Rescuing the aberrant sex development of H3K9 demethylase Jmjd1a-deficient mice by modulating H3K9 methylation balance.

Histone H3 lysine 9 (H3K9) methylation is a hallmark of heterochromatin. H3K9 demethylation is crucial in mouse sex determination; The H3K9 demethylase Jmjd1a deficiency leads to increased H3K9 methylation at the Sry locus in embryonic gonads, thereby compromising Sry expression and causing male-to-female sex reversal. We hypothesized that the H3K9 methylation level at the Sry locus is finely tuned by the balance in activities between the H3K9 demethylase Jmjd1a and an unidentified H3K9 methyltransferase to ensure correct Sry expression. Here we identified the GLP/G9a H3K9 methyltransferase complex as the enzyme catalyzing H3K9 methylation at the Sry locus. Based on this finding, we tried to rescue the sex-reversal phenotype of Jmjd1a-deficient mice by modulating GLP/G9a complex activity. A heterozygous GLP mutation rescued the sex-reversal phenotype of Jmjd1a-deficient mice by restoring Sry expression. The administration of a chemical inhibitor of GLP/G9a enzyme into Jmjd1a-deficient embryos also successfully rescued sex reversal. Our study not only reveals the molecular mechanism underlying the tuning of Sry expression but also provides proof on the principle of therapeutic strategies based on the pharmacological modulation of epigenetic balance.

First Japanese Case of Carnitine Palmitoyltransferase II Deficiency with the Homozygous Point Mutation S113L.

Carnitine palmitoyltransferase II (CPT II) deficiency is a rare inherited disorder related to recurrent episodes of rhabdomyolysis. The adult myopathic form of CPT II deficiency is relatively benign and difficult to diagnose. The point mutation S113L in CPT2 is very common in Caucasian patients, whereas F383Y is the most common mutation among Japanese patients. We herein present a case of CPT II deficiency in a Japanese patient homozygous for the missense mutation S113L. The patient showed a decreased frequency of rhabdomyolysis recurrence after the administration of a diet containing medium-chain triglyceride oil and supplementation with carnitine and bezafibrate.

Induction and maintenance of anti-influenza antigen-specific nasal secretory IgA levels and serum IgG levels after influenza infection in adults.

OBJECTIVES: To determine the induction and changes in anti-influenza virus secretory IgA (s-IgA) levels in nasal washes and serum IgG levels in patients with influenza. METHODS: The study recruited 16 patients with influenza aged 35.6 ± 9.6 years in 2007/2008 and 2008/2009 seasons. Nasal washes and serum were obtained throughout the first year. Anti-viral s-IgA levels and neutralization activities in nasal washes, and serum anti-viral IgG levels and hemagglutination inhibition (HI) titers were measured. RESULTS: Anti-viral(H1N1) s-IgA to total IgA ratio and neutralizing antibody titer were low in nasal washes of all patients, whereas serum levels of anti-viral IgG and HI titers varied widely at day 1.4 ± 1.0 postinfection. Both nasal s-IgA and serum IgG levels later increased significantly, reaching peak levels at day 9.6 ± 3.3 postinfection. The induced nasal s-IgA then returned toward the initial levels within 300 days, although the levels at day 143 ± 70 were 3.03-fold of the initial. Individual serum IgG levels also returned toward the initial levels within 300 days, although the mean levels remained high probably because of re-infection in a subgroup of patients. Although influenza A (H3N2) was a minor epidemic subtype in both flu seasons, a significant rise in nasal anti-viral (H3N2) s-IgA levels and a slightly increase in serum IgG levels were noted. CONCLUSION: Low levels of nasal anti-viral s-IgA and neutralizing antibody were noted compared with a wide range of serum anti-viral IgG and HI titers at the onset of infection. Elevated s-IgA and IgG returned toward the initial levels within 300 days of infection with minor exceptions.

Thermolabile CPT II variants and low blood ATP levels are closely related to severity of acute encephalopathy in Japanese children.

Despite the decrease in Reye syndrome after the discontinuation of aspirin, acute encephalopathy (non-Reye syndrome type) has been continually reported in Japan. Recent studies suggested that the thermolabile phenotype of carnitine palmitoyltransferase II (CPT II) variation [F352C] was closely related to the pathomechanism of influenza-associated encephalopathy (IAE) in Japanese, causing mitochondrial ATP utilization failure during periods of high fever, resulting in brain edema. So, we analyzed CPT II polymorphism and peripheral blood ATP levels as a signal of "energy crisis" in 12 and 10 patients with acute encephalopathy, respectively. Out of the 12 patients with acute encephalopathy, six showed thermolabile CPT II variants [F352C], and of these six, two patients died in spite of intensive care. In contrast, the remaining six patients with no thermolabile CPT II variant [F352C] showed a relatively mild clinical course. Blood ATP levels of the 10 patients in the acute phase of encephalopathy were significantly lower than those during the convalescent phase and also those of patients with febrile seizure status. Our data suggest that the thermolabile F352C CPT II variant, found only in Japanese, might be one of the predisposing factors to trigger the pathomechanism of acute encephalopathy in the Japanese population, and that it is causally related to the severity of disease. The decreased blood ATP level seems to reflect systemic mitochondrial dysfunction including the blood brain barrier during the acute phase of encephalopathy.

Characterization of compound missense mutation and deletion of carnitine palmitoyltransferase II in a patient with adenovirus-associated encephalopathy.

BACKGROUND: In mammals, carnitine palmitoyltransferase (CPT) system is a pivotal component of energy metabolism through mitochondrial fatty acid oxidation. The majority of patients with fatal or handicapped influenza-associated encephalopathy exhibit thermolabile compound homo/heterozygous mutations of CPT II. OBJECTIVE: Compound CPT II mutations, [c.647A>G (p.Q216R)], [c.1102G>A (p.V368I)], [c.1939A>G (p.M647V)] and [c.745delG (p.G249EfsX16)], were found in a patient with adenovirus-associated encephalopathy and his family. The properties of these CPT II mutations were analyzed in COS-7 cells. METHODS: CPT II mutations in the patient and his family were expressed in COS-7 cells and their molecular masses, enzyme activities, thermal instabilities and half-lives were analyzed. RESULTS: We identified two novel CPT II mutations in the patient, [c.647A>G (p.Q216R)] and [c.745delG (p.G249EfsX16)]. The CPT II Q216R mutation showed mild reduction of activity, thermal instability and short half-life but compound mutations with Q216R+V368I+M647V showed further enhancement of these disabilities, although mutations V368I and M647V had no such effects. CPT II mutation [c.745delG (p.G249EfsX16)] abolished enzyme activity and showed short half-life. CONCLUSION: The thermal instability and short half-life of the novel CPT II mutations, [c.647A>G (p.Q216R)] and [c.745delG (p.G249EfsX16)], could play important roles in energy crisis in the pathogenesis of virus-associated encephalopathy.

Bezafibrate upregulates carnitine palmitoyltransferase II expression and promotes mitochondrial energy crisis dissipation in fibroblasts of patients with influenza-associated encephalopathy.

Influenza-associated encephalopathy (IAE) is characterized by persistently high fever, febrile convulsions, severe brain edema and high mortality. We reported previously that a large proportion of patients with disabling or fatal IAE exhibit a thermolabile phenotype of compound variants for [1055T>G/F352C] and [1102G>A/V368I] of carnitine palmitoyltransferase II (CPT II) and mitochondrial energy crisis during high fever. In the present study, we studied the effect of bezafibrate, a hypolipidemic pan-agonist of peroxisome proliferator-activated receptor (PPAR), on CPT II expression and mitochondrial energy metabolism in fibroblasts of IAE patients and wild type (WT) fibroblasts from a healthy volunteer at 37°C and 41°C. Although heat stress markedly upregulated CPT II, CPT IA and PPAR-δ mRNA expression levels, CPT II activity, ß-oxidation and ATP levels in WT and IAE fibroblasts at 41°C were paradoxically downregulated probably due to the thermal instability of the corresponding enzymes. Bezafibrate significantly enhanced the expression levels of the above mRNAs and cellular functions of these enzymes in fibroblasts at 37°C. Bezafibrate-induced increase in CPT II activity also tended to restore the downregulated ATP levels, though moderately, and improved mitochondrial membrane potential even at 41°C to the levels at 37°C in fibroblasts of IAE patients. L-carnitine, a substrate of CPT II, boosted the effects of bezafibrate on cellular ATP levels in WT and IAE fibroblasts, even in severe IAE fibroblasts with thermolabile compound variations of F352C+V368I at 37°C and 41°C. The results suggest the potential usefulness of bezafibrate for the treatment of IAE.

Thermal instability of compound variants of carnitine palmitoyltransferase II and impaired mitochondrial fuel utilization in influenza-associated encephalopathy.

Influenza-associated encephalopathy (IAE) is characterized by persistent high fever, febrile convulsions, severe brain edema, and high mortality in otherwise apparently healthy individuals. We have reported that a large proportion of patients suffering from disabling or fatal IAE, with transiently elevated serum acylcarnitine during high fever, exhibit a thermolabile phenotype of compound homo-/heterozygous variants of carnitine palmitoyltransferase II (CPT II, gene symbol CPT2). We characterized the enzymatic properties of five single and three compound CPT II variants in patients with IAE. The kinetic characteristics of WT and variant CPT IIs, expressed in COS-7 cells, indicated that the variants exert a dominant-negative effect on the homotetrameric protein of the enzyme. Among the variants, three compound variations found in patients with severe encephalopathy; [c.1055T>G (p.Phe352Cys); c.1102G>A (p.Val368Ile)], [c.1511C>T (p.Pro504Leu); c.1813G>C (p.Val605Leu)], and [c.1055T>G (p.Phe352Cys); c.1102G>A (p.Val368Ile); c.1813G>C (p.Val605Leu)], showed reduced activities, thermal instability, and short half-lives compared with the WT. Like other disease-causing mutant proteins, these variant proteins were poly-ubiquitinated and rapidly degraded by a lactacystin-sensitive proteasome pathway. COS-7 cells transfected with the compound variants had their fatty acid beta-oxidation decreased to 30-59% and intracellular ATP levels to 48-79%, and a marked reduction of mitochondrial membrane potential at 41 degrees C, compared with control cells transfected with WT at 37 degrees C. The unstable CPT II variants with decreased enzymatic activities may bring mitochondrial fuel utilization below the phenotypic threshold during high fever, and thus may play an important etiopathological role in the development of brain edema of IAE.