Genetic testing for familial hypercholesterolaemia: practical and ethical issues

Coronary artery disease (CAD) has a strong genetic component, but is also greatly influenced by environmental factors such as diet and smoking, and disorders such as diabetes mellitus and hypertension. This interaction makes prediction of CAD risk generally difficult. However, in familial hypercholesterolaemia (FH), risk of early CAD is considerably increased by the mutation of a single gene, and genetic testing may be appropriate. We summarize current knowledge concerning DNA-based tests in the identification and management of FH, and propose specific recommendations for genetic testing and further research. The major value of DNA tests for FH is in genetic tracing programs to identify and treat affected individuals. DNA testing is appropriate for: (a) diagnosis of FH when physical signs or family history are equivocal or absent (important given the increased risk of CAD associated with FH compared to other hypercholesterolaemias); (b) detection of a mutation causing FH in immediate family members (particularly children) where there is a family history of premature CAD. A positive DNA-based test for a mutation is especially useful in children, in whom plasma lipid levels may not be diagnostic. Current clinical practice is to test relatives for raised cholesterol. Testing for mutation carriers in distant relatives, although feasible, is not currently recommended. Research projects should now be started to address two issues: (i) whether genetic tests for FH better predict clinical outcome than does measurement of plasma lipid levels; (ii) whether genetic testing for FH confers overall benefit both to the patient and their relatives, and to the NHS. Answers to these questions will guide the subsequent development and implementation of genetic tests for CAD risk in general, if and when the considerably more complex genetic causes of CAD are identified.      

Prestorage white cell reduction in saline-adenine-glucose-mannitol red cells by use of an integral filter: evaluation of storage values and invivo recovery

BACKGROUND : Prestorage white cell (WBC) reduction in blood components may decrease the incidence of adverse reactions and improve component quality. A bottom-and-top system with an integral third-generation WBC- reduction filter has been studied. STUDY DESIGN AND METHODS : Whole blood was collected from 30 healthy donors: from 20 by using a blood container system with an integral filter and from 10 controls by using a standard blood container system. Ten test units were buffy coat- depleted, stored for 72 hours at 4 degrees C, and then filtered, while an additional 10 test units were buffy coat-depleted and filtered at room temperature within 8 hours of collection. All units were stored at 4 degrees C for 42 days and sampled weekly. RESULTS : The mean WBC content of the 72-hour, 4 degrees C units was 0.33 × 10(6), that of the room-temperature units was 2.6 × 10(6), and that of the buffy coat- depleted controls was 460 × 10(6) (p < 0.0005). No significant differences were found among lactate, glucose, sodium, potassium, and plasma hemoglobin levels in the three groups. ATP and 2,3 DPG levels were significantly better preserved in control units than in 72-hour, 4 degrees C units (p = 0.016 and p = 0.032, respectively), but not better than in the room-temperature units. Significant differences were observed between pH values in filtered units and both groups of test units (p = 0.016). In biologic terms however, these differences were small. Red cells from an additional eight healthy volunteer donors were processed by an 8-hour room-temperature method and stored for 35 days. Studies in vivo 24-hour recovery of autologous red cells were performed by transfusing a radiolabeled (51Cr plus 131I-albumin) aliquot after 35 days' storage. Good recovery (mean > 80%) was found by both the single- and double-isotope-label methods. Recovery was significantly greater when calculated by the single-isotope method (p = 0.02). CONCLUSION : The combination of buffy coat removal and filtration in the blood container system with an integral filter achieved effective WBC reduction (> or = 3 log10 reduction from whole blood) without biologically significant detriment to in vitro or in vivo storage values.     

Coordinated inhibition of actin-induced platelet aggregation by plasma gelsolin and vitamin D-binding protein

Actin is an abundant intracellular protein that is released into the blood during tissue injury and its injection into rats causes microthrombi to form in the vasculature. This report and others have shown that actin filaments are able to aggregate platelets in an adenosine diphosphate (ADP)-dependent manner. The effects on this process of two plasma actin-binding proteins, vitamin D-binding protein (DBP) and gelsolin, were examined separately and together. The addition of DBP, a monomer-binding protein, to actin filaments did not affect their ability to induce platelet aggregation. However, severing of actin filaments with gelsolin resulted in an increased degree of platelet aggregation. Preincubation of F-actin with both gelsolin and DBP resulted in a significant inhibition of aggregation. The effects of DBP and gelsolin on actin-induced aggregation paralleled their effects on exchange of actin-bound adenine nucleotides. DBP inhibited 1, N6- ethenoadenosine 5' triphosphate (epsilon-ATP) exchange with G-actin but not with F-actin. Gelsolin increased epsilon-ATP exchange with F-actin, which was largely abrogated by the addition of DBP. These results suggest that gelsolin's severing (and subsequent capping) of actin filaments not only results in an increase in the number of pointed filament ends but also in the dissociation of actin monomers containing ADP. Phalloidin, which stabilizes actin filaments while decreasing both monomer and nucleotide exchange, inhibited actin-induced aggregation, as well, indicating that depolymerization of actin filaments is not required to inhibit aggregation. Platelet activation by either G- or F- actin may thus be regulated by the local concentrations of the plasma actin-binding proteins gelsolin and DBP. Together, these proteins inhibit platelet aggregation in a manner that can be explained by their effects on actin's filament structure and the accessibility of its bound ADP. Depletion of DBP or gelsolin may allow actin released from injured tissues to stimulate purinergic receptors on platelets, and perhaps other cells, via its bound adenine nucleotides.     

Quantitation of erythropoietin-producing cells in kidneys of mice by in situ hybridization: correlation with hematocrit, renal erythropoietin mRNA, and serum erythropoietin concentration

In situ hybridization was used to quantitate the cells that produce erythropoietin (EP) in the renal cortices of mice with varying severities of acute anemia and of mice recovering from severe, acute anemia. The number of EP-producing cells in the renal cortex increased in an exponential manner as hematocrit was decreased. Individual EP- producing cells had very similar densities of silver grains in autoradiograms regardless of whether they were from normal mice or from slightly, moderately or severely anemic animals. With increasingly severe anemia, total renal EP mRNA levels and serum EP concentrations showed increases that correlated with the number of renal EP-producing cells. These results indicate that as mice become more anemic, additional cells are recruited to produce EP rather than the cells already producing EP being stimulated to increase their individual production. In mildly and moderately anemic animals, small clusters of EP-producing cells were found in the inner cortex with large areas of cortex containing no EP-producing cells. In severely anemic mice, EP- producing cells were found throughout the inner cortex with only a very few found scattered in the outer cortex and outer medulla. The data indicate that only a subset of total renal interstitial cells produce EP. During recovery from severe, acute anemia, the numbers of EP- producing cells decreased exponentially as hematocrits rose and correlated with decreases in total renal EP mRNA and serum EP concentrations. These results suggest that following an acute blood loss and during the recovery from a blood loss, the capacity to deliver oxygen, as represented by hematocrit, is the major regulator of EP production.     

Factor VIII gene inversions in severe hemophilia A: results of an international consortium study

Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).     

Phenylephrine-Induced Ventricular Arrhythmias in Dogs with Inherited Sudden Death

Dogs with Inherited Sudden Death. Introduction: Dogs with an inherited predisposition to sudden death display ventricular arrhythmias having certain characteristics, such as pause dependence, that are suggestive of early afterdepolarization-induced triggered activity. We hypothesized that α-adrenergic stimulation may facilitate the development of these arrhythmias by inducing a reflex bradycardia and by exerting a direct myocardial effect.
Methods and Results: Twenty affected dogs and 7 unaffected dogs were studied. The incidence and severity of ventricular arrhythmias were determined after administration of phenylephrine (0.01 mg/kg IV), with or without pretreatment with propranolol (0.1 to 0.3 mg/kg IV), atropine (0.04 mg/kg IV), or prazosin (0.5 mg/kg IV). Third-degree heart block was induced by AV nodal ablation in 4 affected dogs. Phenylephrine increased ventricular arrhythmias in affected dogs, with or without pretreatment with propranolol, but did not induce ventricular arrhythmias in unaffected dogs. In dogs with intact AV nodal conduction, atropine increased sinus rate, which suppressed baseline and phenylephrine-induced arrhythmias. In dogs with heart block, arrhythmias were increased during baseline and after phenylephrine with or without pretreatment with atropine. Prazosin and overdrive ventricular pacing suppressed phenylephrine-induced arrhythmias.
Conclusion: Phenylephrine increases ventricular arrhythmias in dogs with inherited sudden death via both an induction of reflex bradycardia and a direct myocardial effect. Superimposition of heightened α-adrenergic and vagal tone may facilitate the development of sudden death in these animals.     

Humoral and cellular immunity in patients with hepatic alveolar echinococcosis. A 2 year follow-up with and without flubendazole treatment

Summary Parameters of humoral and cellular immunity were assessed in 12 patients with alveolar echinococcosis (AE) of the liver before, during and after discontinuation of treatment with flubendazole (FZ). In infected patients, before any medical treatment values of serum IgG, IgA, total haemolytic complement and C4 were significantly higher than those observed in control subjects; IgA levels were higher in jaundiced patients. Specific antibodies assayed by indirect haemagglutination and immunoelectrophoresis were present only in infected patients and were shown to decrease by the sixth month of treatment; however, similar fluctuations were observed without treatment. The percentage and absolute number of B lymphocytes, and total circulating lymphocytes, were significantly lower in patients with AE. An impairment of functional activity of T cells assayed by the leucocyte migration test, with PPD and Candidin as antigens, was demonstrated despite a normal percentage of SRBC rosettes. The 'score' of migration index still decreased during FZ treatment and returned to initial values after the year of follow-up without treatment. These results suggest that human AE is associated with important immunological disturbances. Changes in humoral immunity can be unequivocally considered to be a consequence of the parasite infection. The primary or secondary nature of the impairment of cellular immune responses and its mechanisms remain to be elucidated. Flubendazole could be responsible for an increase of cellular immune alterations in these patients.     

An Animal Model of Spontaneous Arrhythmic Death

Spontaneous Arrhythmic Death. Ventricular arrhythmias and the proclivity for sudden death have been identified in German shepherd dogs. This disorder is inherited, and affected animals can he consistently produced from an established colony. The arrhythmias are most prevalent in young dogs between 22 and 26 weeks of age, with death most frequent at this same age. Death occurs most frequently during presumed sleep or at rest after exercise or excitement. The QT interval is not prolonged; however, more frequent notching of the T wave exists in affected dogs compared to control dogs. Polymorphic rapid nonsustained ventricular tachycardia occurs most frequently following long RR intervals. Accordingly, perturhations that decrease the heart rate or enhance sinus arrhythmia increase the incidence of ventricular arrhythmias. Because the arrhythmias are age, behavior, and heart rate dependent, the autonomic nervous system may play a role in their generation. As determined by metaiodohenzylguanidine scintigraphy and immunocytochemical staining of tyrosine hydroxylase, cardiac sympathetic innervation is regionally deficient in affected dogs. Evidence suggests that initiation of the ventricular arrhythmias is caused by early after depolarization (EAD)-induced triggered activity originating from left ventricular Purkinje fibers. Alpha₁-adrenergic stimulation provokes EADs in the Purkinje fibers and ventricular arrhythmias in the dogs. The development of EADs may be related to heterogeneity of repolarizing currents (I_to in particular) in affected dogs. From this canine model of spontaneous ventricular arrhythmias, the opportunity exists to investigate the interplay between abnormal development of cardiac innervation and the genesis of lethal ventricular arrhythmias.     

K562 cells produce and respond to human erythroid-potentiating activity

Human erythroid-potentiating activity (EPA) is a 28,000 mol wt glycoprotein that stimulates the growth of erythroid progenitors in vitro and enhances colony formation by the K562 human erythroleukemia cell line. EPA has potent protease inhibitory activity, and is also referred to as tissue inhibitor of metalloproteinases (TIMP). We observed that colony formation by K562 cells in semi-solid medium containing reduced fetal calf serum (FCS) is not directly proportional to the number of cells plated, suggesting production of autostimulatory factors by K562 cells. Using radioimmunoprecipitation and a bioassay for EPA, medium conditioned by K562 cells was found to contain high levels of biologically active EPA; Northern hybridization analysis confirmed the expression of EPA mRNA. Radiolabeled EPA was used to identify cell surface receptors on K562 cells. Together, these results suggest that EPA may act as an autocrine growth factor for K562 cells.