3-methylcrotonyl-CoA carboxylase deficiency: Clinical, biochemical, enzymatic and molecular studies in 88 individuals

ABSTRACT: BACKGROUND: Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine metabolism caused by mutations in MCCC1 or MCCC2 encoding the alpha and beta subunit of MCC, respectively. The phenotype is highly variable ranging from acute neonatal onset with fatal outcome to asymptomatic adults. METHODS: We report clinical, biochemical, enzymatic and mutation data of 88 MCC deficient individuals, 53 identified by newborn screening, 26 diagnosed due to clinical symptoms or positive family history and 9 mothers, identified following the positive newborn screening result of their baby. RESULTS: Fifty-seven percent of patients were asymptomatic while 43% showed clinical symptoms, many of which were probably not related to MCC deficiency but due to ascertainment bias. However, 12 patients (5 of 53 identified by newborn screening) presented with acute metabolic decompensations. We identified 15 novel MCCC1 and 16 novel MCCC2 mutant alleles. Additionally, we report expression studies on 3 MCCC1 and 8 MCCC2 mutations and show an overview of all 132 MCCC1 and MCCC2 variants known to date. CONCLUSIONS: Our data confirm that MCC deficiency, despite low penetrance, may lead to a severe clinical phenotype resembling classical organic acidurias. However, neither the genotype nor the biochemical phenotype is helpful in predicting the clinical course.     

Implication of CD38 gene in podocyte epithelial-to-mesenchymal transition and glomerular sclerosis

CD38 is a multifunctional protein involving in a number of signalling pathways. Given that the lack of CD38 is considered as a dedifferentiation marker of lymphocytes and other cells, we hypothesized that CD38 and its signalling pathway may participate in the epithelial-to-mesenchymal transition (EMT) process of podocytes and thereby regulates the integrity of glomerular structure and function. Western blot analysis and RT-PCR demonstrated that renal tissue CD38 expression was lacking in CD38(-/-) mice or substantially reduced in renal CD38 shRNA-transfected WT (CD38-shRNA) mice compared to CD38(+/+) littermates. Confocal fluorescent microscopy demonstrated the reduced expression of epithelial markers (P-Cadherin, ZO-1 and podocin) and increased expression of mesenchymal markers (FSP-1, α-SMA and desmin) in the glomeruli of CD38(-/-) and CD38-shRNA mice compared to CD38(+/+) mice. Morphological examinations showed profound injury in the glomeruli of CD38(-/-) or CD38-shRNA mice compared to CD38(+/+) mice. This enhanced glomerular injury in CD38(-/-) or CD38-shRNA mice was accompanied by increased albuminuria and proteinuria. DOCA/high salt treatment further decreased the expression of epithelial markers and increased the abundance of mesenchymal markers, which were accompanied by more increased glomerular damage index and mean arterial pressure in CD38(-/-) and CD38-shRNA mice than CD38(+/+) mice. In vitro studies showed that inhibition of CD38 enhances the EMT in podocytes. In conclusion, our observations reveal that the normal expression of CD38 importantly contributes to the differentiation and function of podocytes and the defect of this gene expression may be a critical mechanism inducing EMT and consequently resulting in glomerular injury and sclerosis.     

Loss of EphA4 impairs short‐term spatial recognition memory performance and locomotor habituation

EphA4 receptor (EphA4) tyrosine kinase is an important regulator of central nervous system development and synaptic plasticity in the mature brain, but its relevance to the control of normal behavior remains largely unexplored. This study is the first attempt to obtain a behavioral profile of constitutive homozygous and heterozygous EphA4 knockout mice. A deficit in locomotor habituation in the open field, impairment in spatial recognition in the Y‐maze and reduced probability of spatial spontaneous alternation in the T‐maze were identified in homozygous EphA4^?/? mice, while heterozygo us EphA4^+/? mice appeared normal on these tests in comparison with wild‐type (WT) controls. The multiple phenotypes observed in EphA4^?/? mice might stem from an underlying deficit in habituation learning, reflecting an elementary form of nonassociative learning that is in contrast to Pavlovian associative learning, which appeared unaffected by EphA4 disruption. A deficit in motor coordination on the accelerating rotarod was also demonstrated only in EphA4^?/? mice – a finding in keeping with the presence of abnormal gait in EphA4^?/? mice – although they were able to improve performance over training. There was no evidence for substantial changes in major neurochemical markers in various brain regions rich in EphA4 as shown by post‐mortem analysis. This excludes the possibility of major neurochemical compensation in the brain of EphA4^?/? mice. In summary, we have demonstrated for the first time the behavioral significance of EphA4 disruption, supporting further investigation of EphA4 as a possible target for behavioral interventions where habituation deficits are prominent.     

L-arginine supplementation improves responses to injury and inflammation in dextran sulfate sodium colitis

Inflammatory bowel disease (IBD), consisting of Crohn's disease and ulcerative colitis (UC), results in substantial morbidity and is difficult to treat. New strategies for adjunct therapies are needed. One candidate is the semi-essential amino acid, L-arginine (L-Arg), a complementary medicine purported to be an enhancer of immunity and vitality in the lay media. Using dextran sulfate sodium (DSS) as a murine colonic injury and repair model with similarities to human UC, we assessed the effect of L-Arg, as DSS induced increases in colonic expression of the y(+) cationic amino acid transporter 2 (CAT2) and L-Arg uptake. L-Arg supplementation improved the clinical parameters of survival, body weight loss, and colon weight, and reduced colonic permeability and the number of myeloperoxidase-positive neutrophils in DSS colitis. Luminex-based multi-analyte profiling demonstrated that there was a marked reduction in proinflammatory cytokine and chemokine expression with L-Arg treatment. Genomic analysis by microarray demonstrated that DSS-treated mice supplemented with L-Arg clustered more closely with mice not exposed to DSS than to those receiving DSS alone, and revealed that multiple genes that were upregulated or downregulated with DSS alone exhibited normalization of expression with L-Arg supplementation. Additionally, L-Arg treatment of mice with DSS colitis resulted in increased ex vivo migration of colonic epithelial cells, suggestive of increased capacity for wound repair. Because CAT2 induction was sustained during L-Arg treatment and inducible nitric oxide (NO) synthase (iNOS) requires uptake of L-Arg for generation of NO, we tested the effect of L-Arg in iNOS(-/-) mice and found that its benefits in DSS colitis were eliminated. These preclinical studies indicate that L-Arg supplementation could be a potential therapy for IBD, and that one mechanism of action may be functional enhancement of iNOS activity.     

A single post-exposure oxime RS194B treatment rapidly reactivates acetylcholinesterase and reverses acute symptoms in macaques exposed to diethylphosphorothioate parathion and chlorpyrifos insecticides

Millions of individuals globally suffer from inadvertent, occupational or self-harm exposures from organophosphate (OP) insecticides, significantly impacting human health. Similar to nerve agents, insecticides are neurotoxins that target and inhibit acetylcholinesterase (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include administration of atropine with an oxime to reactivate the OP-inhibited AChE. However, animal model studies and recent clinical trials using insecticide-poisoned individuals have shown minimal clinical benefits of the currently approved oximes and their efficacy as antidotes has been debated. Currently used oximes either reactivate poorly, do not readily cross the blood–brain barrier (BBB), or are rapidly cleared from the circulation and must be repeatedly administered. Zwitterionic oximes of unbranched and simplified structure, for example RS194B, have been developed that efficiently cross the BBB resulting in reactivation of OP-inhibited AChE and dramatic reversal of severe clinical symptoms in mice and macaques exposed to OP insecticides or nerve agents. Thus, a single IM injection of RS194B has been shown to rapidly restore blood AChE and butyrylcholinesterase (BChE) activity, reverse cholinergic symptoms, and prevent death in macaques following lethal inhaled sarin and paraoxon exposure. The present macaque studies extend these findings and assess the ability of post-exposure RS194B treatment to counteract oral poisoning by highly toxic diethylphosphorothioate insecticides such as parathion and chlorpyrifos. These OPs require conversion by P450 in the liver of the inactive thions to the active toxic oxon forms, and once again demonstrated RS194B efficacy to reactivate and alleviate clinical symptoms within 60 mins of a single IM administration. Furthermore, when delivered orally, the Tmax of RS194B at 1–2 h was in the same range as those administered IM but were maintained in the circulation for longer periods greatly facilitating the use of RS194B as a non-invasive treatment, especially in isolated rural settings.

     

Paternal origins of complete hydatidiform moles proven by whole genome single-nucleotide polymorphism haplotyping

Complete hydatidiform moles (CHMs) are diploid tumors that result from fertilization of an empty ovum by a haploid 23,X sperm. In most cases, the resulting duplication of the genome gives rise to a 46,XX genotype and is thought to be androgenetic in origin. If this hypothesis is correct, then the genotypes of all polymorphic markers in CHMs should be homozygous. We used a dense set of single-nucleotide polymorphism (SNP) markers, evenly spaced throughout the genome, to definitively test this hypothesis. We genotyped genomic DNA samples from five CHMs and their corresponding maternal samples with 1494 SNP markers using high-density microarrays (HuSNP). As predicted, the maternal samples were heterozygous at >25% of the markers, which is consistent with the expected average heterozygosity of this panel of SNPs. In contrast, the five CHM samples were heterozygous at <0.75% of the SNP markers, which shows that these diploid tumors consist of a duplicated set of chromosomes. Because the CHM genotypes represent the haplotypes of their genomes, our results show that long-range haplotypes can be obtained easily with this resource and that a collection of such samples is a simple way to obtain reference haplotypes for association studies in various populations.     

Insulin and Endothelin in the Acute Regulation of Adiponectin in Vivo in Humans

Objective: In vitro, insulin and endothelin (ET) both modulate adiponectin secretion from adipocyte cell lines. The current studies were performed to assess whether endogenous ET contributes to the acute action of insulin infusions on adiponectin levels in vivo in humans. Research Methods and Procedures: We studied 17 lean and 20 obese subjects (BMI 21.8 ± 2.2 and 34.0 ± 5.0 kg/m², respectively). Hyperinsulinemic euglycemic clamp studies were performed using insulin infusion rates of 10, 30, or 300 mU/m² per minute alone or with concurrent infusion of BQ123, an antagonist of type A ET receptors. Circulating adiponectin levels were assessed at baseline and after achievement of steady‐state glucose with the insulin infusion. Results: Adiponectin levels were lower in obese than lean subjects (6.76 ± 3.66 vs. 8.37 ± 2.79 μg/mL, p = 0.0148 adjusted for differences across gender). Insulin infusions suppressed adiponectin by a mean of 7.8% (p < 0.0001). In a subset of 13 lean and 14 obese subjects for whom data with and without BQ123 were available, there was no evident effect of BQ123 to modulate clamp‐associated suppression of adiponectin (p = 0.16). Surprisingly, there was no evident relationship between steady‐state insulin concentrations and adiponectin suppression (r = 0.14, p = 0.30), and again no effect of BQ123 to modify this relationship was seen. Discussion: Despite baseline differences in adiponectin levels, we observed equal suppression of adiponectin with insulin infusions in lean and obese subjects. ET receptor antagonism with BQ123 did not modulate this effect, suggesting that endogenous ET does not have a role in modifying the acute effects of insulin on adiponectin production and/or disposition.     

Reexamining Obesigenic Families: Parents’ Obesity‐related Behaviors Predict Girls’ Change in BMI

Objective: It has been shown that girls from families in which mothers and fathers had high dietary intake and low physical activity (i.e., obesigenic families) were at increased risk of obesity from ages 5 to 7 years. This follow‐up study uses additional data collected when girls were 9 and 11 years old to examine whether girls from obesigenic families continued to show greater increases in BMI over time and reported unhealthy dietary and activity patterns. Research Methods and Procedures: Families from the original cohort were reexamined when girls were 9 and 11 years of age. Parents’ and girls’ BMI, dietary intake, and physical activity and girls’ percentage body fat and television viewing were assessed. Results: In comparison with girls from non‐obesigenic families, girls from obesigenic families showed greater increases in BMI and BMI z score from ages 5 to 7 years that were maintained across ages 7 to 11 years. Furthermore, girls from obesigenic families had higher percentage body fat at ages 9 and 11 years. These results were independent of parents’ BMI. Additional findings showed that girls from obesigenic families had diets higher in percentage fat and had higher levels of television viewing than girls from non‐obesigenic families. Discussion: The environment that parents create, by way of their own dietary and physical activity behaviors, may have a lasting negative effect on children's weight trajectories and their emerging obesity risk behaviors, such as their dietary patterns. These findings further highlight the importance of the family in establishing children's obesity risk and the necessity of targeting parents of young children in obesity prevention efforts.