Showing papers by "Children's Hospital Oakland Research Institute published in 2009"

Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis.

Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disorder Ten percent of cases are inherited; most involve unidentified genes We report here 13 mutations in the fused in sarcoma/translated in liposarcoma (FUS/TLS) gene on chromosome 16 that were specific for familial ALS The FUS/TLS protein binds to RNA, functions in diverse processes, and is normally located predominantly in the nucleus In contrast, the mutant forms of FUS/TLS accumulated in the cytoplasm of neurons, a pathology that is similar to that of the gene TAR DNA-binding protein 43 (TDP43), whose mutations also cause ALS Neuronal cytoplasmic protein aggregation and defective RNA metabolism thus appear to be common pathogenic mechanisms involved in ALS and possibly in other neurodegenerative disorders

Consuming fructose-sweetened, not glucose-sweetened, beverages increases visceral adiposity and lipids and decreases insulin sensitivity in overweight/obese humans

Abstract: Studies in animals have documented that, compared with glucose, dietary fructose induces dyslipidemia and insulin resistance. To assess the relative effects of these dietary sugars during sustained consumption in humans, overweight and obese subjects consumed glucose- or fructose-sweetened beverages providing 25% of energy requirements for 10 weeks. Although both groups exhibited similar weight gain during the intervention, visceral adipose volume was significantly increased only in subjects consuming fructose. Fasting plasma triglyceride concentrations increased by approximately 10% during 10 weeks of glucose consumption but not after fructose consumption. In contrast, hepatic de novo lipogenesis (DNL) and the 23-hour postprandial triglyceride AUC were increased specifically during fructose consumption. Similarly, markers of altered lipid metabolism and lipoprotein remodeling, including fasting apoB, LDL, small dense LDL, oxidized LDL, and postprandial concentrations of remnant-like particle-triglyceride and -cholesterol significantly increased during fructose but not glucose consumption. In addition, fasting plasma glucose and insulin levels increased and insulin sensitivity decreased in subjects consuming fructose but not in those consuming glucose. These data suggest that dietary fructose specifically increases DNL, promotes dyslipidemia, decreases insulin sensitivity, and increases visceral adiposity in overweight/obese adults.

Population Analysis of Large Copy Number Variants and Hotspots of Human Genetic Disease

Abstract: Copy number variants (CNVs) contribute to human genetic and phenotypic diversity. However, the distribution of larger CNVs in the general population remains largely unexplored. We identify large variants in ∼2500 individuals by using Illumina SNP data, with an emphasis on “hotspots” prone to recurrent mutations. We find variants larger than 500 kb in 5%–10% of individuals and variants greater than 1 Mb in 1%–2%. In contrast to previous studies, we find limited evidence for stratification of CNVs in geographically distinct human populations. Importantly, our sample size permits a robust distinction between truly rare and polymorphic but low-frequency copy number variation. We find that a significant fraction of individual CNVs larger than 100 kb are rare and that both gene density and size are strongly anticorrelated with allele frequency. Thus, although large CNVs commonly exist in normal individuals, which suggests that size alone can not be used as a predictor of pathogenicity, such variation is generally deleterious. Considering these observations, we combine our data with published CNVs from more than 12,000 individuals contrasting control and neurological disease collections. This analysis identifies known disease loci and highlights additional CNVs (e.g., 3q29, 16p12, and 15q25.2) for further investigation. This study provides one of the first analyses of large, rare (0.1%–1%) CNVs in the general population, with insights relevant to future analyses of genetic disease.

Antiparallel beta-sheet: a signature structure of the oligomeric amyloid beta-peptide.

Abstract: AD (Alzheimer's disease) is linked to Abeta (amyloid beta-peptide) misfolding. Studies demonstrate that the level of soluble Abeta oligomeric forms correlates better with the progression of the disease than the level of fibrillar forms. Conformation-dependent antibodies have been developed to detect either Abeta oligomers or fibrils, suggesting that structural differences between these forms of Abeta exist. Using conditions which yield well-defined Abeta-(1-42) oligomers or fibrils, we studied the secondary structure of these species by ATR (attenuated total reflection)-FTIR (Fourier-transform infrared) spectroscopy. Whereas fibrillar Abeta was organized in a parallel beta-sheet conformation, oligomeric Abeta displayed distinct spectral features, which were attributed to an antiparallel beta-sheet structure. We also noted striking similarities between Abeta oligomers spectra and those of bacterial outer membrane porins. We discuss our results in terms of a possible organization of the antiparallel beta-sheets in Abeta oligomers, which may be related to reported effects of these highly toxic species in the amyloid pathogenesis associated with AD.

Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster

Abstract: We constructed Drosophila melanogaster BAC libraries with 21-kb and 83-kb inserts in the P(acman) system. Clones representing 12-fold coverage and encompassing more than 95percent of annotated genes were mapped onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using Phi C31 integrase to rescue mutations. They can be modified through recombineering, for example to incorporate protein tags and assess expression patterns.

Definitions of the phenotypic manifestations of sickle cell disease

Abstract: Sickle cell disease (SCD) is a pleiotropic genetic disorder of hemoglobin that has profound multiorgan effects. The low prevalence of SCD ( approximately 100,000/US) has limited progress in clinical, basic, and translational research. Lack of a large, readily accessible population for clinical studies has contributed to the absence of standard definitions and diagnostic criteria for the numerous complications of SCD and inadequate understanding of SCD pathophysiology. In 2005, the Comprehensive Sickle Cell Centers initiated a project to establish consensus definitions of the most frequently occurring complications. A group of clinicians and scientists with extensive expertise in research and treatment of SCD gathered to identify and categorize the most common complications. From this group, a formal writing team was formed that further reviewed the literature, sought specialist input, and produced definitions in a standard format. This article provides an overview of the process and describes 12 body system categories and the most prevalent or severe complications within these categories. A detailed Appendix provides standardized definitions for all complications identified within each system. This report proposes use of these definitions for studies of SCD complications, so future studies can be comparably robust and treatment efficacy measured. Use of these definitions will support greater accuracy in genotype-phenotype studies, thereby achieving a better understanding of SCD pathophysiology. This should nevertheless be viewed as a dynamic rather than final document; phenotype descriptions should be reevaluated and revised periodically to provide the most current standard definitions as etiologic factors are better understood, and new diagnostic options are developed.

Living with iron (and oxygen): questions and answers about iron homeostasis.

Abstract: Mechanisms to regulate iron homeostasis are very likely billions of years older than those for oxygen homeostasis since contemporary microbes regulate iron in the absence of oxygen and may model ancient organisms that lived before atmospheric oxygen appeared.1 Moreover, proteins that manage iron and oxidants such as the mini-ferritins in contemporary bacteria, also called Dps (DNA protection during stress) proteins, are expressed in anaerobic archea.2 Mini-ferritins are 12 subunit protein cages from archea and bacteria that contrast with maxi-ferritins, the 24 subunit cages from bacteria, animals and plants, which use iron and dioxygen as substrates, by consuming iron and hydrogen peroxide as substrates to make the mineral inside protein nanocages. Such peroxide-consuming ferritins may be progenitors of modern ferritins and may have contributed to the transition from anaerobic to aerobic life with iron and oxygen. Iron and oxygen homeostasis, in animals, integrate DNA/mRNA controls; regulation of these two processes intersect along many pathways. Iron homeostasis occurs within cells and between cells to confer balance throughout tissues and the organism. Each cell or tissue type will have a different iron set point for homeostasis that reflects the specific role of each cell type. For example, animal red blood cells need much more iron than epithelial cells because of the synthesis of hemoglobin.3 Similarly, in plants, leaves contain much more iron than flowers because of the synthesis of ferredoxins important in photosynthesis.4 Growing animals or plants will also have different requirements for iron than aging animals or senescent plants. Thus, the conditions that cause changes in iron homeostasis may vary depending on age or specialized function, explaining, in part, quantitatively different results that can be obtained with cultured cell lines derived from different tissues. Metal ion homeostasis has common features shared by all the metal ions that include cell uptake, cell efflux, and intracellular transport. However, the chemical properties of iron require additional iron–specific homeostatic features compared to other metals, because of the hydrolytic properties of ferric ion under physiological condition. Hydrated ferric ions are relatively strong acids; protons in water coordinated to ferric ions have a pKa ≈ 3. The conjugate bases of hydrated ferric ions form multinuclear species rapidly, accounting for the low solubility of aqueous ferric ions (10−18 M) and for rust formation. In addition, living cells and organisms use much more iron than other metals. For example, the human body contains ~3.5 g of iron compared to 100 mg of copper. Iron concentrations in cells are much higher than the solubility of free ferric ions in aqueous solution in air. Average iron concentrations in cells are ~10−4 M requiring, since the solubility of free ferric ions in water/plasma is 10−18 M, a concentration gradient of 100 trillion between aqueous, external environments and intracellular environments. The concentration difference is achieved using transporters (membrane), intracellular carriers and concentrators, cellular exporters, and, in multicellular organisms, extracellular carriers. Except for iron concentrators such as ferritins, the uses of receptors, transporters, and intracellular carriers is shared with other metal ions, such as copper, manganese, etc. The ferritins, which concentrate and store intracellular iron at concentrations far above the solubility of the free ion, are unique for iron homeostasis, compared to other metal ions. Many different chemical species of iron exist in the environment that are available for biological absorption. Examples include iron–protoporphyrin IX, iron chelates, inorganic iron salts, polynuclear iron, and, at least in animals, iron minerals stored in ferritin.5,6 Iron acquisition related to homeostasis is discussed elsewhere in this issue. Studies on iron homeostasis are rarely related to the chemical species of iron used. The working model of iron in transit is Fe(II) bound to chaperone or carrier sites of varying kinetic and equilibrium stabilities. Thus, this review will focus on iron homeostasis independently of iron speciation.

High-resolution, high-throughput HLA genotyping by next-generation sequencing

Abstract: The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome. Hematopoietic stem cell transplantation requires allele-level HLA typing at multiple loci to select the best matched unrelated donors for recipient patients. In current methods for HLA typing, both alleles of a heterozygote are amplified and typed or sequenced simultaneously, often making it difficult to unambiguously determine the sequence of the two alleles. Next-generation sequencing methods clonally propagate in parallel millions of single DNA molecules, which are then also sequenced in parallel. Recently, the read lengths obtainable by one such next-generation sequencing method (454 Life Sciences, Inc.) have increased to >250 nucleotides. These clonal read lengths make possible setting the phase of the linked polymorphisms within an exon and thus the unambiguous determination of the sequence of each HLA allele. Here we demonstrate this capacity as well as show that the throughput of the system is sufficiently high to enable a complete, 7-locus HLA class I and II typing for 24 or 48 individual DNAs in a single GS FLX sequencing run. Highly multiplexed amplicon sequencing is facilitated by the use of sample-specific internal sequence tags (multiplex identification tags or MIDs) in the primers that allow pooling of samples yet maintain the ability to assign sequences to specific individuals. We have incorporated an HLA typing software application developed by Conexio Genomics (Freemantle, Australia) that assigns HLA genotypes for these 7 loci (HLA-A, -B, -C, DRB1, DQA1, DQB1, DPB1), as well as for DRB3, DRB4, and DRB5 from 454 sequence data. The potential of this HLA sequencing system to analyze chimeric mixtures is demonstrated here by the detection of a rare HLA-B allele in a mixture of two homozygous cell lines (1/100), as well as by the detection of the rare nontransmitted maternal allele present in the blood of a severe combined immunodeficiency disease syndrome (SCIDS) patient.

Genomic expression profiling across the pediatric systemic inflammatory response syndrome, sepsis, and septic shock spectrum*

Abstract: The systemic inflammatory response syndrome (SIRS) is common in critically ill children (1). SIRS is not a specific disease and recognition of SIRS does not necessarily drive clinical decision processes (2). SIRS is defined by alterations of three fundamental physiologic variables and one basic laboratory variable (3). Although the clinical utility of SIRS is debatable (4, 5), at a minimum, the SIRS concept serves to broadly describe the clinical and heterogeneous systemic inflammatory response of critically ill patients to a variety of insults, including trauma, surgery, and pancreatitis (2). When critically ill patients meet criteria for SIRS, in the context of infection, they are said to have “sepsis.” Critically ill patients with sepsis, who progress to the development of cardiovascular and other organ failure, are said to have “septic shock” or “severe sepsis.” Although sepsis and septic shock are also highly heterogeneous clinical syndromes, the recognition of sepsis and septic shock does drive clinical decision processes and dictates specific therapeutic strategies (2, 6). Thus, in critically ill children, there exists a clinically relevant, conceptual spectrum of increasing illness severity and specificity ranging from SIRS to sepsis to septic shock. In an ongoing translational research program based on genome-wide expression profiling, we have generated novel observations in children with septic shock (7–9). An important caveat to our observations surrounds the issue of specificity. That is, all of our previous genome-wide expression studies have focused exclusively on children with septic shock, while ignoring the categories of SIRS and sepsis. Thus, an important question to address is: are the genome-wide expression signatures that we have derived for pediatric septic shock, and the resulting hypotheses, specific to septic shock, or are they generalizable to other populations of critically ill children? We recently addressed this question, in part, by formally validating our previous observations in a separate validation septic shock cohort (10). In the current study, we have addressed this question by directly comparing the genome-wide expression signatures of children with SIRS, sepsis, or septic shock.

Binding of Complement Factor H (fH) to Neisseria meningitidis Is Specific for Human fH and Inhibits Complement Activation by Rat and Rabbit Sera

Abstract: Complement factor H (fH), a molecule that downregulates complement activation, binds to Neisseria meningitidis and increases resistance to serum bactericidal activity. We investigated the species specificity of fH binding and the effect of human fH on downregulating rat (relevant for animal models) and rabbit (relevant for vaccine evaluation) complement activation. Binding to N. meningitidis was specific for human fH (low for chimpanzee fH and not detected with fH from lower primates). The addition of human fH decreased rat and rabbit C3 deposition on the bacterial surface and decreased group C bactericidal titers measured with rabbit complement 10- to 60-fold in heat-inactivated sera from human vaccinees. Administration of human fH to infant rats challenged with group B strain H44/76 resulted in an fH dose-dependent increase in CFU/ml of bacteria in blood 8 h later (P < 0.02). At the highest fH dose, 50 μg/rat, the geometric mean number of CFU per ml was higher than that in control animals (1,050 versus 43 [P < 0.005]). The data underscore the importance of binding of human fH for survival of N. meningitidis in vitro and in vivo. The species specificity of binding of human fH adds another mechanism toward our understanding of why N. meningitidis is strictly a human pathogen.

A prospective study of ventilator-associated pneumonia in children.

Abstract: OBJECTIVE. We conducted a prospective, observational study in a tertiary care pediatric center to determine risk factors for the development of and outcomes from ventilator-associated pneumonia. METHODS. From November 2004 to June 2005, all NICU and PICU patients mechanically ventilated for >24 hours were eligible for enrollment after parental consent. The primary outcome measure was the development of ventilator-associated pneumonia, which was defined by both Centers for Disease Control and Prevention/National Nosocomial Infections Surveillance criteria and clinician diagnosis. Secondary outcome measures were length of mechanical ventilation, hospital and ICU length of stay, hospital cost, and death. RESULTS. Fifty-eight patients were enrolled. The median age was 6 months, and 57% were boys. The most common ventilator-associated pneumonia organisms identified were Gram-negative bacteria (42%), Staphylococcus aureus (22%), and Haemophilus influenzae (11%). On multivariate analysis, female gender, postsurgical admission diagnosis, presence of enteral feeds, and use of narcotic medications were associated with ventilator-associated pneumonia. Patients with ventilator-associated pneumonia had greater need for mechanical ventilation (12 vs 22 median ventilator-free days), longer ICU length of stay (6 vs 13 median ICU-free days), higher total median hospital costs ($308534 vs $252652), and increased absolute hospital mortality (10.5% vs 2.4%) than those without ventilator-associated pneumonia. CONCLUSIONS. In mechanically ventilated, critically ill children, those with ventilator-associated pneumonia had a prolonged need for mechanical ventilation, a longer ICU stay, and a higher mortality rate. Female gender, postsurgical diagnosis, the use of narcotics, and the use of enteral feeds were associated with an increased risk of developing ventilator-associated pneumonia in these patients.

118 SNPs of folate-related genes and risks of spina bifida and conotruncal heart defects

Abstract: Folic acid taken in early pregnancy reduces risks for delivering offspring with several congenital anomalies. The mechanism by which folic acid reduces risk is unknown. Investigations into genetic variation that influences transport and metabolism of folate will help fill this data gap. We focused on 118 SNPs involved in folate transport and metabolism. Using data from a California population-based registry, we investigated whether risks of spina bifida or conotruncal heart defects were influenced by 118 single nucleotide polymorphisms (SNPs) associated with the complex folate pathway. This case-control study included 259 infants with spina bifida and a random sample of 359 nonmalformed control infants born during 1983–86 or 1994–95. It also included 214 infants with conotruncal heart defects born during 1983–86. Infant genotyping was performed blinded to case or control status using a designed SNPlex assay. We examined single SNP effects for each of the 118 SNPs, as well as haplotypes, for each of the two outcomes. Few odds ratios (ORs) revealed sizable departures from 1.0. With respect to spina bifida, we observed ORs with 95% confidence intervals that did not include 1.0 for the following SNPs (heterozygous or homozygous) relative to the reference genotype: BHMT (rs3733890) OR = 1.8 (1.1–3.1), CBS (rs2851391) OR = 2.0 (1.2–3.1); CBS (rs234713) OR = 2.9 (1.3–6.7); MTHFD1 (rs2236224) OR = 1.7 (1.1–2.7); MTHFD1 (hcv11462908) OR = 0.2 (0–0.9); MTHFD2 (rs702465) OR = 0.6 (0.4–0.9); MTHFD2 (rs7571842) OR = 0.6 (0.4–0.9); MTHFR (rs1801133) OR = 2.0 (1.2–3.1); MTRR (rs162036) OR = 3.0 (1.5–5.9); MTRR (rs10380) OR = 3.4 (1.6–7.1); MTRR (rs1801394) OR = 0.7 (0.5–0.9); MTRR (rs9332) OR = 2.7 (1.3–5.3); TYMS (rs2847149) OR = 2.2 (1.4–3.5); TYMS (rs1001761) OR = 2.4 (1.5–3.8); and TYMS (rs502396) OR = 2.1 (1.3–3.3). However, multiple SNPs observed for a given gene showed evidence of linkage disequilibrium indicating that the observed SNPs were not individually contributing to risk. We did not observe any ORs with confidence intervals that did not include 1.0 for any of the studied SNPs with conotruncal heart defects. Haplotype reconstruction showed statistical evidence of nonrandom associations with TYMS, MTHFR, BHMT and MTR for spina bifida. Our observations do not implicate a particular folate transport or metabolism gene to be strongly associated with risks for spina bifida or conotruncal defects.

Mechanisms and Function of DUOX in Epithelia of the Lung

Abstract: The human lung produces considerable amounts of H2O2 In the normal uninflamed epithelium of both the airways and the alveoli, mucosal release of H2O2 is readily detected both in cell cultures in vitro and in the exhaled breath of humans The dual oxidases DUOX1 and DUOX2 are the H2O2-producing isoforms of the NADPH oxidase family found in epithelial cells The DUOXs are prominently expressed at the apical cell pole of ciliated cells in the airways and in type II cells of the alveoli Recent studies focused on the functional consequences of H2O2 release by DUOX into the lung lining fluid In the airways, a major function of DUOX is to support lactoperoxidase (LPO) to generate bactericidal OSCN−, and there are indications that the DUOX/LPO defense system is critically dependent on the function of the CFTR Cl− channel, which provides both SCN− (for LPO function) and HCO3− (for pH adjustment) to the airway surface liquid Although DUOX is also functional in the alveolar epithelium, no comparable heme peroxidase is present in the alveolus, and thus DUOX-mediated H2O2 release by alveolar cells may have other functions, such as cellular signaling Antioxid Redox Signal 11, 2453–2465

Orofacial clefts in the National Birth Defects Prevention Study, 1997–2004

Abstract: Orofacial clefts are among the most common types of birth defects, but their clinical presentation has not been well described in a geographically diverse US population. To describe the birth prevalence and phenotype of nonsyndromic clefts, we used data from the National Birth Defects Prevention Study (NBDPS), a multi-site, population-based, case-control study aimed at identifying genetic and environmental risk factors for birth defects. Included in the study were infants born during 1997-2004 with a cleft lip (CL), cleft lip with cleft palate (CLP), or cleft palate (CP). Infants with clefts associated with recognized single-gene disorders, chromosome abnormalities, holoprosencephaly, or amniotic band sequence were excluded. A total of 3,344 infants with nonsyndromic orofacial clefts were identified, including 751 with CL, 1,399 with CLP, and 1,194 with CP, giving birth prevalence estimates of 0.3, 0.5, and 0.4/1,000 live births, respectively. Among infants with CLP where cleft laterality was specified, about twice as many had unilateral vs. bilateral involvement, while for CL there were over 10 times as many with unilateral versus bilateral involvement. Involvement was most often left-sided. About one-quarter of infants with CP had Pierre Robin sequence. Over 80% of infants had an isolated orofacial cleft. Among infants with CL or CLP, heart, limb, and other musculoskeletal defects were most commonly observed, while heart, limb, and central nervous system defects were most common among infants with CP. Better understanding of the birth prevalence and phenotype may help guide clinical care as well as contribute to an improved understanding of pathogenesis.

Potential application of mesenchymal stem cells in acute lung injury

Abstract: Despite extensive research into the pathogenesis of acute lung injury and the acute respiratory distress syndrome (ALI/ARDS), mortality remains high at approximately 40%. Current treatment is primarily supportive, with lung-protective ventilation and a fluid conservative strategy. Pharmacologic therapies that reduce the severity of lung injury in experimental studies have not yet been translated into effective clinical treatment options. Therefore, innovative therapies are needed. Recent studies have suggested that bone-marrow-derived multipotent mesenchymal stem cells (MSC) may have therapeutic applications in multiple clinical disorders including myocardial infarction, diabetes, sepsis, hepatic and acute renal failure. Recently, MSC have been studied in several in vivo models of lung disease. This review focuses on first describing the existing experimental literature that has tested the use of MSC in models of ALI/ARDS, and then the potential mechanisms underlying their therapeutic use with an emphasis on secreted paracrine soluble factors. The review concludes with a discussion of future research directions required for potential clinical trials.

Newborn Screening for Hemoglobinopathies in California

Abstract: Background. Newborn screening (NBS) for hemoglobinopathies facilitates early identification of affected individuals to ensure the prompt institution of comprehensive medical care for affected newborns in California. When linked to extensive follow-up and education, NBS has been shown to significantly reduce mortality in children with sickle cell disease. Due to changing immigration patterns from Asia and Latin America, the State of California has witnessed an increased prevalence of clinically significant hemoglobin (Hb) disorders, including those resulting from novel genotypes. In 1999, newborn screening for Hb H disorders was incorporated in the statewide hemoglobinopathy screening program. Procedure. Primary screening for hemoglobin variants was performed using high performance liquid chromatography. Confirmatory testing on hemoglobinopathy mutations was performed by electropheresis techniques and genotyping methods. Results. Of

FOXE1 association with both isolated cleft lip with or without cleft palate, and isolated cleft palate

Abstract: Nonsyndromic orofacial clefts are a common complex birth defect caused by genetic and environmental factors and/or their interactions. A previous genome-wide linkage scan discovered a novel locus for cleft lip with or without cleft palate (CL/P) at 9q22-q33. To identify the etiologic gene, we undertook an iterative and complementary fine mapping strategy using family-based CL/P samples from Colombia, USA and the Philippines. Candidate genes within 9q22-q33 were sequenced, revealing 32 new variants. Concurrently, 397 SNPs spanning the 9q22-q33 2-LOD-unit interval were tested for association. Significant SNP and haplotype association signals (P = 1.45E - 08) narrowed the interval to a 200 kb region containing: FOXE1, C9ORF156 and HEMGN. Association results were replicated in CL/P families of European descent and when all populations were combined the two most associated SNPs, rs3758249 (P = 5.01E - 13) and rs4460498 (P = 6.51E - 12), were located inside a 70 kb high linkage disequilibrium block containing FOXE1. Association signals for Caucasians and Asians clustered 5' and 3' of FOXE1, respectively. Isolated cleft palate (CP) was also associated, indicating that FOXE1 plays a role in two phenotypes thought to be genetically distinct. Foxe1 expression was found in the epithelium undergoing fusion between the medial nasal and maxillary processes. Mutation screens of FOXE1 identified two family-specific missense mutations at highly conserved amino acids. These data indicate that FOXE1 is a major gene for CL/P and provides new insights for improved counseling and genetic interaction studies.

Small cell undifferentiated variant of hepatoblastoma: adverse clinical and molecular features similar to rhabdoid tumors.

Abstract: Background Small cell undifferentiated (SCU) histology in patients with stage I hepatoblastoma (HB) predicts an increased risk of relapse. We sought to determine the significance of SCU histology in patients with unresectable HB. Procedure Patients enrolled on the pediatric Intergroup (INT0098) trial for HB and patients from the personal consultation files of two of the authors (MF, LG) were reviewed for cases with SCU histology. These patients were compared with SCU HB patients identified by literature review. Results Eleven patients were studied. All patients with reported AFP results exhibited normal or minimally increased serum AFP levels. None of the patients survived: 10 died of disease progression, and 1 died from treatment complications. Immunostaining revealed that tumors from six of six patients tested were INI1 negative. Cytogenetic and molecular abnormalities in one patient (and two patients from the literature review) were similar to those described in rhabdoid tumors. Comparison with patients from the literature review revealed similar results except that 4 of 29 patients survived without evidence of disease. Conclusions SCU histology in HB patients is associated with an adverse outcome. These tumors appear to be biologically different from non-SCU HB. Evaluation of patient characteristics and outcomes for children with SCU HB and/or those with low AFP levels should be determined from large cooperative group studies. In the meantime, we suggest patients with unresectable HB containing SCU elements have careful cytogenetic, molecular, and immunohistochemical evaluation to ascertain rhabdoid features and receive treatment that differs from that provided for other HB patients. Pediatr Blood Cancer 2009;52:328–334. © 2008 Wiley-Liss, Inc.

The value of avian genomics to the conservation of wildlife

Abstract: Genomic studies in non-domestic avian models, such as the California condor and white-throated sparrow, can lead to more comprehensive conservation plans and provide clues for understanding mechanisms affecting genetic variation, adaptation and evolution. Developing genomic tools and resources including genomic libraries and a genetic map of the California condor is a prerequisite for identification of candidate loci for a heritable embryonic lethal condition. The white-throated sparrow exhibits a stable genetic polymorphism (i.e. chromosomal rearrangements) associated with variation in morphology, physiology, and behavior (e.g., aggression, social behavior, sexual behavior, parental care). In this paper we outline the utility of these species as well as report on recent advances in the study of their genomes. Genotyping of the condor resource population at 17 microsatellite loci provided a better assessment of the current population's genetic variation. Specific New World vulture repeats were found in the condor genome. Using condor BAC library and clones, chicken-condor comparative maps were generated. A condor fibroblast cell line transcriptome was characterized using the 454 sequencing technology. Our karyotypic analyses of the sparrow in combination with other studies indicate that the rearrangements in both chromosomes 2m and 3a are complex and likely involve multiple inversions, interchromosomal linkage, and pleiotropy. At least a portion of the rearrangement in chromosome 2m existed in the common ancestor of the four North American species of Zonotrichia, but not in the one South American species, and that the 2m form, originally thought to be the derived condition, might actually be the ancestral one. Mining and characterization of candidate loci in the California condor using molecular genetic and genomic techniques as well as linkage and comparative genomic mapping will eventually enable the identification of carriers of the chondrodystrophy allele, resulting in improved genetic management of this disease. In the white-throated sparrow, genomic studies, combined with ecological data, will help elucidate the basis of genic selection in a natural population. Morphs of the sparrow provide us with a unique opportunity to study intraspecific genomic differences, which have resulted from two separate yet linked evolutionary trajectories. Such results can transform our understanding of evolutionary and conservation biology.

High-density SNP screening of the major histocompatibility complex in systemic lupus erythematosus demonstrates strong evidence for independent susceptibility regions.

Abstract: A substantial genetic contribution to systemic lupus erythematosus (SLE) risk is conferred by major histocompatibility complex (MHC) gene(s) on chromosome 6p21. Previous studies in SLE have lacked statistical power and genetic resolution to fully define MHC influences. We characterized 1,610 Caucasian SLE cases and 1,470 parents for 1,974 MHC SNPs, the highly polymorphic HLA-DRB1 locus, and a panel of ancestry informative markers. Single-marker analyses revealed strong signals for SNPs within several MHC regions, as well as with HLA-DRB1 (global p = 9.99 x 10(-16)). The most strongly associated DRB1 alleles were: *0301 (odds ratio, OR = 2.21, p = 2.53 x 10(-12)), *1401 (OR = 0.50, p = 0.0002), and *1501 (OR = 1.39, p = 0.0032). The MHC region SNP demonstrating the strongest evidence of association with SLE was rs3117103, with OR = 2.44 and p = 2.80 x 10(-13). Conditional haplotype and stepwise logistic regression analyses identified strong evidence for association between SLE and the extended class I, class I, class III, class II, and the extended class II MHC regions. Sequential removal of SLE-associated DRB1 haplotypes revealed independent effects due to variation within OR2H2 (extended class I, rs362521, p = 0.006), CREBL1 (class III, rs8283, p = 0.01), and DQB2 (class II, rs7769979, p = 0.003, and rs10947345, p = 0.0004). Further, conditional haplotype analyses demonstrated that variation within MICB (class I, rs3828903, p = 0.006) also contributes to SLE risk independent of HLA-DRB1*0301. Our results for the first time delineate with high resolution several MHC regions with independent contributions to SLE risk. We provide a list of candidate variants based on biologic and functional considerations that may be causally related to SLE risk and warrant further investigation.

Neuronal mitochondrial amelioration by feeding acetyl-L-carnitine and lipoic acid to aged rats

Abstract: Brain function declines with age and is associated with diminishing mitochondrial integrity. The neuronal mitochondrial ultrastructural changes of young (4 months) and old (21 months) F344 rats supplemented with two mitochondrial metabolites, acetyl-L-carnitine (ALCAR, 0.2%[wt/vol] in the drinking water) and R-α-lipoic acid (LA, 0.1%[wt/wt] in the chow), were analysed using qualitative and quantitative electron microscopy techniques. Two independent morphologists blinded to sample identity examined and scored all electron micrographs. Mitochondria were examined in each micrograph, and each structure was scored according to the degree of injury. Controls displayed an age-associated significant decrease in the number of intact mitochondria (P = 0.026) as well as an increase in mitochondria with broken cristae (P < 0.001) in the hippocampus as demonstrated by electron microscopic observations. Neuronal mitochondrial damage was associated with damage in vessel wall cells, especially vascular endothelial cells. Dietary supplementation of young and aged animals increased the proliferation of intact mitochondria and reduced the density of mitochondria associated with vacuoles and lipofuscin. Feeding old rats ALCAR and LA significantly reduced the number of severely damaged mitochondria (P = 0.02) and increased the number of intact mitochondria (P < 0.001) in the hippocampus. These results suggest that feeding ALCAR with LA may ameliorate age-associated mitochondrial ultrastructural decay and are consistent with previous studies showing improved brain function.

Population admixture associated with disease prevalence in the Boston Puerto Rican health study

Abstract: Older Puerto Ricans living in the continental U.S. suffer from higher rates of diabetes, obesity, cardiovascular disease and depression compared to non-Hispanic White populations. Complex diseases, such as these, are likely due to multiple, potentially interacting, genetic, environmental and social risk factors. Presumably, many of these environmental and genetic risk factors are contextual. We reasoned that racial background may modify some of these risk factors and be associated with health disparities among Puerto Ricans. The contemporary Puerto Rican population is genetically heterogeneous and originated from three ancestral populations: European settlers, native Taino Indians, and West Africans. This rich-mixed ancestry of Puerto Ricans provides the intrinsic variability needed to untangle complex gene-environment interactions in disease susceptibility and severity. Herein, we determined whether a specific ancestral background was associated with either of four major disease outcomes (diabetes, obesity, cardiovascular disease, and depression). We estimated the genetic ancestry of 1,129 subjects from the Boston Puerto Rican Health Study based on genotypes of 100 ancestry informative markers (AIMs). We examined the effects of ancestry on tests of association between single AIMs and disease traits. The ancestral composition of this population was 57.2% European, 27.4% African, and 15.4% Native American. African ancestry was negatively associated with type 2 diabetes and cardiovascular disease, and positively correlated with hypertension. It is likely that the high prevalence rate of diabetes in Africans, Hispanics, and Native Americans is not due to genetic variation alone, but to the combined effects of genetic variation interacting with environmental and social factors.

Pharmacogenomic Discovery Using Cell-Based Models

Abstract: Quantitative variation in response to drugs in human populations is multifactorial; genetic factors probably contribute to a significant extent. Identification of the genetic contribution to drug response typically comes from clinical observations and use of classic genetic tools. These clinical studies are limited by our inability to control environmental factors in vivo and the difficulty of manipulating the in vivo system to evaluate biological changes. Recent progress in dissecting genetic contribution to natural variation in drug response through the use of cell lines has been made and is the focus of this review. A general overview of current cell-based models used in pharmacogenomic discovery and validation is included. Discussion includes the current approach to translate findings generated from these cell-based models into the clinical arena and the use of cell lines for functional studies. Specific emphasis is given to recent advances emerging from cell line panels, including the International HapMap Project and the NCI60 cell panel. These panels provide a key resource of publicly available genotypic, expression, and phenotypic data while allowing researchers to generate their own data related to drug treatment to identify genetic variation of interest. Interindividual and interpopulation differences can be evaluated because human lymphoblastoid cell lines are available from major world populations of European, African, Chinese, and Japanese ancestry. The primary focus is recent progress in the pharmacogenomic discovery area through ex vivo models.

Conformational flexibility of metazoan fatty acid synthase enables catalysis

Abstract: The metazoan cytosolic fatty acid synthase (FAS) contains all of the enzymes required for de novo fatty acid biosynthesis covalently linked around two reaction chambers Although the three-dimensional architecture of FAS has been mostly defined, it is unclear how reaction intermediates can transfer between distant catalytic domains Using single-particle EM, we have identified a near continuum of conformations consistent with a remarkable flexibility of FAS The distribution of conformations was influenced by the presence of substrates and altered by different catalytic mutations, suggesting a direct correlation between conformation and specific enzymatic activities We interpreted three-dimensional reconstructions by docking high-resolution structures of individual domains, and they show that the substrate-loading and condensation domains dramatically swing and swivel to access substrates within either reaction chamber Concomitant rearrangement of the beta-carbon-processing domains synchronizes acyl chain reduction in one chamber with acyl chain elongation in the other

Local Suppression of T Cell Responses by Arginase-Induced L-Arginine Depletion in Nonhealing Leishmaniasis

Abstract: The balance between T helper (Th) 1 and Th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized Th1 or Th2 responses are not sufficient to account for healing or nonhealing. Here we show that high arginase activity, a hallmark of nonhealing disease, is primarily expressed locally at the site of pathology. The high arginase activity causes local depletion of L-arginine, which impairs the capacity of T cells in the lesion to proliferate and to produce interferon-γ, while T cells in the local draining lymph nodes respond normally. Healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. Moreover, competitive inhibition of arginase as well as supplementation with L-arginine restored T cell effector functions and reduced pathology and parasite growth at the site of lesions. These results demonstrate that in nonhealing leishmaniasis, arginase-induced L-arginine depletion results in impaired T cell responses. Our results identify a novel mechanism in leishmaniasis that contributes to the failure to heal persistent lesions and suggest new approaches to therapy.

Predicting Phenotype and Emerging Strains among Chlamydia trachomatis Infections

Abstract: Chlamydia trachomatis is a global cause of blinding trachoma and sexually transmitted infections (STIs). We used comparative genomics of the family Chlamydiaceae to select conserved housekeeping genes for C. trachomatis multilocus sequencing, characterizing 19 reference and 68 clinical isolates from 6 continental/subcontinental regions. There were 44 sequence types (ST). Identical STs for STI isolates were recovered from different regions, whereas STs for trachoma isolates were restricted by continent. Twenty-nine of 52 alleles had nonuniform distributions of frequencies across regions (p<0.001). Phylogenetic analysis showed 3 disease clusters: invasive lymphogranuloma venereum strains, globally prevalent noninvasive STI strains (ompA genotypes D/Da, E, and F), and nonprevalent STI strains with a trachoma subcluster. Recombinant strains were observed among STI clusters. Single nucleotide polymorphisms (SNPs) were predictive of disease specificity. Multilocus and SNP typing can now be used to detect diverse and emerging C. trachomatis strains for epidemiologic and evolutionary studies of trachoma and STI populations worldwide.

Mitochondrial Decay in the Brains of Old Rats: Ameliorating Effect of Alpha-Lipoic Acid and Acetyl-L-carnitine

Abstract: To investigate the mitochondrial decay and oxidative damage resulting from aging, the activities/kinetics of the mitochondrial complexes were examined in the brains of young and old rats as well as in old rats fed R-α-lipoic acid plus acetyl-l-carnitine (LA/ALC). The brain mitochondria of old rats, compared with young rats, had significantly decreased endogenous antioxidants and superoxide dismutase activity; more oxidative damage to lipids and proteins; and decreased activities of complex I, IV and V. Complex I showed a decrease in binding affinity (increase in Km) for substrates. Feeding LA/ALC to old rats partially restored age-associated mitochondrial dysfunction to the levels of the young rats. These results indicate that oxidative mitochondrial decay plays an important role in brain aging and that a combination of nutrients targeting mitochondria, such as LA/ALC, could ameliorate mitochondrial decay through preventing mitochondrial oxidative damage.