Recém-nascido com deficiência de glicerol quinase: um caso clínico / Neonate with glycerol kinase deficiency: A clinical case

OBJETIVOS: Relatar o caso de um recém-nascido com deficiência de glicerol quinase, no qual foi identificada uma mutação isolada ainda não descrita no gene GK. DESCRIÇÃO DO CASO: Um recém-nascido com 10 dias de vida foi trazido ao serviço de urgência por recusa alimentar com 24 horas de evolução. Ao exame físico apresentava perda de 31% do peso de nascimento e sinais de desidratação. Os exames laboratoriais constataram presença de acidose metabólica com anion gap aumentado, creatinina 2,41mg/dL, ureia 306 mg/dL, hipernatremia (182mEq/L), hipercalemia (6,8mEq/L), hipercloremia (151mEq/L), transaminase glutâmico-oxalacética 879U/L, transaminase glutâmico-pirúvica 243U/L, triglicerídeos 725mg/dL. A cromotagrafia de ácidos orgânicos revelou hiperglicerolemia e glicerolúria compatíveis com deficiência de glicerol quinase. O estudo genético revelou uma mutação ainda não descrita: c.187T>C (p.S63P) em hemizigotia no gene GK. CONCLUSÕES: A causa mais frequente de desidratação hipernatrêmica no período neonatal é a hipogalatia materna. Nos casos mais graves de desidratação outras etiologias devem ser consideradas, incluindo causas metabólicas como a deficiência de glicerol quinase. Neste caso foi encontrada uma mutação no gene GK ainda não descrita.

AIMS: To report the case of a newborn with glycerol kinase deficiency, in which an isolated mutation not yet described in the GK gene was identified. CASE DESCRIPTION: A neonate with 10 days of age was brought to the emergency department for refusal to feed with 24 hours of evolution. Physical examination showed a loss of 31% of birth weight and signs of dehydration. Laboratory tests revealed a metabolic acidosis with increased anion gap, creatinine 2.41mg/dL, urea 306mg/dL, hypernatremia (182mEq/L), hyperkalemia (6.8mEq/L), hyperchloremia (151mEq/L), glutamic-oxalacetic transaminase 879U/L, glutamic-pyruvic transaminase 243U/L, triglycerides 725mg/dL. Chromotagraphy of organic acids revealed hyperglycerolemia and glyceroluria compatible with glycerol kinase deficiency. The genetic study revealed a mutation not yet described: c.187T>C (p.S63P) as hemizygote status in the GK gene. CONCLUSIONS: The most frequent cause of hypernatremic dehydration in the neonatal period is maternal hypogalactia. In more severe cases of dehydration, other etiologies should be considered, including metabolic causes such as glycerol kinase deficiency. In this case a mutation not yet described in the GK gene was found.

Using Pichia pastoris to produce recombinant glycerol kinase / Produção de glycerol quinase recombinante utilizando o sistema Pichia pastoris

The methylotrophic yeast Pichia pastoris has been developed into an efficient expression system for the production of recombinant protein under the tight control of the methanol-induced alcohol oxidase promoter (pAOX1). In this study, a 2.5-liter culture system was developed for the growth of a P. pastoris strain bearing the GUT1 gene from Saccharomyces cerevisiae for the expression of recombinant glycerol kinase (GK). The best culture conditions to produce high levels of secreted GK were investigated by growing the recombinant strain of P. pastoris in shake flasks and a fermenter. Cell growth and enzyme production were found to be optimal after two days of growth. Enzyme production was affected by the nitrogen source, Difco peptone being the most appropriate for this purpose. Three different rates of air flow (1 to 3 L/min) were tested to observe their effect on cell growth and the secretion of GK into a medium containing 1% methanol as the sole carbon source. Increasing the rate of air bubbling in the culture medium enhanced both cell growth and GK activity, reaching a dry biomass of 7.84 mg/mL, cell viability of 98.4% and a maximal GK activity of 1.57 U/mL, at a flow rate of 2.0 L/minute, at 30° C and pH 6.0. Moreover, the enzyme activity in the P. pastoris culture medium was 2.3 times higher under these conditions than in the shake-flask culture, demonstrating the significant influence of aeration on biomass production and GK activity secreted by P. pastoris...

A levedura metilotrófica Pichia pastoris possui um sistema de expressão eficiente para a produção de proteínas recombinantes. A indução da produção da proteína de interesse é feita com metanol, que é capaz de ativar a transcrição do gene de interesse clonado sob controle do promotor do gene AOX1. Um meio de cultura de 2.5 litros foi elaborado para o crescimento da cepa Pichia pastoris construída com o gene GUT1 de Saccharomyces cerevisiae para expressar a enzima recombinante glicerol quinase (GK). As condições ideais de cultura, para alcançar altos níveis de expressão de GK foram investigados em crescimentos realizados em frascos e fermentador. Crescimento celular e produção de enzima atingiram valores ótimos em dois dias de cultura. A produção enzimática foi afetada pela fonte de nitrogênio no meio. Peptona da marca Difco foi a fonte de nitrogênio mais adequada para a expressão destaenzima. Três diferentes concentrações (1-3 L / min) defluxo de ar foram analisados em ensaios de crescimento celular e secreção da GK, no meio contendo 1 % demetanol como única fonte de carbono. O aumento do fluxo do ar no meio de cultura produziu melhores resultados para o crescimento celular e atividade da GK, atingindo 7,84 mg / mL de biomassa seca e 98,4%de viabilidade. A máxima atividade de GK foi de 1,57U / mL, com a concentração de fluxo de ar de 2,0 L/ minuto a 30 ° C e pH 6.0. O aumento da atividade enzimática foi 2,3 vezes maior no meio de cultura da Pichia pastoris nestas condições, revelando a influência deste parâmetro na produção de biomassa e atividade da GK...

The preparation and identification of diagnostic recombinant glycerol kinase / 生物医学工程学杂志

In order to establish an efficient and low-cost production procedure of recombinant glycerol kinase (r-GK), we expressed the r-GK gene at high level in E. coli by induction with lactose on a large-scale fermentation of 300L. The results showed that the biomass concentration reached OD600 of 42 and the expression of r-GK in E. coli accounted for about 30% of total soluble protein. The cell-free extract was processed by selective thermo-denaturation and then purified with Ni sepharose FF column chromatography. Finally, highly purified r-GK was obtained and its purity reached 97% by using analysis on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), polyacrylamide gel electrophoresis (PAGE) and gradient polyacrylamide gel electrophoresis (Gradient PAGE). Further identification study showed that the molecular weight of r-GK was 120kDa with two subunit of 58kDa. Contaminants of NADH oxidase and catalase were not detected in the sample pool of r-GK. The purified r-GK was able to retain about 85% of its initial activity at 4 degrees C for 30 days. After lyophilized, it can retain 93% of its initial activity at 4 degrees C for one year.

Central precocious puberty in a patient with X-linked adrenal hypoplasia congenita and Xp21 contiguous gene deletion syndrome

X-linked adrenal hypoplasia congenita is caused by the mutation of DAX-1 gene (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1), and can occur as part of a contiguous gene deletion syndrome in association with glycerol kinase (GK) deficiency, Duchenne muscular dystrophy and X-linked interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) gene deficiency. It is usually associated with hypogonadotropic hypogonadism, although in rare cases, it has been reported to occur in normal puberty or even central precocious puberty. This study addresses a case in which central precocious puberty developed in a boy with X-linked adrenal hypoplasia congenita who had complete deletion of the genes DAX-1, GK and IL1RAPL1 (Xp21 contiguous gene deletion syndrome). Initially he was admitted for the management of adrenal crisis at the age of 2 months, and managed with hydrocortisone and florinef. At 45 months of age, his each testicular volumes of 4 mL and a penile length of 5 cm were noted, with pubic hair of Tanner stage 2. His bone age was advanced and a gonadotropin-releasing hormone (GnRH) stimulation test showed a luteinizing hormone peak of 8.26 IU/L, confirming central precocious puberty. He was then treated with a GnRH agonist, as well as steroid replacement therapy. In Korea, this is the first case of central precocious puberty developed in a male patient with X-linked adrenal hypoplasia congenita.

Central precocious puberty in a patient with X-linked adrenal hypoplasia congenita and Xp21 contiguous gene deletion syndrome

X-linked adrenal hypoplasia congenita is caused by the mutation of DAX-1 gene (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1), and can occur as part of a contiguous gene deletion syndrome in association with glycerol kinase (GK) deficiency, Duchenne muscular dystrophy and X-linked interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) gene deficiency. It is usually associated with hypogonadotropic hypogonadism, although in rare cases, it has been reported to occur in normal puberty or even central precocious puberty. This study addresses a case in which central precocious puberty developed in a boy with X-linked adrenal hypoplasia congenita who had complete deletion of the genes DAX-1, GK and IL1RAPL1 (Xp21 contiguous gene deletion syndrome). Initially he was admitted for the management of adrenal crisis at the age of 2 months, and managed with hydrocortisone and florinef. At 45 months of age, his each testicular volumes of 4 mL and a penile length of 5 cm were noted, with pubic hair of Tanner stage 2. His bone age was advanced and a gonadotropin-releasing hormone (GnRH) stimulation test showed a luteinizing hormone peak of 8.26 IU/L, confirming central precocious puberty. He was then treated with a GnRH agonist, as well as steroid replacement therapy. In Korea, this is the first case of central precocious puberty developed in a male patient with X-linked adrenal hypoplasia congenita.

Construction of a lentiviral vector for RNA interference of glycerol kinase gene in human hepatocytes in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a lentiviral vector for RNA interference (RNAi) of human glycerol kinase (GK) gene to stably down-regulate GK expression in human hepatocytes.</p><p><b>METHODS</b>The sequence of siRNA for GK interference were cloned into the pSicoR vector. Following packaging in 293T cells, the lentivirus was titrated using fluorescence activated cell sorting. Human hepatocyte L02 cells was infected with the lentivirus and the expression of GK was analyzed using Western blotting.</p><p><b>RESULTS</b>The lentiviral particle pSicoR-GK was successfully packaged with a virus titer reaching 3×10(7) pfu/ml. The expression level of GK protein was down-regulated to 20% of the control level in L02 cells infected with the lentivirus.</p><p><b>CONCLUSION</b>The lentiviral vector for RNAi of human GK gene has been successfully constructed, which can significantly down-regulate GK expression in human hepatocytes.</p>

Complex glycerol kinase deficiency in three children / 中国当代儿科杂志

Glycerol kinase deficiency (GKD), a rare X-linked recessive disorder, is classified into two types: isolated and complex. Complex GKD is an Xp21 contiguous gene deletion involving the glycerol kinase locus together with the adrenal hypoplasia congenita (AHC) or Duchenne muscular dystrophy (DMD) loci or both. Its clinical features depend on the involved loci. GKD can be confirmed by an elevated urinary glycerol concentration tested by gas chromatography mass spectrometry (GC/MS). The three cases reported here were all male, presenting symptoms from neonatal period. The predominant clinical profile was characterized by hypoadrenocorticism, glyceroluria and Duchenne muscular dystrophy. After receiving a low fat diet and glucocorticoid replacement, they improved with relieved symptoms of hypoadrenocorticism. But they had significant developmental delays and myasthenia. In the follow-up two of them died of adrenal crisis.

Two Cases of Xp21 Contiguous Gene Deletion Syndrome

On chromosome Xp21 region, several genes such as glycerol kinase (GK) gene, adrenal hypoplasia congenita gene and Duchenne muscular dystrophy gene are located contiguously. Xp21 contiguous gene deletion syndrome involves the glycerol kinase gene deletion together with the adrenal hypoplasia congenita and/or Duchenne muscular dystrophy gene. The clinical features of a patient with a Xp21 contiguous gene deletion syndrome are sum of each disease,psychomotor retardation and lethargy for glycerol kinase deficiency, hyperpigmentation and salt wasting dehydration for congenital adrenal hypoplasia and muscular weakness and hypotonia for Duchenne muscular dystrophy. We experienced and reviewed two cases of Xp21 contiguous gene deletion syndrome with literatures.

Complex glycerol kinase deficiency: an X-linked disorder associated with adrenal hypoplasia congenita.

Complex glycerol kinase deficiency (GKD) results from the contiguous deletion on Xp21 of all or part of the gene for glycerol kinase together with that for adrenal hypoplasia congenita (AHC) and /or Duchenne muscular dystrophy (DMD). The authors present the case of a newborn whose initial issues were refractory hypoglycaemia along with hyponatremia and hyperkalemia. He also had low serum cortisol levels and raised urinary excretion of glycerol and required steroid supplementation. His creatinine phosphokinase (CPK) levels were normal. Molecular studies revealed a contiguous Xp21 deletion. Therapy in such cases must be prompt and includes correction of hypoglycaemia and dyselectrolytemia, a low fat diet and steroid replacement.

Application of New Pathogenesis on the Drug Treatment of Diabetes

Patients with type 2 diabetes mellitus are associated with insulin resistance and/or impaired insulin secretion. Previous observations indicate that patients with type 2 diabetes tend to have an impaired insulin response after a glycemic load. Recently it has been reported that hyperglycemia after a glucose load is itself a risk factor for the development of cardiovascular complications in the absence of elevated fasting plasma glucose. There are several points to be addressed for the application of new pathogenesis to diabetes treatment. One of them is the association between postprandial hyperglycemia and mortality from cardiovascular diseases. For the management of postprandial hyperglycemia inhibitors ofglucosidase and rapidacting insulin secretagogues have beneficial effects. Alphaglucosidase inhibitors in combination with diet therapy ameliorate insulin resistance and reduce the blood sugar level. A rapidly acting insulin secretagogue, such as repaglinide, lowers postprandial glucose levels without asignificant gain of body weight. These drugs may protect pancreaticcells from postprandial glucose toxicity and prevent the progression of diabetes. Both metformin and thiazolidinedione derivative (TZDs) improve insulin resistance, the major pathogenetic background of type 2 diabetes, and decrease blood glucose levels without stimulating, insulin secretion. Metformin inhibits glucose output from the liver, while TZDs increase glucose utilization in the peripheral tissues. In addition, it has been indicated that these agents ameliorate the metabolic syndrome beyond lowering the glucose level. Molecular targets for these agents have recentl been revealed ; AMPactivated protein kinase for metfOrnin and adiponectin, while PPAR for TZDs that induce gene expression of adipocyte glycerol kinase and adiponectin. Insulin-sensitizing agents are clinically useful for obese diabetic patients with insulin resistance. However, periodical examinations are necessary to avoid serious adverse effects such as lactic acidosis, although rare, by metformin and liver injury by TZDs. The advantage of insulin therapy for type 2 diabetic patients is still controversial. However, in many intervention studies, the intensive insulin therapy provided promising effects on preventing cardiovascular diseases. Moreover, insulin has been shown to stimulate nitric oxide production by cultured endothelial cells and to suppress the expression of intercellular adhesion molecule1 at least in vitro. In view of this antiinflammatory effect, longterm insulin therapy may potentially have an antiatherogenic effect.

A Case of Xp21 Contiguous Gene Deletion Syndrome with Hyperglycerolemia, Congenital Adrenal Hypoplasia and Duchenne Muscular Dystrophy

On Xp21 region several genes such as adrenal hypoplasia congenita(AHC) gene, glycerol kinase (GK) gene and Duchenne muscular dystrophy(DMD) gene are located contiguously. If there is a long deletion in that region, various combination of genetic defect can be occurred from one kind of genetic defect to all three kinds of genetic defect simultaneously. In case of more than two genetic defects simultaneously, we call it contiguous gene deletion syndrome. The major clinical manifestations of the Xp21 contiguous gene deletion syndrome are sum of each diseases, electrolyte imbalance and hyperpigmentation for adrenal hypoplasia congenita, psychomotor retardation, letharginess and convulsion for glycerol kinase deficiency and muscle weakness and hypotonia for Duchenne muscular dystrophy. Goals of the treatment are control of each disorders, glucocorticoid and mineralocorticoid for adrenal hypoplasia congenita, low fat diet and prevention of fasting and hypercatabolic status for glycerol kinase deficiency and physiotherapy for Duchenne muscular dystrophy. In case of hyponatremia and hyperkalemia combined with hyperpigmentation, adrenal hypoplasia congenita could be suspected. In glycerol kinase deficiency, markedly elevated glycerol excretion can be detected on urine organic acid analysis by gaschromatography with mass spectrometry. On Duchenne muscular dystrophy, creatinine kinase is markedly elevated on chemistry. We report here first Korean case of Xp21 contiguous gene deletion syndrome of adrenal hypoplasia congenita, glycerol kinase deficiency and Duchenne muscular dystrophy.