Mechanistic basis of an epistatic interaction reducing age at onset in hereditary spastic paraplegia.

Many genetic neurological disorders exhibit variable expression within affected families, often exemplified by variations in disease age at onset. Epistatic effects (i.e. effects of modifier genes on the disease gene) may underlie this variation, but the mechanistic basis for such epistatic interactions is rarely understood. Here we report a novel epistatic interaction between SPAST and the contiguous gene DPY30, which modifies age at onset in hereditary spastic paraplegia, a genetic axonopathy. We found that patients with hereditary spastic paraplegia caused by genomic deletions of SPAST that extended into DPY30 had a significantly younger age at onset. We show that, like spastin, the protein encoded by SPAST, the DPY30 protein controls endosomal tubule fission, traffic of mannose 6-phosphate receptors from endosomes to the Golgi, and lysosomal ultrastructural morphology. We propose that additive effects on this pathway explain the reduced age at onset of hereditary spastic paraplegia in patients who are haploinsufficient for both genes.

Sinus node dysfunction in patients with Fontan circulation: could heart rate variability be a predictor for pacemaker implantation?

Sinus node dysfunction (SND) causes significant morbidity in patients after Fontan surgery. Heart rate variability (HRV) reflects the autonomic regulation of the heart, and changes in HRV have been associated with SND in adults. We aimed to study whether changes in HRV could be detected in 24-h electrocardiographic (ECG) recordings in Fontan patients with SND. We compared HRV results from two patient groups; patients with Fontan circulation who later required a pacemaker due to severe SND (n = 12) and patients with Fontan circulation and SND, without indication for pacemaker treatment (n = 11), with two control groups; patients with Fontan circulation without SND (n = 90) and healthy controls (n = 66). The Poincaré plot index SD2 (representing changes in heart rate over 24-h) and the very low-frequency (VLF) HRV component were significantly higher in both SND groups, both compared with healthy controls and patients with Fontan circulation without SND. In SND patients with pacemakers, SD2 and VLF were slightly reduced compared to SND patients without pacemaker (p = 0.06). In conclusion, in Fontan patients with SND the HRV is significantly higher compared to healthy controls and Fontan patients without SND. However, in patients with severe SND requiring pacemaker, SD2 and VLF tended to be lower than in patients with SND without pacemaker, which could indicate a reduced diurnal HRV in addition to the severe bradycardia. This is a small study, but our results indicate that HRV analysis might be a useful method in the follow-up of Fontan patients regarding development of SND.

Interplay among distinct Ca2+ conductances drives Ca2+ sparks/spontaneous transient outward currents in rat cerebral arteries.

KEY POINTS: Distinct Ca2+ channels work in a coordinated manner to grade Ca2+ spark/spontaneous transient outward currents (STOCs) in rat cerebral arteries. The relative contribution of each Ca2+ channel to Ca2+ spark/STOC production depends upon their biophysical properties and the resting membrane potential of smooth muscle. Na+ /Ca2+ exchanger, but not TRP channels, can also facilitate STOC production. ABSTRACT: Ca2+ sparks are generated in a voltage-dependent manner to initiate spontaneous transient outward currents (STOCs), events that moderate arterial constriction. In this study, we defined the mechanisms by which membrane depolarization increases Ca2+ sparks and subsequent STOC production. Using perforated patch clamp electrophysiology and rat cerebral arterial myocytes, we monitored STOCs in the presence and absence of agents that modulate Ca2+ entry. Beginning with CaV 3.2 channel inhibition, Ni2+ was shown to decrease STOC frequency in cells held at hyperpolarized (-40 mV) but not depolarized (-20 mV) voltages. In contrast, nifedipine, a CaV 1.2 inhibitor, markedly suppressed STOC frequency at -20 mV but not -40 mV. These findings aligned with the voltage-dependent profiles of L- and T-type Ca2+ channels. Furthermore, computational and experimental observations illustrated that Ca2+ spark production is intimately tied to the activity of both conductances. Intriguingly, this study observed residual STOC production at depolarized voltages that was independent of CaV 1.2 and CaV 3.2. This residual component was insensitive to TRPV4 channel modulation and was abolished by Na+ /Ca2+ exchanger blockade. In summary, our work highlights that the voltage-dependent triggering of Ca2+ sparks/STOCs is not tied to a single conductance but rather reflects an interplay among multiple Ca2+ permeable pores with distinct electrophysiological properties. This integrated orchestration enables smooth muscle to grade Ca2+ spark/STOC production and thus precisely tune negative electrical feedback.

Drip 'n Ship Versus Mothership for Endovascular Treatment: Modeling the Best Transportation Options for Optimal Outcomes.

BACKGROUND AND PURPOSE: There is uncertainty regarding the best way for patients outside of endovascular-capable or Comprehensive Stroke Centers (CSC) to access endovascular treatment for acute ischemic stroke. The role of the nonendovascular-capable Primary Stroke Centers (PSC) that can offer thrombolysis with alteplase but not endovascular treatment is unclear. A key question is whether average benefit is greater with early thrombolysis at the closest PSC before transportation to the CSC (Drip 'n Ship) or with PSC bypass and direct transport to the CSC (Mothership). Ideal transportation options were mapped based on the location of their endovascular-capable CSCs and nonendovascular-capable PSCs. METHODS: Probability models for endovascular treatment were developed from the ESCAPE trial's (Endovascular Treatment for Small Core and Anterior Circulation Proximal Occlusion With Emphasis on Minimizing CT to Recanalization Times) decay curves and for alteplase treatment were extracted from the Get With The Guidelines decay curve. The time on-scene, needle-to-door-out time at the PSC, door-to-needle time at the CSC, and door-to-reperfusion time were assumed constant at 25, 20, 30, and 115 minutes, respectively. Emergency medical services transportation times were calculated using Google's Distance Matrix Application Programming Interface interfaced with MATLAB's Mapping Toolbox to create map visualizations. RESULTS: Maps were generated for multiple onset-to-first medical response times and door-to-needle times at the PSCs of 30, 60, and 90. These figures demonstrate the transportation option that yields the better modeled outcome in specific regions. The probability of good outcome is shown. CONCLUSIONS: Drip 'n Ship demonstrates that a PSC that is in close proximity to a CSC remains significant only when the PSC is able to achieve a door-to-needle time of ≤30 minutes when the CSC is also efficient.

Ca(V)3.2 channels and the induction of negative feedback in cerebral arteries.

RATIONALE: T-type (CaV3.1/CaV3.2) Ca(2+) channels are expressed in rat cerebral arterial smooth muscle. Although present, their functional significance remains uncertain with findings pointing to a variety of roles. OBJECTIVE: This study tested whether CaV3.2 channels mediate a negative feedback response by triggering Ca(2+) sparks, discrete events that initiate arterial hyperpolarization by activating large-conductance Ca(2+)-activated K(+) channels. METHODS AND RESULTS: Micromolar Ni(2+), an agent that selectively blocks CaV3.2 but not CaV1.2/CaV3.1, was first shown to depolarize/constrict pressurized rat cerebral arteries; no effect was observed in CaV3.2(-/-) arteries. Structural analysis using 3-dimensional tomography, immunolabeling, and a proximity ligation assay next revealed the existence of microdomains in cerebral arterial smooth muscle which comprised sarcoplasmic reticulum and caveolae. Within these discrete structures, CaV3.2 and ryanodine receptor resided in close apposition to one another. Computational modeling revealed that Ca(2+) influx through CaV3.2 could repetitively activate ryanodine receptor, inducing discrete Ca(2+)-induced Ca(2+) release events in a voltage-dependent manner. In keeping with theoretical observations, rapid Ca(2+) imaging and perforated patch clamp electrophysiology demonstrated that Ni(2+) suppressed Ca(2+) sparks and consequently spontaneous transient outward K(+) currents, large-conductance Ca(2+)-activated K(+) channel mediated events. Additional functional work on pressurized arteries noted that paxilline, a large-conductance Ca(2+)-activated K(+) channel inhibitor, elicited arterial constriction equivalent, and not additive, to Ni(2+). Key experiments on human cerebral arteries indicate that CaV3.2 is present and drives a comparable response to moderate constriction. CONCLUSIONS: These findings indicate for the first time that CaV3.2 channels localize to discrete microdomains and drive ryanodine receptor-mediated Ca(2+) sparks, enabling large-conductance Ca(2+)-activated K(+) channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.

Swedish children with hereditary angioedema report good overall health and quality of life despite symptoms.

AIM: Few studies have been published on children with hereditary angioedema (HAE), an autosomal dominant disease caused by mutations on chromosome 11. This study explored various aspects of the disease in the Swedish paediatric population. METHODS: A retrospective questionnaire was sent to all 36 Swedish children known to have HAE, and a physician carried out follow-up telephone interviews. RESULTS: Most of the questionnaires were completed by the parents of 31 (86%) children with HAE, with or without their input, at a median age of nine years (range 1-17), and the physician also interviewed 29. HAE symptoms were experienced by 23 children, including abdominal attacks (96%), skin swelling (78%) and swelling in the mouth and/or upper airways (52%). Psychological stress was the most common trigger for abdominal attacks and trauma and sports triggered skin swelling. The majority (n = 19) had access to complement-1 esterase inhibitor concentrate at home. Current health and quality of life were generally rated as good, independent of whether the child had experienced HAE symptoms or not. CONCLUSION: Most children with HAE had experienced abdominal attacks and skin swelling, but their overall health and quality of life were generally perceived to be good.

Elevated Troponin Levels in Acute Stroke Patients Predict Long-term Mortality.

BACKGROUND: Elevated plasma levels of troponin in acute stroke patients are common and have in several studies been shown to predict in-hospital and short-term mortality. Little is, however, known about the long-term prognosis of these patients. The aim of this study was to determine patient characteristics and 5-year mortality in patients with acute stroke and troponin elevation on admission. METHODS: A retrospective cohort study of all consecutive patients with acute stroke and a plasma troponin I (TnI) analyzed on admission to Danderyd Hospital between January 1, 2005, and January 1, 2006 (n = 247). Patient characteristics were obtained from the Swedish National Stroke Register, Riksstroke, as well as hospital records. Mortality data were obtained from the Swedish Cause of Death Register. RESULTS: There were 133 patients (54%) with TnI less than .03 µg/L (normal), 74 patients (30%) with TnI .03-.11 µg/L (low elevation), and 40 patients (16%) with TnI greater than .11 µg/L (high elevation). TnI elevations were associated with a higher age, prior ischemic stroke, chronic heart failure, renal insufficiency, stroke severity, and ST segment elevation or depression on admission. The rate of hyperlipidemia decreased with increasing TnI. Adjusted for age and comorbidity, elevated TnI values on admission had a significantly and sustained increased mortality over the 5-year follow-up, with a hazard ratio of 1.90 (95% confidence interval, 1.33-2.70). CONCLUSIONS: Troponin elevation in patients with acute stroke, even when adjusted for several possible confounders, is associated with an almost 2-fold increased risk of 5-year mortality.

Cri du chat syndrome and primary ciliary dyskinesia: a common genetic cause on chromosome 5p.

Cri du chat syndrome (CdCS) and primary ciliary dyskinesia (PCD) are rare diseases that present with frequent respiratory symptoms. PCD can be caused by hemizygous DNAH5 mutation in combination with a 5p segmental deletion attributable to CdCS on the opposite chromosome. Chronic oto-sino-pulmonary symptoms or organ laterality defects in CdCS should prompt an evaluation for PCD.

A/T mutagenesis in hypermutated immunoglobulin genes strongly depends on PCNAK164 modification.

B cells use translesion DNA synthesis (TLS) to introduce somatic mutations around genetic lesions caused by activation-induced cytidine deaminase. Monoubiquitination at lysine(164) of proliferating cell nuclear antigen (PCNA(K164)) stimulates TLS. To determine the role of PCNA(K164) modifications in somatic hypermutation, PCNA(K164R) knock-in mice were generated. PCNA(K164R/K164R) mutants are born at a sub-Mendelian frequency. Although PCNA(K164R/K164R) B cells proliferate and class switch normally, the mutation spectrum of hypermutated immunoglobulin (Ig) genes alters dramatically. A strong reduction of mutations at template A/T is associated with a compensatory increase at G/C, which is a phenotype similar to polymerase eta (Poleta) and mismatch repair-deficient B cells. Mismatch recognition, monoubiquitinated PCNA, and Poleta likely cooperate in establishing mutations at template A/T during replication of Ig genes.

Rapid cardiovascular effects of growth hormone treatment in short prepubertal children: impact of treatment duration.

OBJECTIVE: Previous studies show that growth hormone (GH) treatment increases cardiac dimensions in short children with GH deficiency (GHD) and has diverse cardiac effects in children with idiopathic short stature (ISS). This study was performed to assess the effect of GH on the cardiovascular system in short children with a broad range of GH secretion and GH sensitivity/responsiveness. DESIGN AND PATIENTS: In this prospective, multicentre study, short prepubertal children diagnosed with isolated GHD (89) or ISS (38) were followed during 2 years of GH treatment. They were randomized to receive either a standard (43 µg/kg/day) or an individualized GH dose (range 17-100 µg/kg/day) based on GH responsiveness estimated by a prediction model and distance to target height. Echocardiography, blood pressure and electrocardiography were performed at baseline, 3, 12 and 24 months. RESULTS: Left ventricular mass (LVM) indexed to body surface area increased significantly during 2 years of GH treatment in both GHD and ISS irrespective of randomized dose. This change was already apparent at 3 months, when standard deviation scores (SDS) of wall thickness and diameter were increased. At 24 months, left ventricular diameter SDS remained increased, whereas myocardial thickness SDS returned to baseline values. There was no impairment of systolic or diastolic function. There was no correlation with treatment dose and LVM SDS at 24 months. CONCLUSIONS: Irrespective of GH status, there was a rapid increase in LVM during GH treatment in short children. At 3 months, wall thickness and diameter were increased, whereas only diameter remained increased at 24 months.

Different thresholds of tissue-specific dose-responses to growth hormone in short prepubertal children.

BACKGROUND: In addition to stimulating linear growth in children, growth hormone (GH) influences metabolism and body composition. These effects should be considered when individualizing GH treatment as dose-dependent changes in metabolic markers have been reported. HYPOTHESIS: There are different dose-dependent thresholds for metabolic effects in response to GH treatment. METHOD: A randomized, prospective, multicentre trial TRN 98-0198-003 was performed for a 2-year catch-up growth period, with two treatment regimens (a) individualized GH dose including six different dose groups ranging from 17-100 µg/kg/day (n=87) and (b) fixed GH dose of 43 µg/kg/day (n=41). The individualized GH dose group was used for finding dose-response effects, where the effective GH dose (ED 50%) required to achieve 50% Δ effect was calculated with piecewise linear regressions. RESULTS: Different thresholds for the GH dose were found for the metabolic effects. The GH dose to achieve half of a given effect (ED 50%, with 90% confidence interval) was calculated as 33(±24.4) µg/kg/day for Δ left ventricular diastolic diameter (cm), 39(±24.5) µg/kg/day for Δ alkaline phosphatase (µkat/L), 47(±43.5) µg/kg/day for Δ lean soft tissue (SDS), 48(±35.7) µg/kg/day for Δ insulin (mU/L), 51(±47.6) µg/kg/day for Δ height (SDS), and 57(±52.7) µg/kg/day for Δ insulin-like growth factor I (IGF-I) SDS. Even though lipolysis was seen in all subjects, there was no dose-response effect for Δ fat mass (SDS) or Δ leptin ng/ml in the dose range studied. None of the metabolic effects presented here were related to the dose selection procedure in the trial. CONCLUSIONS: Dose-dependent thresholds were observed for different GH effects, with cardiac tissue being the most responsive and level of IGF-I the least responsive. The level of insulin was more responsive than that of IGF-I, with the threshold effect for height in the interval between.

Reduced conduction reserve in the diabetic rat heart: role of iPLA2 activation in the response to ischemia.

Hearts from streptozotocin (STZ)-induced diabetic rats have previously been shown to have impaired intercellular electrical coupling, due to reorganization (lateralization) of connexin43 proteins. Due to the resulting reduction in conduction reserve, conduction velocity in diabetic hearts is more sensitive to conditions that reduce cellular excitability or intercellular electrical coupling. Diabetes is a known risk factor for cardiac ischemia, a condition associated with both reduced cellular excitability and reduced intercellular coupling. Activation of Ca(2+)-independent phospholipase A(2) (iPLA(2)) is known to be part of the response to acute ischemia and may contribute to the intercellular uncoupling by causing increased levels of arachidonic acid and lysophosphatidyl choline. Normally perfused diabetic hearts are known to exhibit increased iPLA(2) activity and may thus be particularly sensitive to further activation of these enzymes. In this study, we used voltage-sensitive dye mapping to assess changes in conduction velocity in response to acute global ischemia in Langendorff-perfused STZ-induced diabetic hearts. Conduction slowing in response to ischemia was significantly larger in STZ-induced diabetic hearts compared with healthy controls. Similarly, slowing of conduction velocity in response to acidosis was also more pronounced in STZ-induced diabetic hearts. Inhibition of iPLA(2) activity using bromoenol lactone (BEL; 10 µM) had no effect on the response to ischemia in healthy control hearts. However, in STZ-induced diabetic hearts, BEL significantly reduced the amount of conduction slowing observed beginning 5 min after the onset of ischemia. BEL treatment also significantly increased the time to onset of sustained arrhythmias in STZ-induced diabetic hearts but had no effect on the time to arrhythmia in healthy control hearts. Thus, our results suggest that iPLA(2) activation in response to acute ischemia in STZ-induced diabetic hearts is more pronounced than in control hearts and that this response is a significant contributor to arrhythmogenic conduction slowing.

Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting.

OBJECTIVE: We sought to evaluate a multiplexed massively parallel shotgun sequencing assay for noninvasive trisomy 21 detection using circulating cell-free fetal DNA. STUDY DESIGN: Sample multiplexing and cost-optimized reagents were evaluated as improvements to a noninvasive fetal trisomy 21 detection assay. A total of 480 plasma samples from high-risk pregnant women were employed. RESULTS: In all, 480 prospectively collected samples were obtained from our third-party storage site; 13 of these were removed due to insufficient quantity or quality. Eighteen samples failed prespecified assay quality control parameters. In all, 449 samples remained: 39 trisomy 21 samples were correctly classified; 1 sample was misclassified as trisomy 21. The overall classification showed 100% sensitivity (95% confidence interval, 89-100%) and 99.7% specificity (95% confidence interval, 98.5-99.9%). CONCLUSION: Extending the scope of previous reports, this study demonstrates that plasma DNA sequencing is a viable method for noninvasive detection of fetal trisomy 21 and warrants clinical validation in a larger multicenter study.

High sensitivity of BRCA1-deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs.

Whereas target-specific drugs are available for treating ERBB2-overexpressing and hormone receptor-positive breast cancers, no tailored therapy exists for hormone receptor- and ERBB2-negative ("triple-negative") mammary carcinomas. Triple-negative tumors account for 15% of all breast cancers and frequently harbor defects in DNA double-strand break repair through homologous recombination (HR), such as BRCA1 dysfunction. The DNA-repair defects characteristic of BRCA1-deficient cells confer sensitivity to poly(ADP-ribose) polymerase 1 (PARP1) inhibition, which could be relevant to treatment of triple-negative tumors. To evaluate PARP1 inhibition in a realistic in vivo setting, we tested the PARP inhibitor AZD2281 in a genetically engineered mouse model (GEMM) for BRCA1-associated breast cancer. Treatment of tumor-bearing mice with AZD2281 inhibited tumor growth without signs of toxicity, resulting in strongly increased survival. Long-term treatment with AZD2281 in this model did result in the development of drug resistance, caused by up-regulation of Abcb1a/b genes encoding P-glycoprotein efflux pumps. This resistance to AZD2281 could be reversed by coadministration of the P-glycoprotein inhibitor tariquidar. Combination of AZD2281 with cisplatin or carboplatin increased the recurrence-free and overall survival, suggesting that AZD2281 potentiates the effect of these DNA-damaging agents. Our results demonstrate in vivo efficacy of AZD2281 against BRCA1-deficient breast cancer and illustrate how GEMMs of cancer can be used for preclinical evaluation of novel therapeutics and for testing ways to overcome or circumvent therapy resistance.

DOSE EVALUATION AND PROPOSAL OF LOCAL DIAGNOSTIC REFERENCE LEVELS FOR PAEDIATRIC CARDIAC CATHETERIZATIONS PERFORMED ON A HIGH-SENSITIVITY ANGIOGRAPHIC SYSTEM ALLOWING LOW-DOSE IMAGING.

Radiation doses from paediatric cardiac catheterizations were analysed based on procedure type and patient weight, and local diagnostic reference levels (LDRLs) were proposed. The procedures were performed on a Siemens Artis Q.zen biplane system with high-sensitivity detectors allowing low-dose imaging. Good radiological practice, e.g. adapting dose level and frame rate continuously and minimizing the x-ray field with collimators, was routine during procedures. The median total dose-area-product (DAP) value was 58 µGym2 for diagnostic catheterizations, 48 µGym2 for interventional catheterizations and 33 µGym2 for myocardial biopsies. The median DAP per body weight was 4.0 µGym2 kg-1. The median total fluoroscopy time varied from 6.0 min for myocardial biopsies, to 9.7 and 10.5 min, respectively, for diagnostic and interventional catheterizations. The third quartile of the DAP values in each weight group was proposed as LDRL. LDRLs for fluoroscopy time were determined solely based on procedure type, due to the absence of weight dependence.

Quantification of fetal DNA by use of methylation-based DNA discrimination.

BACKGROUND: Detection of circulating cell-free fetal nucleic acids in maternal plasma has been used in noninvasive prenatal diagnostics. Most applications rely on the qualitative detection of fetal nucleic acids to determine the genetic makeup of the fetus. This method leads to an analytic dilemma, because test results from samples that do not contain fetal DNA or are contaminated with maternal cellular DNA can be misleading. We developed a multiplex approach to analyze regions that are hypermethylated in placenta relative to maternal blood to evaluate the fetal portion of circulating cell-free DNA isolated from maternal plasma. METHODS: The assay used methylation-sensitive restriction enzymes to eliminate the maternal (unmethylated) fraction of the DNA sample. The undigested fetal DNA fraction was then coamplified in the presence of a synthetic oligonucleotide to permit competitive PCR. The amplification products were quantified by single-base extension and MALDI-TOF MS analysis. RESULTS: Using 2 independent markers, (sex determining region Y)-box 14 (SOX14) and T-box 3 (TBX3), we measured a mean of 151 copies of fetal DNA/mL plasma and a mean fetal fraction of 0.13 in samples obtained from pregnant women. We investigated 242 DNA samples isolated from plasma from pregnant and nonpregnant women and observed an analytical sensitivity and specificity for the assay of 99% and 100%, respectively. CONCLUSIONS: By investigating several regions in parallel, we reduced the measurement variance and enabled quantification of circulating cell-free DNA. Our results indicate that this multiplex methylation-based reaction detects and quantifies the amount of fetal DNA in a sample isolated from maternal plasma.

Colorimetric quantification of mRNA expression in rare tumour cells amplified by multiple ligation-dependent probe amplification.

An enzyme-linked oligonucleotide assay (ELONA) for quantification of mRNA expression of five genes involved in breast cancer, extracted from isolated rare tumour cells and amplified by multiplex ligation-dependent probe amplification (MLPA) is presented. In MLPA, a multiplex oligonucleotide ligation assay is combined with a PCR reaction in which all ligation products are amplified by use of a single primer pair. Biotinylated probes complementary to each of the target sequences were immobilised on the surface of a streptavidin-coated microtitre plate and exposed to single-stranded MLPA products. A universal reporting probe sequence modified with horseradish peroxidase (URP-HRP) and complementary to a universal primer used during the MLPA step was further added to the surface-bound duplex as a reporter probe. Simultaneous addition of anchoring probe and target, followed by addition of reporter probe, rather than sequential addition, was achieved with no significant effect on sensitivity and limits of detection, but considerably reduced the required assay time. Detection limits as low as 20 pmol L(-1), with an overall assay time of 95 min could be achieved with negligible cross-reactivity between probes and non-specific targets present in the MLPA-PCR product. The same MLPA-PCR product was analysed using capillary electrophoresis, the technique typically used for analysis of MLPA products, and good correlation was observed. The assay presented is easy to carry out, relatively inexpensive, rapid, does not require sophisticated instrumentation, and enables quantitative analysis, making it very promising for the analysis of MLPA products.

Experimental and theoretical ventricular electrograms and their relation to electrophysiological gradients in the adult rat heart.

The electrical activity of adult mouse and rat hearts has been analyzed extensively, often as a prerequisite for genetic engineering studies or for the development of rodent models of human diseases. Some aspects of the initiation and conduction of the cardiac action potential in rodents closely resemble those in large mammals. However, rodents have a much higher heart rate and their ventricular action potential is triangular and very short. As a consequence, an interpretation of the electrocardiogram in the mouse and rat remains difficult and controversial. In this study, optical mapping techniques have been applied to an in vitro left ventricular adult rat preparation to obtain patterns of conduction and action potential duration measurements from the epicardial surface. This information has been combined with previously published mathematical models of the rat ventricular myocyte to develop a bidomain model for action potential propagation and electrogram formation in the rat left ventricle. Important insights into the basis for the repolarization waveform in the ventricular electrogram of the adult rat have been obtained. Notably, our model demonstrated that the biphasic shape of the rat ventricular repolarization wave can be explained in terms of the transmural and apex-to-base gradients in action potential duration that exist in the rat left ventricle.

REEP1 mutation spectrum and genotype/phenotype correlation in hereditary spastic paraplegia type 31.

Mutations in the receptor expression enhancing protein 1 (REEP1) have recently been reported to cause autosomal dominant hereditary spastic paraplegia (HSP) type SPG31. In a large collaborative effort, we screened a sample of 535 unrelated HSP patients for REEP1 mutations and copy number variations. We identified 13 novel and 2 known REEP1 mutations in 16 familial and sporadic patients by direct sequencing analysis. Twelve out of 16 mutations were small insertions, deletions or splice site mutations. These changes would result in shifts of the open-reading-frame followed by premature termination of translation and haploinsufficiency. Interestingly, we identified two disease associated variations in the 3'-UTR of REEP1 that fell into highly conserved micro RNA binding sites. Copy number variation analysis in a subset of 133 HSP index patients revealed a large duplication of REEP1 that involved exons 2-7 in an Irish family. Clinically most SPG31 patients present with a pure spastic paraplegia; rare complicating features were restricted to symptoms or signs of peripheral nerve involvement. Interestingly, the distribution of age at onset suggested a bimodal pattern with the appearance of initial symptoms of disease either before the age of 20 years or after the age of 30 years. The overall mutation rate in our clinically heterogeneous sample was 3.0%; however, in the sub-sample of pure HSP REEP1 mutations accounted for 8.2% of all patients. These results firmly establish REEP1 as a relatively frequent autosomal dominant HSP gene for which genetic testing is warranted. We also establish haploinsufficiency as the main molecular genetic mechanism in SPG31, which should initiate and guide functional studies on REEP1 with a focus on loss-of-function mechanisms. Our results should be valid as a reference for mutation frequency, spectrum of REEP1 mutations, and clinical phenotypes associated with SPG31.