce useful elastograms in the clinical setting. Consequently, we are exploring other imaging methods to solve this potential problem. This study evaluated the quality of elastograms produced with SA imaging, plane-wave (PW) imaging, and compounded-plane-wave (CPW) imaging. We performed studies on simulated and physical vessel phantoms, and the carotid artery of a healthy volunteer. All echo imaging was performed with a linear transducer array that contained 128 elements, operating at 5 MHz. In SA imaging, 7 elements were fired during transmission, but all 128 elements were active during reception. In PW imaging, all 128 elements were active during both transmission and reception. We created CPW images by steering the acoustic beam within the range of -15° to 15° in increments of 5°. SA radial and circumferential strain elastograms were comparable to those produced using PW and CPW imaging. Additionally, side-lobe levels incurred during SA imaging were 20 dB lower than those produced during PW imaging, and 10 dB lower than those computed using CPW imaging. Overall, SA imaging performs well in vivo; therefore, we plan to improve the technique and perform preclinical studies.

Background: Rare diseases often present in the first days and weeks of life and may require complex management in the setting of a neonatal intensive care unit (NICU). Exhaustive consultations and traditional genetic or metabolic investigations are costly and often fail to arrive at a final diagnosis when no recognizable syndrome is suspected. For this pilot project, we assessed the feasibility of next-generation sequencing as a tool to improve the diagnosis of rare diseases in newborns in the NICU. Methods: We retrospectively identified and prospectively recruited newborns and infants admitted to the NICU of the Children’s Hospital of Eastern Ontario and the Ottawa Hospital, General Campus, who had been referred to the medical genetics or metabolics inpatient consult service and had features suggesting an underlying genetic or metabolic condition. DNA from the newborns and parents was enriched for a panel of clinically relevant genes and sequenced on a MiSeq sequencing platform (Illumina Inc.). The data were interpreted with a standard informatics pipeline and reported to care providers, who assessed the importance of genotype–phenotype correlations. Results: Of 20 newborns studied, 8 received a diagnosis on the basis of next-generation sequencing (diagnostic rate 40%). The diagnoses were renal tubular dysgenesis, SCN1A-related encephalopathy syndrome, myotubular myopathy, FTO deficiency syndrome, cranioectodermal dysplasia, congenital myasthenic syndrome, autosomal dominant intellectual disability syndrome type 7 and Denys–Drash syndrome. Interpretation: This pilot study highlighted the potential of next-generation sequencing to deliver molecular diagnoses rapidly with a high success rate. With broader use, this approach has the potential to alter health care delivery in the NICU.

The authors develop a new classroom experimental game to illustrate environmental issues by using the production-possibility frontier in an introductory economics course. Waste evolves as a byproduct of the production of widgets. Environmental cleanup is produced by reallocating scarce resources away from the production of the dirty good. In addition to the description of the game and classroom discussion, the authors illustrate how the students’ experience with the game can be used as the basis for exercises on production decisions and environmental regulation.

Dejerine-Sottas disease (DSD) is a particular phenotype of the Charcot-Marie-Tooth (CMT) disease spectrum that is genetically heterogeneous. It represents a severe form of hypertrophic axonal and demyelinating neuropathy. Although it is predominantly inherited as an autosomal recessive condition, autosomal dominant inheritance has also been described. To date, the autosomal recessive forms of DSD are classified into several CMT type 4 (CMT4) subclasses based on allelic heterogeneity. We present a 7-year-old boy with a severe form of CMT disease consistent with the autosomal recessive phenotype of DSD. He was found to be a compound heterozygote for mutations in the PMP22 gene resulting in homozygous deletion of exons 2 and 3. The maternally inherited allele was the typical 1.5 Mb deletion involving PMP22 seen with hereditary neuropathy with liability to pressure palsy (HNPP). The paternally inherited allele was a deletion of exons 2 and 3. Both parents presented with a typical clinical picture of HNPP. To our knowledge, this is the first patient reported with large deletions involving both PMP22 alleles. Our patient has also developed severe gastroesophageal reflux disease (GERD), a clinical feature not previously reported with CMT or DSD. The correlation of the phenotype and the molecular defects observed in this patient may set a new subcategory in the classification of DSD.

Purpose: The aim of the study was to determine the actual GJB2 and GJB6 mutation frequencies in North America after several years of generalized testing for autosomal recessive nonsyndromic sensorineural hearing loss to help guide diagnostic testing algorithms, especially in light of molecular diagnostic follow-up to universal newborn hearing screening. Methods: Mutation types, frequencies, ethnic distributions, and genotype-phenotype correlations for GJB2 and GJB6 were assessed in a very large North American cohort. Results: GJB2 variants were identified in 1796 (24.3%) of the 7401 individuals examined, with 399 (5.4%) homozygous and 429 (5.8%) compound heterozygous. GJB6 deletion testing was performed in 12.0% (888/7401) of all cases. The >300-kb deletion was identified in only nine individuals (1.0%), all of whom were compound heterozygous for mutations in GJB2 and GJB6. Among a total of 139 GJB2 variants identified, 53 (38.1%) were previously unreported, presumably representing novel pathogenic or benign variants. Conclusions: The frequency and distribution of sequence changes in GJB2 and GJB6 in North America differ from those previously reported, suggesting a considerable role for loci other than GJB2 and GJB6 in the etiology of autosomal recessive nonsyndromic sensorineural hearing loss, with minimal prevalence of the GJB6 deletion.

Transforming growth factor–β1 (TGF-β1) and plasminogen activator inhibitor–type 1 (PAI-1) might play a role in the development of fibrosis and stenosis of hemodialysis vascular accesses. We studied polymorphisms in the TGFβ1 (869T>C; 915G>C), and PAI-1 (4G/5G) genes in 416 hemodialysis patients (107 access thrombosis cases, 309 controls), to determine if they are related to vascular access thrombosis. Three TGF-β1 production haplotypes (low, intermediate, and high) were defined according to the combination of polymorphisms found. The adjusted odds ratio (OR) and 95% confidence interval (CI) for access thrombosis in low TGF-β1 producers was 7.31 (2.15-24.88; P = .001). The interaction between low TGF-β1 production haplotype and the 4G/4G PAI-1 genotype was strongly associated with access thrombosis (adjusted OR 19.3; 95% CI 2.82-132.40; P = .003). Mean access thrombosis–free survival times in years (95% CI) were 14.65 (12.05-17.25), 11.96 (8.67-15.25), and 4.94 (3.06-6.83) in high, intermediate, and low TGF-β1 producers, respectively (P = .044). Analysis of the synergy index and the case-only cross-product supported the presence of an interaction. We concluded that low TGF-β1 production haplotype is a risk factor for hemodialysis access thrombosis and that in the presence of the 4G/4G PAI-1 genotype there is an additional increase in risk.

To determine whether carotid intima media thickness is increased in children with primary hypertension, the current study compared carotid intima media thickness in hypertensive children with that of normotensive control subjects matched closely for body mass index and determined the relationship between carotid intima media thickness and hypertension severity determined by ambulatory blood pressure monitoring. Children with newly diagnosed office hypertension (n=28) had carotid intima media thickness, left ventricular mass index, and ambulatory blood pressure monitoring performed. Carotid intima media thickness was performed in normotensive control subjects (n=28) matched pairwise to hypertensive subjects for age (±1 year), gender, and body mass index (±10%). Eighty-two percent of subjects were overweight or obese (body mass index ≥85th percentile). The median carotid intima media thickness of hypertensive subjects was greater than that of matched controls (0.67 versus 0.63 mm; P =0.045). In the hypertensive subjects, carotid intima media thickness correlated strongly with several ambulatory blood pressure monitoring parameters, with the strongest correlation for daytime systolic blood pressure index ( r =0.57; P =0.003). In the hypertensive group, the prevalence of left ventricular hypertrophy was 32%, but unlike carotid intima media thickness, left ventricular mass index did not correlate with ambulatory blood pressure monitoring. Together, the findings that hypertensive subjects had increased carotid intima media thickness compared with matched controls and that higher carotid intima media thickness correlated with more severe hypertension by ambulatory blood pressure monitoring provide strong evidence that carotid intima media thickness is increased in childhood primary hypertension, independent of the effects of obesity.

Bernard-Soulier syndrome (BSS) is a rare inherited bleeding disorder due to quantitative or qualitative abnormalities in the platelet glycoprotein (GP) Ib/IX/V complex, the major von Willebrand factor receptor. The complex comprises four subunits, each encoded by a separate gene. Several mutations have been described for each of the subunits, except for GPV, as a cause of BSS. We describe here the genetic basis of the disorder in a child with BSS. Flow-cytometric analysis of the patient’s platelets showed a markedly reduced surface expression of all three glycoproteins of the GPIb/IX/V complex. DNA sequencing analysis showed the patient to be a compound heterozygote for two mutations in the GPIX gene, a novel nine-nucleotide deletion starting at position 1952 of the gene that changes asparagine 86 for alanine and eliminates amino acids 87, 88, and 89 (arginine, threonine, and proline) and a previously reported point mutation that changes the codon asparagine (AAC) for serine (AGC) at residue 45. Her mother was heterozygous for the Asn45Ser mutation, and her father, for the nine-nucleotide deletion. Our findings suggest that the additive effects of both mutations in the GPIX gene are responsible for the BSS phenotype of the patient. Am. J. Hematol. 78:41–48, 2005. a 2004 Wiley-Liss, Inc.

We compared patient outcomes using two protocols: one routinely and the other selectively evaluating the calves completely during sonographic assessment of the lower extremities in patients with suspected deep venous thrombosis.In this randomized prospective study, patients were assigned to two groups. In one group, the deep calf veins were routinely evaluated in their entirety, and in the other group the calf was not evaluated unless the patient had symptoms or physical signs in the calf, in which case only the areas of symptoms or physical signs were evaluated. Patients were followed up for 3 months by medical record review, physician surveys, and telephone calls. An adverse outcome was a propagated deep venous thrombosis into the thigh or a pulmonary embolus. Examination times were recorded when possible.Of the 235 patients in the group in which the deep calf veins were routinely evaluated, we saw no adverse outcomes (0.0%; 97.5% one-sided confidence interval [CI], 0.6-1.6%). Of the 261 patients in the group in which the calf was only evaluated if there were signs or symptoms, we saw two adverse outcomes (0.8%; 95% CI, 0.1-2.7%).We found no significant difference in adverse outcomes in patients undergoing a protocol in which the deep calf veins were routinely evaluated or a protocol in which the calf was evaluated only if physical signs or symptoms were present.

SummaryThe role of ABO blood type as a risk factor for recurrent venous thromboembolism (VTE) in patients with a first unprovoked VTE who complete oral anticoagulation therapy is unknown. The aim of this study was to determine if non-OO blood type is a risk factor for recurrent VTE in patients with a first unprovoked VTE who completed 5–7 months of anticoagulant therapy. In an ongoing cohort study of patients with unprovoked VTE who discontinued oral anticoagulation after 5–7 months of therapy, six single nucleotide polymorphisms sites were tested to determine ABO blood type using banked DNA. The main outcome was objectively proven recurrent VTE. Mean follow-up for the cohort was 4.19 years (SD 2.16). During 1,553 patient-years of follow-up, 101 events occurred in 380 non-OO patients (6.5 events per 100 patient years; 95% CI 5.3–7.7) compared to 14 events during 560 patient years of follow-up in 129 OO patients (2.5 per 100 patient years; 95% CI 1.2–3.7), the adjusted hazard ratio was 1.98 (1.2–3.8). In conclusion, non-OO blood type is associated with a statistically significant and clinically relevant increased risk of recurrent VTE following discontinuation of anticoagulant therapy for a first episode of unprovoked VTE.

Congenital deafness occurs in approximately 1 in 1,000 live births, and 50% of these cases are hereditary. Connexin mutations have been identified as the most common cause of hereditary hearing loss in many populations. The prevalence of this mutation in African patients has not been adequately studied. The objective of this study was to determine the prevalence of connexin 26 and 30 mutations in a population of hearing-impaired patients from Uganda.This is an observational study.Coding regions of both GJB2 and GJB6, noncoding exon 1 of GJB2, and 30 nucleotides of intronic sequence bordering the exons were analyzed in 126 subjects from Uganda with confirmed bilateral, severe-to-profound sensorineural hearing loss. All variants were analyzed for possible clinical significance using a combination of database searches and in silico tools.Complete sequence data were obtained on 115/126 individuals; 11 had only partial or no results. Only one reported pathogenic variant was found in GJB2 (c.208CG; p.Pro70Ala) and none in GJB6. Three reported variants and two novel variants within intron 1 of GJB2 and two variants within exon 3 of GJB6 were also found.None of the most common types of deletions in the GJB2 gene (c.35delG, c.167delT or c.235delC) were found in this large cohort of deaf children from Uganda. This prompts a search for genetic causes of deafness among this and other previously studied African populations.

Background Rare disease can present in the first days and weeks of life and is associated with significant morbidity and mortality. The genetic and clinical heterogeneity of these conditions can pose a significant challenge for diagnosis. Objective To evaluate the diagnostic utility of the Illumina TruSightTM One panel, we performed Next-generation sequencing (NGS) for a series of 20 newborns presenting with features suggestive of a Mendelian disease in the Neonatal Intensive Care Unit (NICU). Design/methods Twenty patients were recruited from the NICU at the Children’s Hospital of Eastern Ontario. Inclusion criteria required a complex medical presentation (congenital anomalies, abnormalities in growth and/or neurological features). We used a family-based trio approach. Target enrichment was performed with the Illumina TruSightTM One Sequencing Panel kit. This panel targets 4,813 genes that are deemed clinically-relevant and referred to as the “clinome”. Sequencing was performed on the Illumina MiSeq. NextGene software (v2.3.4.4) was used for analyses. Time from sample acquisition to data analysis was possible in 7 working days. Results/conclusion To date, 13 trios were sequenced and analysed. A molecular diagnosis was made in 5 of 13 patients, comparable to the rate obtained by whole-exome sequencing from the literature. Positive cases included bi-allelic mutations in WDR19 and ACE, an X-linked mutation in MTM1, homozygous mutations in FTO, and a de novo mutation in SCN1A. Clinome sequencing has the potential to improve our ability to efficiently diagnose rare diseases in the NICU by providing a cost-effective tool to evaluate the clinically relevant portion of the genome in a week’s time.

Myotonic dystrophy is an autosomal dominant disorder characterized by myotonia, progressive muscle wasting, and cataracts. There are two forms identified: myotonic dystrophy type 1 (DM1), caused by an expansion of a CTG repeat in the 3′ untranslated region of the myotonin-protein kinase (DMPK) gene on chromosome 19, and myotonic dystrophy type 2 (DM2), caused by an expansion of a CCTG repeat in intron 1 of the cellular nucleic acid–binding protein (CNBP) gene on chromosome 3. There is no single method that can identify all ranges of repeats in both disorders. Protocols in this unit describe the analysis of PCR-amplified CTG repeats from the DMPK gene and CCTG repeats from the CNBP gene, respectively, using a fluorescent-labeled primer followed by capillary electrophoresis. An additional protocol describes the analysis of genomic DNA by Southern blot and hybridization for DM1, while yet another describes a similar technique to analyze the repeat in DM2 using field-inversion gel electrophoresis. Both techniques identify 100% of cases of these two disorders. Curr. Protoc. Hum. Genet. 61:9.6.1-9.6.19. © 2009 by John Wiley & Sons, Inc. Keywords: myotonic dystrophy type 1; myotonic dystrophy type 2; DMPK; CNBP

A new game illustrating the trade-off between production of dirty goods and environmental clean-up is developed. Student feedback from playing the game has been very positive. To test the effectiveness of the game a multiple choice questionnaire on the production possibility frontier was administered in two introductory economics courses as a pre-test (prior to the topic presentation) and as a post-test (after the topic was delivered) to measure value added. Results indicate no statistically significant value added of using the game relative to the "chalk and talk" method.

Objective: To collect data on the practices of molecular genetic testing (MGT) laboratories for the development of national and international policies for quality assurance (QA). Methods: A web-based survey of MGT laboratory directors (n = 827; response rate 63%) in 18 countries on 3 continents. QA and reporting indices were developed and calculated for each responding laboratory. Results: Laboratory setting varied among and within countries, as did qualifications of the directors. Respondents in every country indicated that their laboratory receives specimens from outside their national borders (64%, n = 529). Pair-wise comparisons of the QA index revealed a significant association with the director having formal training in molecular genetics (p < 0.005), affiliation with a genetics unit (p = 0.003), accreditation of the laboratory (p < 0.005) and participation in proficiency testing (p < 0.005). Research labs had a lower mean report score compared to all other settings (p < 0.05) as did laboratories accessioning Conclusion: MGT is provided under widely varying conditions and regulatory frameworks. The data provided here may be a useful guide for policy action at both governmental and professional levels.

Glycogen storage disease, type II (GSDII; Pompe disease; acid maltase deficiency) is an autosomal recessive disease caused by mutations of the GAA gene that lead to deficient acid alpha-glucosidase enzyme activity and accumulation of lysosomal glycogen. Although measurement of acid alpha-glucosidase enzyme activity in fibroblasts remains the gold standard for the diagnosis of GSDII, analysis of the GAA gene allows confirmation of clinical or biochemical diagnoses and permits predictive and prenatal testing of individuals at risk of developing GSDII. We have developed a clinical molecular test for the detection of GAA mutations based on cycle sequencing of the complete coding region. GAA exons 2-20 are amplified in six independent PCR using intronic primers. The resulting products were purified and sequenced. Preliminary studies using this protocol were conducted with DNA from 21 GSDII-affected individuals from five centers across Canada. In total, 41 of 42 mutations were detected (96.7% detection rate). Mutations spanned intron 1 through exon 19 and included nine novel mutations. Haplotype analysis of recurrent mutations further suggested that three of these mutations are likely to have occurred independently at least twice. Additionally, we report the identification of the c.-32-13T>G GAA mutation in an individual with infantile variant GSDII, despite reports of this mutation being associated almost exclusively with late-onset forms of the disease. The development of a clinical molecular test provides an important tool for the management and counseling of families and individuals with GSDII, and has provided useful information about the GAA mutation spectrum in Canada.

Objective We sought to compare needle visualization and diagnostic yield rates resulting from the use of several commercially available treated needles in sonographically guided biopsies. Methods We conducted a randomized prospective study in which 61 patients (42 women and 19 men; mean age, 57 years; range, 19-84 years) were assigned to undergo biopsies (37 thyroid and 24 liver, 2 passes per patient) with 1 of 4 different 22-gauge needles: Teflon coated, etched tip, echogenic polymer coated, and untreated. Two blinded radiologists independently scored needle shaft and tip visualization from 0 (no visualization) to 4 (excellent visualization). After blinded cytologic evaluation, the individual passes were rated as adequate or inadequate for establishing a tissue diagnosis. Results The echogenic polymer-coated needle had the highest mean score +/- SD for visualization of both the shaft (3.4 +/- 0.90) and tip (3.5 +/- 0.87) compared with the untreated (shaft, 2.2 +/- 0.77; P = 0.003; tip, 2.8 +/- 0.92; P = 0.01), Teflon-coated (shaft, 2.7 +/- 0.94; tip, 3.1 +/- 0.75), and etched tip (shaft, 3.0 +/- 0.82; tip, 3.0 +/- 0.56) needles. Diagnostic yield rates for the 4 different needle types were 75.0% for the echogenic polymer-coated, 64.7% for the Teflon-coated, 56.3% for the etched tip, and 75.0% for the untreated needles (no significant difference). Conclusions The echogenic polymer-coated needle was the best visualized of all needles evaluated, both treated and untreated. No significant difference was found in diagnostic yield rates, but that may be reflective of the relatively small sample size.

SummaryThe deletion/deletion (D/D) genotype of the angiotensin converting enzyme (ACE) has been purported to be a risk for postoperative thrombosis. This D/D genotype has not been evaluated as a risk factor for idiopathic venous thromboembolism (VTE).The primary objective of the present study was to determine whether the D/D genotype of ACE is independently associated with the occurrence of idiopathic venous thromboembolic disease.We prospectively enrolled consecutive patients with at least one objectively confirmed idiopathic VTE. Friends of cases were recruited as controls and matched to cases by sex, ethnicity, and age. Patients were tested for the ACE I/D polymorphism in addition to factor V Leiden, prothrombin G20210A, and factor VIII levels.Three hundred cases and 300 controls were enrolled; 97% were Caucasian. There were 148 females and 152 males in each group with a mean age of 56.21 years (SD=15.33). The ACE D/D genotype was present in 25.3% of cases and 32.4% of controls for an adjusted odds ratio of 0.66 (95% CI = 0.433 to 0.997). We can conclude that the ACE D/D genotype is protective against idiopathic venous thromboembolism.

We assessed the usefulness of the resistive index (RI) and renal length in predicting a significant renal artery stenosis (RAS) and evaluated the effect of captopril on the RI in kidneys with and without a significant RAS.The RIs and renal lengths of both kidneys were measured in 39 patients who were referred for captopril renography for suspected renovascular hypertension. The difference in RIs (delta RI), the smaller RI (SRI), the difference in lengths (delta L), and the shorter length (SL) of the patient's two kidneys were determined. The accuracy of each of these parameters was calculated using captopril renography (n = 39) and arteriography (n = 9) as the gold standards.There was a significant difference in the delta RI (P.05), SRI (p.001), and delta L (p.05) in patients with a positive captopril renogram for a significant RAS. Captopril increased delta RI (p = .052) in patients with a positive captopril renogram (n = 6). Use of an SRI threshold of less than .55 resulted in ultrasound being as accurate as captopril renography in predicting an angiographically documented stenosis of greater than or equal to 50%.The RI and renal length are useful in detecting a significant RAS. In this preliminary study, captopril was shown to increase delta RI in patients with a significant RAS, but larger prospective studies are necessary to further assess the value of captopril sonography in detecting a significant RAS.

Abstract 1142 Background Pregnancy is associated with a hypercoagulable state, with the highest risk of venous thromboembolism (VTE) occurring during the postpartum period. This risk is further increased by the presence of inherited thrombophilic states, including Factor V Leiden (FVL) or the prothrombin gene variant (PGV), such that many clinicians provide routine postpartum thromboprophylaxis for this group. Publications examining the absolute incidence of pregnancy associated VTE in carriers of FVL and PGV report a rate of only 0–1% over the combined antepartum and 6–12 week postpartum periods. However, given that at least 50% of pregnancy associated VTE occur postpartum, the daily risk of VTE may be sufficiently high to warrant thromboprophylaxis during this period. Objectives 1. To determine the absolute rate of postpartum VTE among a prospectively enrolled cohort of FVL or PGV positive women. 2. To determine the pooled proportion of postpartum VTE in this population through a systematic review of published prospective cohort studies. Methods We performed a substudy analysis of the prospective Ottawa and Kingston (OaK) birth cohort study, in which women presenting for routine antenatal care were followed throughout pregnancy until delivery. Genotyping for FVL and PGV by polymerase chain reaction was performed for all participants after completion of the postpartum period. 336 women who enrolled through the participating Ottawa hospitals and identified as FVL or PGV positive were eligible for the current analysis. Data relating to the occurrence of VTE during the 6-week post partum period were obtained for 201/336 women through medical record review or direct contact by a study investigator. The main outcome measure was the proportion of objectively confirmed VTE within 6 weeks of delivery among participants with heterozygous FVL or PGV. Eligible articles were identified through a comprehensive search of the MEDLINE database. Inclusion criteria included a) prospective cohort design of women completing pregnancy during the study period; b) assessment of all participants for FVL and/or PGV; c) observation of participants throughout the postpartum period; d) report of objectively confirmed VTE during the postpartum period. Studies limited to women with previous pregnancy complications or previous VTE were excluded. The pooled proportion of postpartum VTE was calculated from combination of reported postpartum VTE rates and the current study results. Results No postpartum VTE occurred among the 188 participants heterozygous for FVL or PGV alone. The proportion of VTE among the population with isolated heterozygous FVL and/or PGV was 0% (95% CI 0 to 2.0). One postpartum event was documented in a heterozygous PGV carrier with coexisting protein C deficiency. No events were documented among 12 women with homozygous or compound heterozygous defects. Superficial vein thrombosis was diagnosed in two carriers of FVL. Ten publications examining the rate of postpartum VTE among FVL or PGV carriers were identified in the systematic review. ( Table 1 ) The pooled proportion of postpartum VTE among those who did not receive postpartum prophylaxis was 0.6% (95% CI 0.2 to 1.3) for heterozygous carriers of FVL and 0% (95% CI 0 to 1.6) for PGV heterozygotes. Conclusion The absolute risk of VTE among asymptomatic carriers of FVL or PGV appears sufficiently low to withhold postpartum prophylaxis. Table 1 . Pooled Proportions of Postpartum VTE Study Proportion Receiving Prophylaxis VTE Proportion: No prophylaxis (95% CI) VTE Proportion: Prophylaxis or unknown prophylaxis (95% CI) FVL - Heterozygous Dizon-Townson 1997 NR – 0/13 Lindqvist 1999 4/263 2/259 1/4 Eichinger 1999 None 0/11 – Murphy 2000 NR – 0/16 Middledorp 2001 9/17 0/8 0/9 Salomen 2004 None 0/23 – Dizon-Townson 2005 None 0/134 – Clark 2008 NR – 0/142 Kjellberg 2010 128/472 3/344 0/128 Current Study 14/139 0/125 0/14 Pooled Proportion 5/904 = 0.6% (0.2–1.3) 1/326 = 0.3% (0.1–1.7) PGV - Heterozygous Salomen 2004 None 0/27 – Silver 2010 None 0/156 – Current Study * 1/48 0/47 0/1 Pooled Proportion 0/230 = 0% (0–1.6) Insufficient data for pooled proportion FVL: factor V Leiden, PGV: prothrombin gene variant, NR: not reported. * Data regarding postpartum prophylaxis unavailable in one case. Disclosures: Ramsay: Heart and Stroke Foundation of Canada: Research Funding; Canadian Institutes of Health Research: Research Funding. Rodger: Canadian Institutes of Health Research: Research Funding; Heart and Stroke Foundation of Canada: Research Funding.

We evaluated the changes in the down-stream Doppler waveforms caused by a proximal stenosis in the main renal artery of dogs.Renal parenchymal arterial waveforms downstream from mild (50%), moderate (50-75%), and severe (76-95%) stenoses were compared with nonstenotic baseline waveforms in five mongrel dogs. Waveform shapes were categorized as biphasic or monophasic. The percentage of biphasic and monophasic waveforms was determined for each stenosis. The acceleration index (AI) and the acceleration time (AT) were determined using traditional and modified calculations (AI' and AT'). Late systolic deceleration (DS), diastolic deceleration (DD), and the resistive index (RI) also were measured.AT, AI', and AT' demonstrated significant differences between the severe stenoses and nonstenotic baselines (p.05); however, there was no difference between the mild and moderate stenoses versus baselines. The percentage distribution of monophasic and biphasic waveforms was highly correlated with the degree of stenosis. Monophasic waveforms increased on average from 22.5% of baseline waveforms to 76.5% of waveforms in the severe stenoses. Biphasic waveforms decreased on average from 69.9% of baseline waveforms to 18.7% of waveforms in the severe stenoses.Quantitative evaluation of the downstream waveform parameters (AI, AT, AI', AT', DS, DD, and RD in the dog kidney is not sufficiently accurate, but calculation of the percentage of the monophasic and biphasic waveforms present may be useful to predict a hemodynamically significant renal artery stenosis (or = 50%).

Background. Transforming growth factor-β1 (TGF-β1) is involved in cell growth and differentiation and it plays an important role in the genesis of fibrosis through the stimulation of neointima proliferation and accumulation of components of the extracellular matrix and it has been suggested that polymorphisms (polym) in its gene contribute to determining the patency of the vascular access (VA) in patients (pts) on hemodialysis (HD) by contributing to both atherogenesis and VA thrombosis. On the other hand it has been demonstrated that polym in the gene encoding the plasminogen activator inhibitor type-1 (PAI-1) are a risk factor for ischemic heart disease and possibly stroke although their role in other vascular territories is unknown. It is also known that TGF-β1 is an important up-regulator of the PAI-1 gene. Methods. We conducted a case-control study to determine the relationship between TGF-β1 polym of the signal sequence (869 T>C; 915 G>C) and VA thrombosis in 416 HD pts. (107 with VA thrombosis, 309 controls had no thrombosis). We also explored for possible interactions with the 4G/5G polym of the PAI-1 gene. TGF-β1 and PAI-1 polym were amplified using PCR and genotyped using an ABI PRISM 3100 Genetic Analyzer. TGF-β1 producing haplotypes (haplo) were defined as low, intermediate or high as previously reported. All pts were also tested for thrombophilia. Statistical analysis was done using univariate and multivariate logistic regression adjusted for thrombophilia, age, access type, etc. Results. Frequencies for low, intermediate and high TGF-β1 producing haplo were 9.3, 26.2 and 64.5% in cases and 2.6, 22.3 and 75.1% in controls. Odds of thrombosis for TGF-β1 haplotypes Haplotype Crude OR (95% CI) p Adjusted OR (95% CI) p High producing haplotype is reference category Low 5.11 (1.93, 13.5) 0.001 7.31 (2.15, 24.88) 0.001 Intermediate 1.30 (0.74, 2.29) 0.36 1.39 (0.70, 2.75) 0.35 Figure Figure Frequencies for 5G/5G, 4G/5G and 4G/4G PAI-1 polym. were 21.5, 56.1 and 22.4% in cases and 25.6, 50.2 and 24.3% in controls respectively. When we explored the interaction between both gene polym we found a highly significant result for the interaction between the low TGF-β1 producers and the 4G/4G PAI-1 polym (adjusted OR 19.3; 95% CI 2.82, 132.40; p=0.003). Conclusions. Our results show that intermediate and high producing TGF-β1 haplo have a protective effect against VA thrombosis in HD patients that is not modified by PAI-1 polym and also suggest that the interaction between low TGF-β1 producing haplo and the 4G/4G PAI-1 polym might be an important contributor to thrombosis of the VA in HD pts. Further studies are ongoing to determine the relationship between TGF-β1 haplo, TGF-β1 level, and PAI-1 polym with thrombosis in this and other populations as well as to clarify the mechanisms underlying this apparently paradoxical effect.

Absence of congemla1 myotomc dystrophy (CDM) in a baby wth 1000 DMPK CTG repeats born afler a slblmg with CDM

Type I spinal muscular atrophy is a devastating neurodegenerative disease with onset by 6 months of age and death by the age of two years, usually due to respiratory infection. Homozygous deletions of the SMNtel, but not the SMNcen, gene have been identified in 95% of SMA patients. These genes map to chromosomes 5p13 and are within a large inverted repeat which includes the NAIP gene, the telomeric copy of which is also deleted in a large proportion of SMA type I patients. We have investigated a family, in which there had been an affected child who was homozygously deleted for SMNtel and NAIP exon 5, to determine the disease status of a fetus. Analysis of cultured amniocytes indicated that the fetus is not homozygously deleted for SMNtel or NAIP. Several markers in the SMN/NAIP region were used to rule out maternal contamination of the amniocytes. During this process, it was noted that the fetus and the affected child had similar haplotypes but the paternal chromosome in the affected child carried a recombination. This recombination is associated with a deletion of several alleles of two multiallelic markers, AG1 and CATT. Dosage analysis revealed that the mother is an SMA earner with only one copy of SMNtel. In contrast, the father has 2 copies of SMNtel, suggesting that he is not a carrier of an SMNtel deletion. Rather, the paternal allele in the affected child probably resulted from a recombination event during which there was simultaneous deletion of the SMNtel gene along with some AG1 and CATT alleles. These data significantly lower the SMA recurrence risk for this family. This case highlights the necessity of using dosage information when assessing recurrence risk in SMA families.