Using human genetics to discover new therapeutic targets for plasma lipids.

Genetic variation arises through multiple different alleles that vary in frequency and severity of effect. Mutations that give rise to Mendelian disorders, such as the LDL receptor (LDLR) mutations that result in familial hypercholesterolaemia, are efficiently winnowed from the population by purifying selection and are almost inevitably rare. Conversely, alleles that are common in the population (such that homozygotes for the minor allele are present even in modest sample sizes) typically have very modest phenotypic effects. Mutations in the gene for proprotein convertase subtilisin/kexin type 9 (PCSK9) represent an unusual but informative exception in that they are relatively common but have large effects on phenotype. Loss-of-function mutations in PCSK9 occur in ~2.5% of African Americans and are associated with large reductions in coronary heart disease (CHD) risk. The development of agents to inhibit PCSK9 demonstrates the utility of translating genetics into clinical therapeutics. Attempts to identify genes responsible for hypercholesterolaemia have used traditional linkage analysis, which requires samples collected from multiple families with defects in the same gene, or genome-wide association, which requires thousands of samples from the population. More recently, whole-exome sequencing studies have revealed loss-of-function mutations in ANGPTL3 associated with pan-hypolipidemia, and in APOC3 that confer protection against CHD. The application of whole-exome sequencing to large populations or to carefully selected patients can streamline the discovery of causal genetic mutations.

Diclofenac plus B vitamins versus diclofenac monotherapy in lumbago: the DOLOR study.

OBJECTIVES: To assess the influence of vitamins B1, B6 and B12 on the analgesia success achieved by diclofenac in subjects with acute lumbago. RESEARCH DESIGN AND METHODS: A randomised, double blind controlled clinical study in parallel groups, in which subjects received twice-daily oral administration of either the combination therapy, Group DB (50 mg diclofenac plus 50 mg thiamine, 50 mg pyridoxine and 1 mg cyanocobalamin) or diclofenac monotherapy, Group D (50mg diclofenac). The study period lasted a maximum of 7 days. If sufficient pain reduction was achieved (defined as Visual Analogue Scale <20 mm and patient's satisfaction), subjects could withdraw from the treatment after 3 or 5 days. All subjects gave written informed consent to participate in the study. MAIN OUTCOME MEASURES: The primary confirmatory study objective was to determine the number of patients with sufficient pain reduction after 3 days of treatment. RESULTS: Three hundred and seventy-two subjects were allocated at random to either treatment group: Group DB - 187 subjects and Group D - 185 subjects. After 3 days of treatment, a statistically significant higher proportion of subjects in Group DB (n = 87; 46.5%) than in Group D (n = 55; 29%) terminated the study due to treatment success (chi(2): 12.06; p = 0.0005). Furthermore, the combination therapy yielded superior results in pain reduction, improvement of mobility and functionality. Drug safety monitoring profile throughout the trial was within the expected safety profile of diclofenac. CONCLUSIONS: The combination of diclofenac with B vitamins was superior to diclofenac monotherapy in lumbago relief after 3 days of treatment. As a study drawback, daily VAS measurements were only recorded until subject withdrawal from treatment, whether after 3, 5, or 7 days. There were no differences in safety profile between the two study groups.

Comparison of closed chamber and open chamber evaporimetry.

BACKGROUND: Recently it has been asserted that a closed chamber evaporimeter, the VapoMeter, offers advantages over standard open chamber devices in measuring transepidermal water loss (TEWL). Purported improvements include the ability to take measurements at any angle, short reading times and insensitivity to external air currents. These claims are compelling, considering that measuring TEWL at diverse skin sites can be tedious, especially with children. The primary aim of this study was to compare the performance of closed and open chamber instruments when they were held at various angles and, secondly to evaluate the ability of the devices to discriminate between test conditions. METHODS: The performance of closed chamber (VapoMeter) and open chamber (DermaLab) evaporimeters were compared by measuring water vapor emitted from IMS Vitro-skin that had been hydrated to a predetermined level. Measurements were taken at three angles from vertical - 0 degrees, 45 degrees, and 90 degrees. Vitro-skin samples were weighed periodically throughout the experimental phase to verify water loss rates. RESULTS: Both the VapoMeter and the DermaLab yielded significantly lower water loss values when held at angles that varied from the vertical (0 degrees) position, indicating that the closed chamber device is no more capable of accurately measuring TEWL at any angle than an open chamber instrument. The DermaLab provided better discrimination than the VapoMeter when the instruments were held vertically, as is the only prescribed testing position for open-chamber instruments. The VapoMeter was easier to use than the DermaLab; however, there was evidence that the sealed chamber could become saturated under high water loss conditions. CONCLUSIONS: Previous assertions that the VapoMeter closed chamber evaporimeter is capable of measuring TEWL regardless of angle were not validated. Each device appeared capable of accurately estimating water loss rates only in the vertical position. Although the VapoMeter was easier to use than the open chamber device, its tendency to become saturated under high water loss conditions could be a disadvantage when assessing dynamic TEWL.

Tackling corruption in the pharmaceutical systems worldwide with courage and conviction.

Poor drug access continues to be one of the main global health problems. Global inequalities in access to pharmaceuticals are caused by a number of variables including poverty, high drug prices, poor health infrastructure, and fraud and corruption--the latter being the subject of this article. There is growing recognition among policy makers that corruption in the pharmaceutical system can waste valuable resources allocated to pharmaceutical products and services. This, in turn, denies those most in need from life-saving or life-enhancing medicines. As a result, international organizations, including the World Health Organization and the World Bank are beginning to address the issue of corruption in the health sector broadly and the pharmaceutical system specifically. This is encouraging news for improving drug access for the global poor who are most harmed by corruption as they tend to purchase less expensive drugs from unqualified or illegal drug sellers selling counterfeit or sub-standard drugs. In our paper, we illuminate what are the core issues that relate to corruption in the pharmaceutical sector. We argue that corruption in the pharmaceutical system can be detrimental to a country's ability to improve the health of its population. Moreover, unless policy makers deal with the issue of corruption, funding allocated to the pharmaceutical system to treat health conditions may simply be wasted and the inequality between rich and poor in access to health and pharmaceutical products will be aggravated.

TRIPS, the Doha Declaration and increasing access to medicines: policy options for Ghana.

There are acute disparities in pharmaceutical access between developing and industrialized countries. Developing countries make up approximately 80% of the world's population but only represent approximately 20% of global pharmaceutical consumption. Among the many barriers to drug access are the potential consequences of the Trade Related Aspects of Intellectual Property Rights (TRIPS) Agreement. Many developing countries have recently modified their patent laws to conform to the TRIPS standards, given the 2005 deadline for developing countries. Safeguards to protect public health have been incorporated into the TRIPS Agreement; however, in practice governments may be reluctant to exercise such rights given concern about the international trade and political ramifications. The Doha Declaration and the recent Decision on the Implementation of Paragraph 6 of the Doha Declaration on the TRIPS Agreement and Public Health may provide more freedom for developing countries in using these safeguards. This paper focuses on Ghana, a developing country that recently changed its patent laws to conform to TRIPS standards. We examine Ghana's patent law changes in the context of the Doha Declaration and assess their meaning for access to drugs of its population. We discuss new and existing barriers, as well as possible solutions, to provide policy-makers with lessons learned from the Ghanaian experience.

Mutations in ATP-cassette binding proteins G5 (ABCG5) and G8 (ABCG8) causing sitosterolemia.

Sitosterolemia is an autosomal recessive disorder caused by mutations in two adjacent genes encoding coordinately regulated ATP binding cassette (ABC) half transporters (ABCG5 and ABCG8). In this paper we describe three novel mutations causing sitosterolemia: 1) a frameshift mutation (c.336-337insA) in ABCG5 that results in premature termination of the protein at amino acid 197; 2) a missense mutation that changes a conserved residue c.1311C>G; N437K) in ABCG5 and 3) a splice site mutation in ABCG8 (IVS1-2A>G). This study expands the spectrum of the ABCG5 and ABCG8 mutations that cause sitosterolemia. Nine nonsynonymous polymorphisms are also reported: I523V, C600Y, Q604E, and M622V in ABCG5; and D19H, Y54C, T400K, A632V, and Y641F in ABCG8.

An apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing.

Comparison of genomic DNA sequences from human and mouse revealed a new apolipoprotein (APO) gene (APOAV) located proximal to the well-characterized APOAI/CIII/AIV gene cluster on human 11q23. Mice expressing a human APOAV transgene showed a decrease in plasma triglyceride concentrations to one-third of those in control mice; conversely, knockout mice lacking Apoav had four times as much plasma triglycerides as controls. In humans, single nucleotide polymorphisms (SNPs) across the APOAV locus were found to be significantly associated with plasma triglyceride levels in two independent studies. These findings indicate that APOAV is an important determinant of plasma triglyceride levels, a major risk factor for coronary artery disease.

Molecular characterization of L-CPT I deficiency in six patients: insights into function of the native enzyme.

Carnitine palmitoyltransferase I (CPT I) catalyzes the formation of acylcarnitine, the first step in the oxidation of long-chain fatty acids in mitochondria. The enzyme exists as liver (L-CPT I) and muscle (M-CPT I) isoforms that are encoded by separate genes. Genetic deficiency of L-CPT I, which has been reported in 16 patients from 13 families, is characterized by episodes of hypoketotic hypoglycemia beginning in early childhood and is usually associated with fasting or illness. To date, only two mutations associated with L-CPT I deficiency have been reported. In the present study we have identified and characterized the mutations underlying L-CPT I deficiency in six patients: five with classic symptoms of L-CPT I deficiency and one with symptoms that have not previously been associated with this disorder (muscle cramps and pain). Transfection of the mutant L-CPT I cDNAs in COS cells resulted in L-CPT I mRNA levels that were comparable to those expressed from the wild-type construct. Western blotting revealed lower levels of each of the mutant proteins, indicating that the low enzyme activity associated with these mutations was due, at least in part, to protein instability. The patient with atypical symptoms had approximately 20% of normal L-CPT I activity and was homozygous for a mutation (c.1436C-->T) that substituted leucine for proline at codon 479. Assays performed with his cultured skin fibroblasts indicated that this mutation confers partial resistance to the inhibitory effects of malonyl-CoA. The demonstration of L-CPT I deficiency in this patient suggests that the spectrum of clinical sequelae associated with loss or alteration of L-CPT I function may be broader than was previously recognized.

Impaired mitochondrial fatty acid oxidative flux in fibroblasts from a patient with malonyl-CoA decarboxylase deficiency.

Malonyl-CoA decarboxylase deficiency is a rare inborn error of metabolism. It has been suggested but never demonstrated that many of the clinical features arise due to inhibition of mitochondrial fatty acid oxidation by accumulated malonyl-CoA. We studied the oxidation of fatty acids in cultured skin fibroblasts from a recently described patient with malonyl-CoA decarboxylase deficiency. There was a marked reduction in the oxidation of palmitic and myristic acids both under baseline conditions and when the cells were cultured in the presence of high concentrations of acetate, a malonyl-CoA precursor. These results suggest that there is inhibition of fatty acid oxidation in malonyl-CoA decarboxylase deficiency and that this inhibition may be related to some of the clinical phenotypes.

Sequence diversity in genes of lipid metabolism.

Elevated plasma lipoprotein levels play a crucial role in the development of coronary artery disease. Genetic factors strongly influence the levels of plasma lipoproteins, but the genes and sequence variations contributing to the most common forms of dyslipidemias are not known. We used GeneChip probe arrays to resequence the coding regions of 10 key genes of lipid metabolism. The sequences of these genes were analyzed in 80 dyslipidemic individuals. Fourteen nonsynonymous and twenty-two synonymous single nucleotide changes were identified that could be confirmed by conventional sequencing. Seven of the fourteen nonsynonymous sequence variants were polymorphisms with allele frequency >1% in the general population. The remaining seven were not found in normolipidemic controls (25 Caucasians and 25 African-Americans). The relationship between nonsynonymous sequence variations and various dyslipidemias was explored in association and family studies. No evidence was found for coding sequence variations in any of the 10 genes contributing to dyslipidemia. Only a single sequence variation, a missense mutation in the low density lipoprotein receptor gene, co-segregated with hyperlipidemia in the proband's family. This study illustrates some of the difficulties associated with identifying sequence variations contributing to complex traits.

Autosomal recessive hypercholesterolemia caused by mutations in a putative LDL receptor adaptor protein.

Atherogenic low density lipoproteins are cleared from the circulation by hepatic low density lipoprotein receptors (LDLR). Two inherited forms of hypercholesterolemia result from loss of LDLR activity: autosomal dominant familial hypercholesterolemia (FH), caused by mutations in the LDLR gene, and autosomal recessive hypercholesterolemia (ARH), of unknown etiology. Here we map the ARH locus to an approximately 1-centimorgan interval on chromosome 1p35 and identify six mutations in a gene encoding a putative adaptor protein (ARH). ARH contains a phosphotyrosine binding (PTB) domain, which in other proteins binds NPXY motifs in the cytoplasmic tails of cell-surface receptors, including the LDLR. ARH appears to have a tissue-specific role in LDLR function, as it is required in liver but not in fibroblasts.

Gene transfer into the fetal primate: evidence for the secretion of transgene product.

In utero adenoviral-mediated transfer of genes via the amniotic fluid results in sustained high-efficiency expression in rodent lung and intestine. Rhesus macaque (Macaca mulatta) fetuses were injected with adenovirus vectors encoding reporter genes at different gestational ages to evaluate feasibility and timing in primates. The fetuses developed normally following gene transfer and no maternal adverse affects were noted. Highly efficient viral uptake and transgene protein expression occurred in the target organs. The lungs exhibited no immune response and transgenic protein was observed up to 30 days postinfection. Unexpectedly, large amounts of reporter gene protein were released, apparently from the lung, into the circulation and accumulated in the renal proximal tubules and bladder. PCR detection for adenovirus DNA was consistently negative in tissues not in contact with the amniotic fluid, such as kidneys, liver, gonads, and eyes. Treatment of primate fetuses at 110 days gestation with an adenovirus expressing the cystic fibrosis transmembrane conductance regulator (cftr) gene resulted in accelerated differentiation of the lung. These studies demonstrate the efficacy of in utero gene therapy in primates and its potential application to genetic diseases.

Cystic fibrosis revisited.

Cystic fibrosis is a pleiotropic disease whose primary defect is thought to be abnormal chloride conductance. Despite intensive study, the role of the protein in the airway and the mechanism for its direct participation in the disease pathology remain unclear. This paper reviews CFTR's cell regulatory functions and data supporting the role of CFTR in secretory epithelial cell development. A hypothesis for CF pathophysiology based on secretory cell differentiation is proposed.

CFTR modulates lung secretory cell proliferation and differentiation.

We have permanently reversed the lethal phenotype in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-deficient (knockout) mouse after in utero gene therapy with an adenovirus containing the cftr gene. The gene transfer targeted somatic stem cells in the developing lung and intestine, and these epithelial surfaces demonstrated permanent developmental changes after treatment. The survival statistics from the progeny of heterozygote-heterozygote matings after in utero cftr gene treatment demonstrated an increased mortality in the homozygous normal pups, indicating that overexpression during development was detrimental. The lungs of these pups revealed accelerated secretory cell proliferation and differentiation. The extent of proliferation and differentiation in the secretory cells of the lung parenchyma after in utero transfer of the cftr gene was evaluated with morphometric and biochemical analyses. These studies provide further support of the regulatory role of the cftr gene in the development of the secretory epithelium.

Premature cardiovascular disease is common in relatives of patients with premature peripheral atherosclerosis.

BACKGROUND: Numerous clinical conditions have been proposed to explain the premature onset of symptomatic peripheral vascular disease (PVD) in young adults, but the role of genetic factors has not been defined. This study was performed to determine the prevalence of cardiovascular disease among family members of patients with premature PVD. METHODS: The prevalence of early cardiovascular events occurring in first-degree relatives of 90 subjects with premature PVD (onset < or =49 years) was determined. The prevalence of occult atherosclerosis was determined by duplex ultrasonography in a cohort of 20 asymptomatic siblings. Reference groups included first-degree relatives of 80 subjects with premature coronary artery disease (CAD) and first-degree relatives of 48 healthy subjects. RESULTS: Cardiovascular events occurred at age 55 years or younger in 28% of the parents of PVD subjects, in 23% of parents of CAD subjects, and in 7% of the parents of healthy controls (P<.001). Cardiovascular events occurred in 24% of siblings of PVD subjects, in 14% of siblings of CAD subjects, and in 7% of siblings of healthy controls (P<.001). Duplex ultrasonography detected early plaques in the lower extremity circulation of 10 (50%) of the asymptomatic siblings of PVD subjects. CONCLUSIONS: Early, symptomatic cardiovascular disease is more common in first-degree relatives of individuals with premature PVD than in relatives of healthy individuals or of probands with premature CAD. Occult vascular disease in the lower extremity is prevalent among asymptomatic siblings of probands with premature PVD. These observations indicate that susceptibility to premature PVD has a familial basis.

Under which conditions do the skin and probe decouple during sinusoidal vibrations?

Previous experiments performed on monkey and human fingertips suggested that the skin surface and stimulus probe decouple for sinusoidal displacements applied perpendicularly to the skin surface. From these observations, it was concluded that sinusoidal vibration may not be a suitable stimulus for understanding and modeling the tactile system. We repeated these experiments on human observers using stimulus frequencies ranging from 0.5 to 240 Hz and with displacement amplitudes up to 1 mm peak-to-peak (p-p). The skin and probe movements were measured in the steady-state using stroboscopic illumination and video microscopy. Contrary to previous conclusions, we found that decoupling did not occur for amplitudes less then 0.25 mm p-p, regardless of stimulus frequency. Decoupling was only observed for stimulus amplitudes greater than 0.25 mm over the stimulus-frequency range investigated. To further investigate this effect, a modified stimulus contactor was used, which permitted the measurement of the skin's movement using reflected light. Measurements were made on both the index fingertip and the thenar eminence. Regardless of body site, no decoupling between the skin and stimulus probe was observed for frequencies ranging from 20 to 100 Hz up to displacements of 0.25 mm p-p. These levels are well within the range used in most human psychophysical experiments performed on these parts of the body. We conclude that sinusoidal vibration can be used reliably to stimulate the tactile system and is an appropriate stimulus for developing models of touch.

Contribution of cholesterol 7alpha-hydroxylase to the regulation of lipoprotein metabolism.

Clinical studies have clearly established a relationship between bile acid synthesis and plasma LDL-cholesterol concentrations. Interruption of the enterohepatic circulation of bile acids leads to increased bile acid synthesis and a reduction in plasma LDL-cholesterol concentrations. New studies indicate that genetic variation in cholesterol 7alpha-hydroxylase activity accounts for a significant fraction of the inter-individual variation in plasma LDL-cholesterol concentrations in the general population, and a specific CYP7A1 allele associated with increased plasma LDL-cholesterol concentrations has been identified. Studies in which cholesterol 7alpha-hydroxylase was transiently overexpressed in hamsters and mice indicate that direct manipulation of cholesterol 7alpha-hydroxylase leads to changes in plasma LDL-cholesterol concentrations. Interestingly, targeted inactivation of the gene encoding cholesterol 7alpha-hydroxylase does not lead to increased plasma LDL-cholesterol concentrations in mice.