Multigenerational Brazilian family with malignant hyperthermia and a novel mutation in the RYR1 gene

Malignant hyperthermia (MH) is a pharmacogenetic disease triggered in susceptible individuals by the administration of volatile halogenated anesthetics and/or succinylcholine, leading to the development of a hypermetabolic crisis, which is caused by abnormal release of Ca2+ from the sarcoplasmic reticulum, through the Ca2+ release channel ryanodine receptor 1 (RyR1). Mutations in the RYR1 gene are associated with MH in the majority of susceptible families. Genetic screening of a 5-generation Brazilian family with a history of MH-related deaths and a previous MH diagnosis by the caffeine halothane contracture test (CHCT) in some individuals was performed using restriction and sequencing analysis. A novel missense mutation, Gly4935Ser, was found in an important functional and conserved locus of this gene, the transmembrane region of RyR1. In this family, 2 MH-susceptible individuals previously diagnosed with CHCT carry this novel mutation and another 24 not previously diagnosed members also carry it. However, this same mutation was not found in another MH-susceptible individual whose CHCT was positive to the test with caffeine but not to the test with halothane. None of the 5 MH normal individuals of the family, previously diagnosed by CHCT, carry this mutation, nor do 100 controls from control Brazilian and USA populations. The Gly4932Ser variant is a candidate mutation for MH, based on its co-segregation with disease phenotype, absence among controls and its location within the protein.

Use of the caffeine-halothane contracture test for the diagnosis of malignant hyperthermia in Brazil

Malignant hyperthermia (MH) is a pharmacogenetic disease triggered by volatile anesthetics and succinylcholine. Deaths due to MH have been reported in Brazil. The first Malignant Hyperthermia Diagnostic and Research Center in Latin America was inaugurated in 1993 at the Federal University of Rio de Janeiro, Brazil. The center followed the diagnostic protocols of the North America MH Group, in which the contractures of biopsies from the vastus lateralis muscle are analyzed after exposure to caffeine and halothane (CHCT). CHCT was performed in individuals who survived, their relatives and those with signs/symptoms somewhat related to MH susceptibility (MHS). Here, we report data from 194 patients collected over 16 years. The Southeast (N = 110) and South (N = 71) represented the majority of patients. Median age was 25 (4-70) years, with similar numbers of males (104) and females (90). MHS was found in 90 patients and 104 patients were normal. Abnormal responses to both caffeine and halothane were observed in 59 patients and to caffeine or halothane in 20 and 11 patients, respectively. The contracture of biopsies from MHS exposed to caffeine and halothane was 1.027 ± 0.075 g (N = 285) and 4.021 ± 0.255 g (N = 226), respectively. MHS was found in patients with either low or high blood creatine kinase and also, with a low score on the clinical grading scale. Thus, these parameters cannot be used with certainty to predict MHS. We conclude that the CHCT protocol described by the North America MH Group contributed to identification of MHS in suspected individuals at an MH center in Brazil with 100% sensitivity and 65.7% specificity.

Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases

Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.

Use of inflammatory molecules to predict the occurrence of fever in onco-hematological patients with neutropenia

Febrile neutropenia remains a frequent complication in onco-hematological patients, and changes in the circulating level of inflammatory molecules (IM) may precede the occurrence of fever. The present observational prospective study was carried out to evaluate the behavior of plasma tumor necrosis factor alpha (TNF-α), soluble TNF-α I and II receptors (sTNFRI and sTNFRII), monocyte chemoattractant protein-1 [MCP-1 or chemokine (c-c motif) ligand 2 (CCL2)], macrophage inflammatory protein-1α (MIP-1α or CCL3), eotaxin (CCL11), interleukin-8 (IL-8 or CXCL8), and interferon-inducible protein-10 (IP-10 or CXCL10) in 32 episodes of neutropenia in 26 onco-hematological patients. IM were tested on enrollment and 24-48 h before the onset of fever and within 24 h of the first occurrence of fever. Eight of 32 episodes of neutropenia did not present fever (control group) and the patients underwent IM tests on three different occasions. sTNFRI levels, measured a median of 11 h (1-15) before the onset of fever, were significantly higher in patients presenting fever during follow-up compared to controls (P = 0.02). Similar results were observed for sTNFRI and CCL2 levels (P = 0.04 for both) in non-transplanted patients. A cut-off of 1514 pg/mL for sTNFRI was able to discriminate between neutropenic patients with or without fever during follow-up, with 65% sensitivity, 87% specificity, and 93% positive predictive value. Measurement of the levels of plasma sTNFRI can be used to predict the occurrence of fever in neutropenic patients.

Relationship between genetic mutation variations and acute-phase reactants in the attack-free period of children diagnosed with familial Mediterranean fever

Familial Mediterranean fever (FMF) is a periodic autoinflammatory disease characterized by chronic inflammation. This study investigated the relationship between acute-phase reactants and gene mutations in attack-free periods of childhood FMF. Patients diagnosed with FMF were divided into four groups based on genetic features: no mutation, homozygous, heterozygous, and compound heterozygous. These groups were monitored for 2 years, and blood samples were collected every 6 months during attack-free periods. Erythrocyte sedimentation rate, C-reactive protein, fibrinogen, and white blood cell count were measured. A disease severity score was determined for each patient. Mean values for erythrocyte sedimentation rate and fibrinogen were significantly different in the homozygous group. White blood cell count and C-reactive protein were similar between the groups. Disease severity score was higher in patients with the M694V mutation than in individuals without the mutation, as well as in those with other mutation groups. Periodic follow-up of patients with FMF MEFV mutations in subjects with acute-phase reactants may be useful in the prevention of morbidity.

Relationship between deep venous thrombosis and inflammatory cytokines in postoperative patients with malignant abdominal tumors

Deep venous thrombosis (DVT) is a common surgical complication in cancer patients and evidence that inflammation plays a role in the occurrence of DVT is increasing. We studied a population of cancer patients with abdominal malignancies with the aim of investigating whether the levels of circulating inflammatory cytokines were associated with postoperative DVT, and to determine the levels in DVT diagnoses. The serum levels of C-reactive protein (CRP), interleukins (IL)-6 and IL-10, nuclear transcription factor-κB (NF-κB) and E-selectin (E-Sel) were determined in 120 individuals, who were divided into 3 groups: healthy controls, patients with and patients without DVT after surgery for an abdominal malignancy. Data were analyzed by ANOVA, Dunnet's T3 test, chi-square test, and univariate and multivariate logistic regression as needed. The CRP, IL-6, NF-κB, and E-Sel levels in patients with DVT were significantly higher than those in the other groups (P<0.05). The IL-10 level was higher in patients with DVT than in controls but lower than in patients without DVT. Univariate analysis revealed that CRP, IL-6, NF-κB, and E-Sel were statistically associated with the risk of DVT (OR=1.98, P=0.002; OR=1.17, P=0.000; OR=1.03, P=0.042; and OR=1.38, P=0.003; respectively), whereas IL-10 had a protective effect (OR=0.94, P=0.011). Multivariate analysis showed that E-Sel was an independent risk factor (OR=1.41, P=0.000). Thus, this study indicated that an increased serum level of E-Sel was associated with increased DVT risk in postoperative patients with abdominal malignancy, indicating that E-Sel may be a useful predictor of diagnosis of DVT.