Philip A. Wood

X-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired NF-kappaB signaling.

The molecular basis of X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) has remained elusive. Here we report hypomorphic mutations in the gene IKBKG in 12 males with EDA-ID from 8 kindreds, and 2 patients with a related and hitherto unrecognized syndrome of EDA-ID with osteopetrosis and lymphoedema (OL-EDA-ID). Mutations in the coding region of IKBKG are associated with EDA-ID, and stop codon mutations, with OL-EDA-ID. IKBKG encodes NEMO, the regulatory subunit of the IKK (IκB kinase) complex, which is essential for NF-κB signaling. Germline loss-of-function mutations in IKBKG are lethal in male fetuses. We show that IKBKG mutations causing OL-EDA-ID and EDA-ID impair but do not abolish NF-κB signaling. We also show that the ectodysplasin receptor, DL, triggers NF-κB through the NEMO protein, indicating that EDA results from impaired NF-κB signaling. Finally, we show that abnormal immunity in OL-EDA-ID patients results from impaired cell responses to lipopolysaccharide, interleukin (IL)-1β, IL-18, TNFα and CD154. We thus report for the first time that impaired but not abolished NF-κB signaling in humans results in two related syndromes that associate specific developmental and immunological defects.

Mitochondrial dysfunction due to long-chain Acyl-CoA dehydrogenase deficiency causes hepatic steatosis and hepatic insulin resistance

Alterations in mitochondrial function have been implicated in the pathogenesis of insulin resistance and type 2 diabetes. However, it is unclear whether the reduced mitochondrial function is a primary or acquired defect in this process. To determine whether primary defects in mitochondrial β-oxidation can cause insulin resistance, we studied mice with a deficiency of long-chain acyl-CoA dehydrogenase (LCAD), a key enzyme in mitochondrial fatty acid oxidation. Here, we show that LCAD knockout mice develop hepatic steatosis, which is associated with hepatic insulin resistance, as reflected by reduced insulin suppression of hepatic glucose production during a hyperinsulinemic-euglycemic clamp. The defects in insulin action were associated with an ≈40% reduction in insulin-stimulated insulin receptor substrate-2-associated phosphatidylinositol 3-kinase activity and an ≈50% decrease in Akt2 activation. These changes were associated with increased PKCε activity and an aberrant 4-fold increase in diacylglycerol content after insulin stimulation. The increase in diacylglycerol concentration was found to be caused by de novo synthesis of diacylglycerol from medium-chain acyl-CoA after insulin stimulation. These data demonstrate that primary defects in mitochondrial fatty acid oxidation capacity can lead to diacylglycerol accumulation, PKCε activation, and hepatic insulin resistance.

Failure to clear persistent vaccine-derived neurovirulent poliovirus infection in an immunodeficient man

BACKGROUND Individuals who chronically excrete neurovirulent poliovirus of vaccine-origin are of considerable concern to the Global Polio Eradication programme. Chronic infection with such polioviruses is a recognised complication of hypogammaglobulinaemia. METHODS We did a series of in-vitro and in-vivo therapeutic studies, with a view to clearing persistent neurovirulent poliovirus infection in an individual with common variable immunodeficiency, using oral immunoglobulin, breast milk (as a source of secretory IgA), ribavirin, and the anti-picornaviral agent pleconaril. We undertook viral quantitation, antibody neutralisation and drug susceptibility assays, and viral gene sequencing. FINDINGS Long-term asymptomatic excretion of vaccine-derived neurovirulent poliovirus 2 was identified in this hypogammaglobulinaemic man, and was estimated to have persisted for up to 22 years. Despite demonstrable in-vitro neutralising activity of immunoglobulin and breast milk, and in-vitro antiviral activity of ribavirin, no treatment was successful at clearing the virus, although in one trial breast milk significantly reduced excretion levels temporarily. During the course of study, the virus developed reduced susceptibility to pleconaril, precluding the in-vivo use of this drug. Sequence analysis revealed the emergence of a methionine to leucine mutation adjacent to the likely binding site of pleconaril in these isolates. INTERPRETATION Chronic vaccine-associated poliovirus infection in hypogammaglobulinaemia is a difficult condition to treat. It represents a risk to the strategy to discontinue polio vaccination once global eradication has been achieved.

Deficiency in short-chain fatty acid β-oxidation affects theta oscillations during sleep

In rodents, the electroencephalogram (EEG) during paradoxical sleep and exploratory behavior is characterized by theta oscillations. Here we show that a deficiency in short-chain acyl-coenzyme A dehydrogenase (encoded by Acads) in mice causes a marked slowing in theta frequency during paradoxical sleep only. We found Acads expression in brain regions involved in theta generation, notably the hippocampus. Microarray analysis of gene expression in mice with mutations in Acads indicates overexpression of Glo1 (encoding glyoxylase 1), a gene involved in the detoxification of metabolic by-products. Administration of acetyl-L-carnitine (ALCAR) to mutant mice significantly recovers slow theta and Glo1 overexpression. Thus, an underappreciated metabolic pathway involving fatty acid β-oxidation also regulates theta oscillations during sleep.

Hyper-IgM syndrome type 4 with a B lymphocyte–intrinsic selective deficiency in Ig class-switch recombination

Hyper-IgM syndrome (HIGM) is a heterogeneous condition characterized by impaired Ig class-switch recombination (CSR). The molecular defects that have so far been associated with this syndrome - which affect the CD40 ligand in HIGM type 1 (HIGM1), CD40 in HIGM3, and activation-induced cytidine deaminase (AID) in HIGM2 - do not account for all cases. We investigated the clinical and immunological characteristics of 15 patients with an unidentified form of HIGM. Although the clinical manifestations were similar to those observed in HIGM2, these patients exhibited a slightly milder HIGM syndrome with residual IgG production. We found that B cell CSR was intrinsically impaired. However, the generation of somatic hypermutations was observed in the variable region of the Ig heavy chain gene, as in control B lymphocytes. In vitro studies showed that the molecular defect responsible for this new HIGM entity (HIGM4) occurs downstream of the AID activity, as the AID gene was induced normally and AID-induced DNA double-strand breaks in the switch micro region of the Ig heavy chain locus were detected during CSR as normal. Thus, HIGM4 is probably the consequence of a selective defect either in a CSR-specific factor of the DNA repair machinery or in survival signals delivered to switched B cells.

Abnormal nonshivering thermogenesis in mice with inherited defects of fatty acid oxidation.

When placed in the cold (4 degreesC), BALB/cByJ mice of both genders rapidly lose body temperature as compared with the control strain, C57BL/6J. This sensitivity to cold resembles that previously described for mice with a defect in nonshivering thermogenesis due to the targeted inactivation of the brown adipocyte-specific mitochondrial uncoupling protein gene, Ucp1. Genetic mapping of the trait placed the gene on chromosome 5 near Acads, a gene encoding the short chain acyl CoA dehydrogenase, which is mutated in BALB/cByJ mice. The analysis of candidate genes in the region indicated a defect only in the expression of Acads. Confirmation of the importance of fatty acid oxidation to thermogenesis came from our finding that mice carrying the targeted inactivation of the long chain acyl CoA dehydrogenase gene (Acadl) are also sensitive to the cold. Both of these mutations attenuate the induction of genes normally responsive to adrenergic signaling in brown adipocytes. These results suggest that the action of fatty acids as regulators of gene expression has been perturbed in the mutant mice. From a clinical perspective, it is important to determine whether defects in thermogenesis may be a phenotype in human neonates with inherited deficiencies in fatty acid beta-oxidation.

Short-Chain Acyl-Coenzyme A Dehydrogenase Deficiency in Mice

ABSTRACT: A murine model for short-chain acyl-coenzyme A dehydrogenase (SCAD) deficiency has been identified and characterized in BALB/cByJ mice. These mice have undetectable SCAD activity, severe organic aciduria; excreting ethylmalonic and methylsuccinic acids and Nbutyrylglycine, and develop a fatty liver upon fasting or dietary fat challenge. The mutant mice develop hypoglycemia after an 18-h fast, and have elevated urinary and muscle butyrylcarnitine concentrations. Most of these findings parallel those of human disorders associated with SCAD deficiency and other β-oxidation defects. This mouse model presents important opportunities to investigate the biology of mammalian fatty acid metabolism and the related human diseases.

Impaired interferon gamma-mediated immunity and susceptibility to mycobacterial infection in childhood

Mendelian susceptibility to poorly virulent mycobacteria such as bacillus Calmette-Guerin (BCG) and environmental nontuberculous mycobacteria is a clinically heterogeneous syndrome. The clinical features of affected children cover a continuous spectrum from disseminated lethal bacillus Calmette-Guerin infection to local recurrent nontuberculous mycobacterial infection. Different types of mutations in four genes (IFNGR1, IFNGR2, IL12B, IL12RB1) have revealed both allelic and nonallelic heterogeneity and result in eight different disorders whose common pathogenic pathway is impaired interferon gamma (IFNγ) mediated immunity. The severity of the clinical phenotype depends on the genotype. Complete IL-12 p40 and IL-12 receptor β1 deficiencies and partial IFNγ receptor 1 (IFNγR1) and IFNγR2 deficiencies generally lead to curable infections at various ages, and antibiotics supplemented with IFNγ if required are likely to be effective. Complete IFNγR1 and IFNγR2 deficiencies predispose to overwhelming infection in early childhood, which may respond to antibiotics but relapse when antibiotics are discontinued. Rapid discrimination between complete IFNγR1 and IFNγR2 deficiency and other defects, therefore, is an important diagnostic step for planning clinical management.

Characterization of carnitine and fatty acid metabolism in the long-chain acyl-CoA dehydrogenase-deficient mouse.

In the present paper, we describe a novel method which enables the analysis of tissue acylcarnitines and carnitine biosynthesis intermediates in the same sample. This method was used to investigate the carnitine and fatty acid metabolism in wild-type and LCAD-/- (long-chain acyl-CoA dehydrogenase-deficient) mice. In agreement with previous results in plasma and bile, we found accumulation of the characteristic C14:1-acylcarnitine in all investigated tissues from LCAD-/- mice. Surprisingly, quantitatively relevant levels of 3-hydroxyacylcarnitines were found to be present in heart, muscle and brain in wild-type mice, suggesting that, in these tissues, long-chain 3-hydroxyacyl-CoA dehydrogenase is rate-limiting for mitochondrial beta-oxidation. The 3-hydroxyacylcarnitines were absent in LCAD-/- tissues, indicating that, in this situation, the beta-oxidation flux is limited by the LCAD deficiency. A profound deficiency of acetylcarnitine was observed in LCAD-/- hearts, which most likely corresponds with low cardiac levels of acetyl-CoA. Since there was no carnitine deficiency and only a marginal elevation of potentially cardiotoxic acylcarnitines, we conclude from these data that the cardiomyopathy in the LCAD-/- mouse is caused primarily by a severe energy deficiency in the heart, stressing the important role of LCAD in cardiac fatty acid metabolism in the mouse.

Resistance to High-fat Diet-induced Obesity and Insulin Resistance in Mice with Very Long-chain Acyl-CoA Dehydrogenase Deficiency

Mitochondrial fatty acid oxidation provides an important energy source for cellular metabolism, and decreased mitochondrial fatty acid oxidation has been implicated in the pathogenesis of type 2 diabetes. Paradoxically, mice with an inherited deficiency of the mitochondrial fatty acid oxidation enzyme, very long-chain acyl-CoA dehydrogenase (VLCAD), were protected from high-fat diet-induced obesity and liver and muscle insulin resistance. This was associated with reduced intracellular diacylglycerol content and decreased activity of liver protein kinase Cvarepsilon and muscle protein kinase Ctheta. The increased insulin sensitivity in the VLCAD(-/-) mice were protected from diet-induced obesity and insulin resistance due to chronic activation of AMPK and PPARalpha, resulting in increased fatty acid oxidation and decreased intramyocellular and hepatocellular diacylglycerol content.